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This is to certify that the
dissertation entitled

INHERITANCE OI= RESISTANCE AND MOLECULAR MAPPING
OF SOYBEAN APHID RESISTANCE GENES IN SOYBEAN PI
567585A; IDENTIFICATION OF APHID RESISTANCE GENES IN
SOYBEAN USING MODIFIED NESTED ASSOCIATION MAPPING
(MNAM)

presented by

Menghan Liu

has been accepted towards fulfillment
of the requirements for the

Ph.D degree in Plant Breeding and Genetics
Crop and Soil Science

 

 

( 0.5 112/- <1 n M...

MajOr Professor’s Signature

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5/08 K:/Prolecc&Pres/ClRC/DateDue.indd

 

INHERITANCE OF RESISTANCE AND MOLECULAR MAPPING OF
SOYBEAN APHID RESISTANCE GENES IN SOYBEAN PI 567585A;
IDENTIFICATION OF APHID RESISTANCE GENES IN SOYBEAN USING
MODIFIED NESTED ASSOCIATION MAPPING (MNAM)

By

Menghan Liu

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment Ofthe requirements
for the degree Of

DOCTOR OF PHILOSOPHY

Plant Breeding and Genetics
Crop and Soil Sciences

2010

ABSTRACT

INHERITANCE OF RESISTANCE AND MOLECULAR MAPPING OF SOYBEAN
APHID RESISTANCE GENES IN SOYBEAN PI 567585A; IDENTIFICATION OF
APHID RESISTANCE GENES IN SOYBEAN USING MODIFIED NESTED
ASSOCIATION MAPPING (MNAM)

By

Menghan Liu

The soybean aphid. Aphis glycine (Matsumura). is a new major pest Of soybean in
the Midwest, including Michigan. Soybean PI 567585A has strong resistance to soybean
aphids. The inheritance of aphid resistance in P1 567585A was determined by crossing
with two susceptible soybean cultivars, Skylla and IA2070. The parents, F1, F3 plants and
F23 families were evaluated for aphid resistance in the field during the summer Of 2008
and 2009. All F . plants exhibited intermediate phenotypes tO soybean aphids. The
Observed segregation ratios in the two F 2 populations, and F23 families fitted a
segregation ratio 1:2:1. These data indicated that the aphid resistance in P1 567585A was
controlled by one major co-dominant gene. Then. the genetic basis of aphid resistance in
PI 567585A was determined. A mapping population Of 158 F45 recombinant inbred lines
(RILs) derived from the cross between PI 567585A and ‘Skylla‘ was evaluated for aphid

resistance in both greenhouse and field in 2009. A single aphid resistance gene was

mapped in an interval between Satt674 and Sct_065 on linkage group .1) using the
composite interval mapping method. The locus explained 93.1% Of the phenotypic
variation in the field trial. and is located in the same genomic region as Rag3. This single
aphid resistance gene in PI 567585A was conflmted in another F3;4 RIL population

derived from a cross between PI 567585A and IA2070.

PI 5675988 was found to possess antibiosis resistance to the soybean aphid. A
modified nested association mapping (MNAM) approach was used to locate resistance
genes in P1 5675988 on the integrated soybean linkage map. PI 5675988 was crossed
with 10 different susceptible cultivars to construct 10 recombinant inbred lines (RILs)
populations. Genomic regions on linkage groups F, G, J and M were found associated
with soybean aphid resistance in MNAM. Linkage analysis of a population of 94 BC [1:435
RILs derived from PI 5675988 and a F45 RIL population derived from E06902 were
used to confirm the MNAM results. The results of linkage analysis showed that genomic

regions on the linkage groups .1, F and N were associated with aphid resistance.

Copyright by
Menghan Liu
2010

. DEDICATION

To myfumily, who has supported so much to bring me to where I am today.

ACKNOWLEDGMENTS

I would like to appreciate the guidance of my major advisor Dr. Dechun Wang, and
the members of my guidance committee, Dr. Jim Kelly, Dr. Amy Iezzoni, and Dr.
Yuehua Cui. I especially like to acknowledge my mentor Dr. Dechun Wang for providing
me with this wonderful opportunity, encouraging me to face challenges. Also, I would
like to thank all of the soybean lab people for their help and support. I would like to give
a special thanks to Dr. Jim Kelly and Dr. Amy Iezzoni for promoting my professional
development in writing, expressing and thinking. Lastly, I would like to acknowledge my
friends in China, the Netherlands. and the US, who help me overcome all kinds of

difficulties past and present.

vi

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. ix

LIST OF FIGURES ................................................................................. xi

INTRODUCTION AND GENERAL OBJECTIVES ........................................... 1

CHAPTER ONE:

LITERATURE REVIEW ........................................................................... 3
THE SOYBEAN ................................................................................... 3
THE SOYBEAN APHID ......................................................................... 4
BIOTYPES OF SOYBEAN APHID ........................................................... 6
CHEMICAL AND NATURAL BIOLOGICAL CONTROL STRATEGIES ............ 7
GENETIC CONTROL BY HOST PLANT INSECT RESISTANCE BREEDING.....8
GENETIC MAPPING OF RESISTANCE GENE(S) IN CROP PLANTS... .............. l4
MOLECULAR BASES FOR RESISTANCE (R) GENES ................................ 22
MOLECULAR BREEDING PRACTICE ................................................... 24
REFERENCES ................................................................................... 28

CHAPTER TWO:

INHERITANCE OF RESISTANCE TO THE SOYBEAN APHID IN

SOYBEAN PI 567585A ....................................................................... 37
ABSTRACT ...................................................................................... 37
INTRODUCTION .............................................................................. 37
MATERIAL AND METHODS ............................................................... 39
RESULTS AND DISCUSSION ............................................................... 41
REFERENCES ................................................................................... 50

CHAPTER THREE:

GENETIC LINKAGE MAPPING OF THE SOYBEAN APHID RESISTANCE GENE

IN PI 567585A ....................................................................................... 53
ABSTRACT ....................................................................................... 53
INTRODUCTION ................................................................................ 54
MATERIAL AND METHODS ................................................................. 56
RESULTS ......................................................................................... 61
DISCUSSION .................................................................................... 63

vii

REFERENCES ................................................................................... 73

CHAPTER FOUR:

IDENTIFICATION OF APHID RESISTANCE GENES IN SOYBEAN

USING MODIFIED NESTED ASSOCIATION MAPPING (MNAM) ..................... 77
ABSTRACT ....................................................................................... 77
INTRODUCTION ................................................................................ 78
MATERIAL AND METHODS ................................................................. 81
RESULTS ......................................................................................... 88
DISCUSSION .................................................................................... 92
REFERENCES .................................................................................. 109

APPENDIX ........................................................................................ 1 l3

viii

LIST OF TABLES

Table 1.1 List of soybean aphid resistance sources available as of 2009 .................... 26

Table 1.2 List of aphid resistance genes identified in soybean accessions available as of
2009 ...................................................................................... 27

Table 2.1 Observed soybean aphid resistance rating for parental and F1 plants 21 days
after aphid inoculation in field in 2008 summer .................................... 46

Table 2.2 Segregation of soybean aphid resistance in F2 populations derived from
different crosses ........................................................................ 47

Table 2.3 Segregation Of F23 progenies from P1 567585A x Skylla and IA2070 F2 plants
for aphid resistance rating ............................................................. 48

Table 3.1 Damage index of soybean aphid in the field in the summer of 2009 for the
parents: PI 567585A, Skylla, and IA2070; 158 F45 RILs derived from
070082-2 validation population (PI 567585A x ‘Skylla’); and 162 PM RILs
derived from 070016-2 mapping population (PI 567585A x IA2070) ............. 67

Table 3.2 X2 test of segregation ratio for the aphid resistance gene (Rag3-1) and seven
SSR markers among 158 F4;5RILs from the Pl 567585A x ‘Skylla’ mapping
population and 94 F34 RILs from the PI 567585A x IA 2070 validation
population .............................................................................. 68

Table 3.3 Summary for aphid resistance loci detected in the mapping population and the
validation population with aphid DI data using the CIM method ............. 69

Table 3.4 Average aphid DI for different genotypes of marker Sct_065 in the field trial
for mapping and validation populations ........................................... 70

Table 4.1 List of primary and secondary populations subjected to MNAM ............ 100

Table 4.2 Consecutive SSR markers significantly associated with aphid resistance on five
linkage groups: 82, F, G, J and M .................................................. 101

Table 4.3 CCPS SSR markers significantly associated with aphid resistance on three
linkage groups: F, G, and M ......................................................... 102

Table 4.4 Summary for aphid resistance locus detected in mapping population and the
validation population with aphid DI data using the CIM/SIM
method ................................................................................... 103

LIST OF FIGURES

Figure 2.1 Frequency distribution of soybean aphid resistance rating scores for F2
populations 070082-1 and 070016-1, respectively ................................. 49

Figure 3.1 Distribution of D1 scores in RIL populations: a) 070082 F45 RILs validation
population; b) 070016 F3;4 RILs mapping population..............................71

Figure 3.2 Linkage maps showing the locations of the soybean aphid resistance genes
from P1 567585A that were mapped on soybean linkage group J. (a) Aphid
resistance gene position in the mapping population for greenhouse and field
trials. (b) The relevant segment of the soybean LG according to the integrated
soybean map of Song et al. (2004). (c) Aphid resistance gene position in the
validation population ................................................................... 72

Figure 4.1 SSR amplification banding patterns of 41 resistant RlLs in the primary
population using primer satt406. Upper and lower bands were the amplification
banding pattern for Titan and PI 5675988 using SSR primer satt406,
respectively .............................................................................. 104

Figure 4.2 Banding patterns of PC R products of the 11 parents in the primary and
secondary populations using SSR marker Satt522 (LG F), Sat_308 (LG G),,
and Satt622 (LG J). The order of the 1 1 parents is: A00-71 1003(1),
A00-711020(2), A02-381100(3), EOOOO3(4), 1A2064(5), RR Titan(6), PI
5675988(7), IA2070(8), IA2072(9), SDXOOR-039-42(10), Skylla(11) ....... 105

Figure 4.3 Locations of soybean aphid resistance locus on LG J using composite interval
mapping method. a map shows the identified resistance locus in BCIF4;5
Population for 3-week and 4-week rating in either greenhouse or field trials:
greenhouse 3-week (GH3W), greenhouse 4-week (GH4W), replication 1 for
3-week rating in field trial (FREP13W), replication 1 for 4-week rating in field
trial (FREP14W), replication 2 for 3-week rating in field trial (FREP23W),
replication 2 for 4-week rating in field trial (FREP24W). b map is the soybean
consensus map for linkage group J. In c map, the filled black bar represents the
locus for the 3-week rating in the field cage trial (FL3W) for F45 RIL
population (070063). In b and 0 map, the LCD threshold is 6.8 and 4.6 for
BCIF4;5 and 070063populations, respectively ..................................... 106

xi

Figure 4.4 PCR products amplified by SSR markers Satt622 (LG J), and Satt529 (LG J)
and Satt463 (LG M), for P1 567543C (1), PI 5675418 (2), PI 5675988 (3), PI
567585A (4), Skylla (5), Titan (6), Dowling (7) .................................. 108

xii

 

 

313

ma!

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INTRODUCTION AND GENERAL OBJECTIVES

The cultivated soybean, Glycine max (L.) Merrill. (2n=2x=40), originated in
eastern Asia, and was first introduced to the United States as a forage crop in the late
17003. Since the 19405, soybean has become the second most important crop in the US.
after com. In 2009, the area planted to soybean was 31.36 million ha, and soybean
production is predicted at 91.45 million tons. Numerous species of insect pests feed on
soybeans. some of which can cause yield loss and even crop failure. The soybean aphid,
Aphis glycines (Matsumura) is a new major pest of soybean in the Midwest, including
Michigan. This insect, native to eastern and southern Asia, has caused significant damage
throughout the soybean growing areas of Michigan and the surrounding states since 2001.

As a component of integrated pest management (IPM) strategy, host plant
resistance has been recognized as an effective and environment-friendly approach to
controlling soybean aphid. Several aphid-resistant soybean accessions have been
identified from soybean germplasm since 2004. Then the inheritance pattern of major
resistance genes in these accessions has been determined by classical genetic studies.
Linkage mapping and association mapping are two widely used methods to localize the
resistance genes in crop plants. To date, only linkage mapping has been used to discover
aphid resistance genes in several soybean accessions. Moreover, nested association
mapping (NAM) has recently been proposed to map gene(s) accurately and efficiently, by

combining the advantages of linkage mapping and association mapping.

1

In this dissertation, the general objective is to identify molecular markers closely
linked to the aphid resistance genes in two accessions, PI 567585A and PI 5675988,
providing breeders with various options to exploit the pyramiding of resistance genes in
cultivar development. PI 567585A is a new aphid resistance source, but the inheritance
pattern is still unknown, and genetic mapping of the resistance gene(s) has not yet been
completed. PI 5675988 has previously been found to possess antibiosis resistance to
soybean aphid, which is controlled by two recessive genes (Mensah et al., 2007). This
research project was divided into three sections, each of which is presented as a chapter
in this dissertation.

Objective 1: Discover the inheritance pattern of aphid resistance in P1 567585A,
including the number and type of genes controlling resistance.

Objective 2: Identify and localize by traditional linkage mapping the aphid resistance
gene(s) in P1 567585A in the soybean genome.

Objective 3: Map aphid resistance genes in P1 5675988 using a new strategy:
modified nested association mapping (MNAM), and compare the results obtained to

those of traditional linkage mapping.

' Lu;

 

CHAPTER 1
LITERATURE REVIEW
THE SOYBEAN

The cultivated soybean, Glycine max (L.) Merrill. (2n=2x=40), is a species of legume
native to eastern Asia. The genus Glycine consists of two subgenera, Soja and Glycine.
The first subgenus Sofa contains three species: Glycine max (L) Merrill., (the cultivated
soybean) and Glycine sofa (L) Sieb and Zucc., (the wild species). Glycine sofa is the most
likely potential progenitor of Glycine max. The second subgenus, Glycine, contains 12
wild. perennial species native to Australia and the Pacific area. The soybean is a highly
self—pollinated species (Singh et al.. 2007). Moreover, the cultivated soybean can be
easily crossed within subgenus Sofa, but not with the subgenus Glycine (Newell and
Hymowitz, 1983). The soybean was first grown in the USA in 1765 (Hymowitz and
Harlan, 1983). Many U.S. germplasm and ancestral genotypes came from China, Korea
and Japan (Li et al.. 2001). During the past half century, the soybean, once an obscure
forage crop. became a major grain crop in the United States (Singh et al., 2007).

The soybean is the second largest source of protein feed and vegetable oil in the
world. The four major soybean-producing countries are the USA, Brazil, Argentina, and
China, accounting for 90% of the global total production in 2005 (Workman, 2007). In
2007, soybean represented 56% of global world oilseed production, 32% of which was

produced in the US. There are four major soybean production regions in the US: North

3

Central (Illinois, Indiana, Iowa, Michigan, Minnesota, Missouri, Ohio and Wisconsin),
Northern Plain (Kansas, Nebraska and South Dakota), Southeast (Kentucky, North
Carolina and Tennessee), and Delta (Arkansas, Louisiana and Mississippi) regions.
Soybeans were planted on 30.6 million hectares in 2008. producing 80.54 million metric

tons of soybeans in the US (Soy Stats. 2008).

THE SOYBEAN APHID

The soybean aphid. Aphis glycines Matsumura (Homoptera: Aphididae), is a native
pest of soybean in eastern Asia. The soybean aphid (winged or Wingless) is a small
(<1/ 16” long when mature), yellow or yellowish green insect with two obvious black
comicles and pale cauda (Ragsdale et al., 2004). The soybean aphid was first reported in
2000 from the area of Wisconsin. northern Illinois. and Michigan. The pest spread over
21 states in the US. and three Canadian provinces (Hartman et al., 2001; Venette et al.,
2004). C urrently, the soybean aphid is one of the most significant pest insects in soybean
production in North America.

The soybean aphid has a heteroecious holocylic life cycle, shuttling between its
primary host. buckthorn (Rhamnus caihariica), and its secondary host, soybean. Soybean
aphids overwinter as the egg stage on buckthorn. In the following spring. the eggs hatch
and produce a few generations until the winged females (alatae) fly to soybeans. During

the summer, soybean aphids commonly produce 15 generations with both Wingless and

4

winged morphs throughout the whole growing season. In the soybean vegetative growth
stages, soybean aphid colonies are found in partially expended young trifoliates, petioles,
and stems. When the soybeans move to the reproductive stages, soybean aphids move
over the whole plant, mature leaves. lateral branches, petioles. and pods. At the same
time. the secreted honeydew on plants results in the production of sooty mold, which
affects the photosynthesis and leads to the yield and seed quality loss. In the autumn
when temperature and photoperiod are reduced. winged females (gynoparae) emerge on
soybeans and disperse to find the primary host buckthorn. After gynoparae settle on the
buckthorn, they develop into oviparae. Meanwhile, males also migrate to buckthorn to
mate with oviparae. Finally they lay overwintering eggs on the buckthorn, starting the
next life cycle in the following year (Ragsdale et al., 2004; Wu et al., 2004).

Severe soybean aphid infestations reduce soybean production directly by causing
plant damage during feeding. such as wrinkled and distorted leaves, lower pod and seed
counts, and reduced seed weight. In addition to the direct plant damage. soybean aphids
have been reported to transmit diseases, including soybean mosaic virus (SMV), soybean
stunt virus. soybean dwarf virus, abaca mosaic, beet mosaic, tobacco vein-banding
mosaic virus. bean. yellow mosaic virus, mungbean mosaic virus, peanut mottle virus,
peanut stripe potty virus. and peanut mosaic virus (Wu et al., 2004 ).

The soybean aphid caused severe yield reduction in several north central states in

2003. An estimated 300,000 ha were affected in Michigan with a loss of $9 million

 

 

(DiFonzo, 2004). Soybean aphid damaged around 1.6 million ha of soybean in Minnesota
with an estimated loss of $80 million (Associated Press, 2003). In Illinois, the estimated

loss due to the infestation of 0.5 million ha soybean was $45 million (Steffey, 2004).

BIOTYPES OF SOYBEAN APHID

Biotype is a term employed to distinguish populations of insects or other organisms,
which show different ability to attack plants due to diverse genetic variants. In many
cases, resistance genes introduced by plant breeding often imposes selection pressure on
insect populations. leading to the development of virulent insect biotypes (Gallum, 1972;
Diehl and Bush, 1984). This means insects evolve to overcome the existing defense
mechanism in plants. For example. the Hessian fly (Mayeiiola destructor) that attacks
wheat has twelve biotypes (Kudagamage et al., 1990; Ratcliffe et al.,, 2000); and the
greenbug (Schizaphis graminu) has eight biotypes (Puterka et al.,, 1988).

The study in Michigan showed that the aphids overcame the resistance in
‘Dowling' and ‘Dowling‘ with the infestation of aphid colonies collected in 2006. PI
5675988 and P15675418 retained the resistance to soybean aphid in greenhouse and field
annual evaluations in Michigan (Mensah et al., 2007). In 2008, the research showed that
there are at least two distinct biotypes of soybean aphid in North America: Ohio and
Illinois isolates. The resistance genes Rag] from ‘Dowling’ and Rag from ‘Jackson’ were

defeated by Ohio isolates. but were resistant to the Illinois isolate. PI 200538 and PI

6

567597C are resistant and PI 5675418 is moderately resistant to both Ohio and Illinois
aphid biotypes (Kim et al., 2008). These results indicated that aphids evolved
spatial-temporally.

Integrated pest management (1PM) is a sustainable approach to manage crop pests
by combining the use ofchemical, natural biological and host plant resistance tactics,

which minimizes economic, health and environmental risks.

CHEMICAL AND NATURAL BIOLOGICAL CONTROL STRATEGIES

There are several foliar insecticides registered for aphid control, which are all
restricted use pesticides. Until now, the application of these insecticides is the only
available and efficient way to control damage of soybean aphid in the commercial field.
However, this approach is evaluated as cost-expensive. time-consuming and
environmentally unfriendly. During the aphid outbreak in 2003, around. 3 million ha of
soybean were sprayed with pesticide to control the soybean aphid in the US (Landis et
al., 2003). In Illinois, the cost of insecticide was $9 - 12 million for aphid control in the
same year (Steffey, 2004).

Though insecticides work efficiently against soybean aphid, it is not recommended
to apply them repeatedly as this can lead to insecticide resistance. In fact. many predators
eat soybean aphids and keep aphid numbers in check naturally in some years preventing

an outbreak. These predators include Asian lady beetle (Harmonia cucyridis), lacewing

7

 

 

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larvae (Neuroptera: Chrysopidae), syrphid fly larvae, minute pirate bugs (Hemiptera:
Anthocoridae) and parasitoid Asian wasp (Aphidius colemani) (Fox et al., 2004). Among
these predators, wasps have the most potential to be the natural biological control of
soybean aphids. The Asian wasp can parasitize on soybean aphid and cause aphid
mummies, is incapable of stinging people, and has a narrow biological host range. Other
predators could not be applied in 1PM because they either sting people or have a broader
host range. As a strain of Asian wasp, Binodoxys communis was released for cage study
in 2007 at 36 sites in seven Midwestern states: including Minnesota, Iowa, South Dakota.
Wisconsin, Illinois. Indiana and Michigan. However, the parasitoid Asian wasp has not
been officially released as natural predator in the field because of the difficulties to
propagate and maintain the wasp number at a certain level under artificial conditions in

each year (Ruth, 2007).

GENETIC CONTROL BY HOST PLANT INSECT RESISTANCE BREEDING
Host plant resistance breeding has enomious benefits as another component of IPM,
in terms of investment return, reduced release of insecticide in the environment, and little
concern about the population fluctuation of natural enemies each year (Li et al.. 2007).
Recently, host plant insect resistance has been developed in some new cultivars of rice,
cotton, and vegetables, resulting in the reduced use of insecticides. Four components are

involved in the host plant insect resistance breeding: 1) discovering host plant resistance

8

mechanisms; 2) determining the genetic inheritance of insect resistance; 3) identifying
the insect resistance gene(s) in host plant; 4) integrating the resistance gene(s) into elite
cultivars.
Discovering host plant insect resistance mechanisms

Insect resistance mechanisms are classified as tolerance, antibiosis and antixenosis
(Painter, 1951). Both antibiosis and antixenosis describe the reaction of an insect to a
plant. while tolerance resistance describes the reactions of a plant to insect attack. In
tolerance mechanism. a plant can survive under the equal infestation pressure that would
kill or severely injure other susceptible plants (Painter, 1951). In the antibiosis
mechanism, the genetic properties of a plant reduce insect abundance by affecting the
growth and production of the insect during feeding. leading to the decreasing plant
damage. For example, first batch of Hessian fly larvae die after they start feeding on
barley cultivars carrying the antibiosis resistance genes (Patterson et al.,, 1994). In the
antixenosis mechanism, the insects feed and oviposit on a plant depending on the plant
morphological characters. including color. leaf angle. odor. taste, and type of pubescence.
For example the blue-green cultivars of peas are more favorable to the pea aphid than the
yellow-green ones (Soroka and Mackay. 1991). Among these three resistance
mechanisms, antibiosis was considered to be the only true form of host resistance because

it involves antibiosis resistance genes in the host.

Both antibiosis and antixenosis deter insect feeding, so it is critical to separate these
two resistance mechanism in insect resistance study. Choice and nonchoice tests have
been extensively used to identify resistance, then the resistance type: either antibiosis or
antixenosis (Mensah et al.. 2005; Hill et al.. 2004). First, a choice test is used to identify
resistance, where aphids feed on their preferred hosts. But the test does not distinguish
between the types of resistance. In nonchoice test. aphid movement is confined to a single
host without preference. It helps distinguish antibiosis from antixenosis, nonhost
preference. Antibiotic resistance source when identified can be used to develop host plant
resistance.

In the US. the first four aphid resistance sources were reported in 2004. After
screening 1,542 soybean accessions, ‘Dowling’. ‘Jackson’ and PI 200538 showed
antibiotic resistance, and PI 71506 had antixenotic resistance (Hill et al., 2004). Both
‘Dowling’ and ‘Jackson’ are late maturity ancestral cultivars. In 2005, Mensah et al.,
evaluated 2,147 soybean germplams in choice tests and identified four new resistant
accessions: PI 5675418, Pl 5675988 PI 567543C. and PI 567597C. The subsequent
nO-choice test showed that PI 5675418 and PI 5675988 possesses antibiotic resistance,
while PI 567543C and PI 567597C have antixenotic resistance (Mensah et al., 2005). In
2006, Diaz-Montano et al. (2006) identified. two antibiotic soybean entries: K1639 and
Pioneer 95897. In the following year, PI 239077 and PI 548664 were identified to have

antibiotic resistance; while PI 595099. PI 436684, ‘Perrin’, and ‘Tracy-M’ have

10

antixenotic resistance (Hesler et al., 2007; Hesler and Dashiell, 2008). The latest aphid
resistance sources were discovered by Mian et al. (2008a) after evaluating nearly 200
soybean genotypes by choice and no—choice tests in the greenhouse and field. PI 243540
showed antibiotic resistance. and PI 5673018 and PI 567324 possessed antixenotic
resistance. The currently available aphid resistance mechanism in different soybean
accessions in the recent years is shown in Table 1.1. However. no commercial soybean
cultivar with either partial or complete aphid resistance is currently available in the USA.
Determining the genetic inheritance of aphid resistance

Information on inheritance of resistance to insects. such as the number of genes and
nature of gene action. can be utilized in selection of appropriate breeding methodology
(pedigree. backcross or population improvement) to transfer resistance genes into elite
cultivars. Classic genetic inheritance studies of insect resistance are based on the
observation of phenotypes (resistance perfomiance) in segregating populations.
Commonly F ] individuals. F2 or backcross populations, and F23 families are used to study
the inheritance of insect resistance. such as whether the gene(s) is dominant or recessive,
and how many genes are involved and whether resistance is qualitative or quantitative.

In several plants, the aphid resistance is mainly controlled by qualitative
dominant/recessive genes. such as barley. cowpea. peach, wheat. and soybean. The aphid
resistance in spring barley (Hordeum vulgare L.) is controlled by two dominant genes

(Momhinweg et al., 2002). The aphid (Aphis craccivora Koch) resistance in cowpea

ll

(Vigna unguiculata L.) involves a single dominant gene. In the peach cultivar ‘Rubira’,
the resistance to the green peach aphid (ii/lyzus persicae) is controlled by a single
dominant gene (Pascal et al., 2002). In wheat (Triticum spp.), eight independent dominant
genes each confer resistance to the Russian wheat aphid (Diuraphis noxia) in different
resistance source, while one recessive gene contributes to resistance in T riticum tauschii
line SQ24 (Liu et al.. 2006). The aphid resistance in ‘Dowling’, ‘Jackson’, PI 243540 and
PI 200538 was found to be controlled by a single dominant gene (Hill et al., 2006a; Hill
et al., 2006b; Kang et al.. 2008: Hill et al.. 2009). The latest genetic inheritance study of
resistance in P15675988 and PI 567541 8 showed that two recessive genes involved in

the aphid resistance (Mensah et al.. 2008).

Identifying the aphid resistance gene(s) in soybean

The identification of aphid resistance genes has been important research challenge
since 2000, due to the destructive soybean aphids. In 2007, two soybean aphid resistance
genes Rag] and Rag were identified on linkage group (LG) M in soybean cultivars
‘Dowling’ and ‘Jackson’ respectively. Rag] was located 4.2cM from the Simple
Sequence Repeat (SSR) marker Satt435 and 7.9cM from Satt463. Rag was mapped
2.1cM from Satt435 and 8.2cM from Satt463 (Li et al., 2007). In 2008, the aphid
resistance gene Rag2 in P1 243540 was positioned in the interval between the SSR

markers Satt334 and Sct_033 on LG F (Mian et al.. 2008b). In 2009, two resistance genes

12

in P1 5675418 were closely linked to marker Satt299 or Satt435 on LG M, and to marker
Satt649 or Satt343 on LG F (Zhang et al.. 2009). The gene on LG F is far away from

RagZ, while the position of the gene on LG M is in the similar region as the Rag or Rag].
Recently, the resistance gene of PI 200538 was mapped to the same region as RagZ (Hill
et al.. 2009), suggesting that PI 200538 may be an additional source of RagZ. The genetic

allelic relationship among these genes on the same linkage group is still an enigma.

Transferring the aphid resistance gene(s) into elite lines

Two general categories ofdisease resistance have been recognized in plant: (1)
qualitative resistance controlled by a single gene with large effect (resistance genes;
R-genes) and (2) quantitative disease resistance (QDR) conditioned by multiple genes on
quantitative trait loci (QTL). each with small individual effects and sensitive to
environments (Poland et al., 2008).

Qualitative genes that provide high levels of resistance, are easily identified in
genetic studies. and integrated into elite lines through pedigree or backcross breeding. For
example, with the assistance of molecular markers linked to Rag]. the resistance gene
was successfully backcrossed into the Midwest-adapted elite soybean lines without yield
reduction (Kim and Diers. 2009). But they are subject to “break-down” due to the
evolution ofpest/pathogen populations. It means that the evolution of insect biotypes

would overcome oligogenic resistance. resulting in a breakdown over a period of time.

13

Now, a total of three biotypes exist in North America: Michigan, Ohio and Illinois
isolates. So it is necessary to consider the durability of an identified resistance resource.
The period of aphid resistance is determined by the genetic inheritance of the resistance
gene(s), such as the number of dominant/recessive gene(s) controlling the resistance.
Generally. the resistance controlled by a single dominant gene is less durable than
resistance controlled by multiple genes. For instance, the two genes-controlled resistance
in P1 5675988 and PI 5675418 is more durable than single gene-controlled resistance in
‘Dowling’ and ‘Jackson’ (Mensah et al.. 2007). In contrast. QDR tends to be more
durable and favorable for breeding durable resistance cultivars. but no QDR was

discovered to confer aphid resistance in soybean.

GENETIC MAPPING OF RESISTANCE GENE(S) IN CROP PLANTS

The goal of genetic mapping is to locate the genetic regions along the chromosomes
which contain sequences that actively cause the phenotypic variation. This procedure
statistically models the observed phenotypes in relation to genotypic information
conveyed by molecular markers. Linkage analysis and association mapping are two

widely used tools for the dissection of complex traits in genetic mapping (Ersoz et al.,

2008; Zhu et al., 2008).

Population construction and phenotypic data

14

Genetic mapping in plants has been dominated by the linkage analysis of designed
bi-parental populations with known pedigree structure. These bi-parental populations are
derived from the cross Of two distinct inbred lines, PI and P2. They produce a
heterozygous but homogenous F1 —offspring. From the F1. different types of population
can be derived, including F2. backcross (BC ), double haploid (DH), or recombinant
inbred line (RIL) populations (Sneller et al.. 2009). Hundreds of linkage analysis studies
have been done by establishing these kinds of bi—parental populations in various plant
species over the past decades (Zhu et al., 2008; Sneller et al., 2009). In soybean aphid
resistance studies. all resistance genes were identified by constructing bi-parental
populations.

However, in principle. phenotypic data obtained from any type of population can
be used for genetic mapping if the genetic variation exists within the population.
Compared to linkage analysis within pedigrees, association mapping exploits the
historical recombination in family based association population or classic association
population (Crepieux et al., 2004; Parisseaux and Bernardo. 2004; Zhang et al.. 2004;
Bernardo and Yu. 2007). resulting in higher power of detection in specific regions of
DNA (Zhu et al., 2007). A family based association population is composed of unrelated
families. which are powerful for gene mapping ofcomplex diseases mainly in human.
Classic association population is equal to a natural population, which is a random sample

ofindividuals from species population. The classic association population is used widely

15

in association mapping of crops to understand the diversity of germplasm, such as maize,
barley, sorghum and wheat (C amus-Kalandalvelu et al., 2006; Murray et al., 2009;
Breseghello and Sorrells, 2006). For example, association mapping provides an efficient
approach to relate genotypes to complex quantitative traits in hexaploid wheat. In 2006,
association mapping of kernel size and milling quality was performed on a selected
sample of 95 elite cultivars from soft winter wheat germplasm by using SSR markers.
The association mapping results showed not only more QTLs, but also agreement with
previous linkage analysis at certain significant SSR marker loci on three chromosomes
(Breseghello and Sorrells, 2006). Later. a population of 44 modern European winter
wheat varieties was studied by association mapping for the association between
Stagonospora nodorum blotch resistance and markers mapped in the region of
QSng.sfr-3BS. The results showed that the association mapping population had at least a
390-fold higher resolution compared to the traditional RIL populations (Tommasini et al.,
2007). The association mapping was also used to assess the genetic diversity of biotypeZ
Russian wheat aphid (RWA2) resistance within 71 bread wheat (T. monococcum)
accessions. New QTLs were identified by association mapping, compared to previous
linkage analysis (Peng et al., 2009). Thus, as the supplementary Of linkage analysis,
association mapping emerged to exploit trait variation with sufficient recombinant events
within a more flexible population construction. such as a natural population, or a diverse

collection of germplasm (Zhu et al., 2008).

16

Genotypic data

There are numerous different types of molecular makers that have been used to
obtain genotypic data in genetic mapping. including Restriction Fragment Length
Polymorphism (RF LP), Amplified Fragment Length Polymorphism (AFLP). Simple
Sequence Repeat (SSR) and Single nucleotide polymorphism. Each type of molecular
marker has its own advantages and disadvantages. For example, SSR markers are most
popular in current soybean genetic studies due to desirable characteristics of
co-dominance. highly polymorphic and sufficient genome coverage. The latest version of
the SSR-based soybean linkage map was released in 2004 (Song et al., 2004). This
integrated genetic map covers 2,523.5 cM of soybean genome across 20 LGs that
contained 1.015 SSR markers. More recently. the appearance of high-throughput and
inexpensive SNP genotyping platforms stimulated the development of SNP markers in
soybean genome. SNPs were first discovered by the resequencing of sequence tagged
sites (STS) by Choi et al.. (2007). As a newly informative genetic marker, SNP includes
single base changes. insertions/deletions (indels). A total of 1.141 sequence-based SNP
markers were used to fill the gaps (>5 cM) in the pre-existing SSR-based map.

The new SSR/SNP based genetic map of soybean genome provides a crucial
resource for quantitative trait locus discovery. map-based cloning. and marker assisted

selection in cultivar improvement. Moreover. the US. Department of Energy Joint

17

Genome Institute (DOE JGI) completed the preliminary assembly and annotation of the
soybean genome. Glycine max. This will accelerate SNP discovery and the construction
of a dense SNP-based soybean genome map (Hyten et al., 2007a. 2007b).

Linkage analysis

Linkage analysis looks for non-random co-segregation of marker alleles and trait
within pedigrees (F 2. BC, DH, RIL populations). For linkage analysis. genotype and
phenotype are integrated into the different statistical models for QTL detection. such as
single-marker regression. simple interval mapping (SIM) (Lander and Botstein, 1986),
composite interval mapping (CIM) (Zeng. 1994) and multiple interval mapping (MIM)
(Kao et al., 1999). The linkage analysis has been widely applied in identification of aphid
resistance genes in soybean since 2000. The linkage mapping populations are F2. F34, F45
RILs populations which were derived from resistant and susceptible parents, where SIM,
CIM and MIM were applied to detect the QTL positions.

However, limited numbers of meioses exist within families and pedigrees in few
generations for linkage analysis. which is suitable to detect QTL in genome-wide with
low resolution at the order Ofmegabases (Thornsberry et al., 2001 ). Until now. a few
number of QTLs identified in linkage analysis were tagged at the gene-level.

Association mapping

1) Linkage disequilibrium

18

Association mapping was established on the basis of linkage disequilibrium (LD)
concept, which is the non-random co-segregation of alleles at two loci. The precision of
association mapping is detemtined by the rate of decay of LD with physical distance. The
rate is highly variable among species. and even among different genomic regions of the
same species, such as coding and non-coding regions. The relationship between LD and
physical distance detemtines the marker density required for genome scan. and the
maximum resolution for phenotype genotype association in the study population
(Veyrieras et al., 2007). As an autogamous crop, the LD structure of soybean has been
analyzed in three genomic regions. and LD ranged from 336 to 574 kb. The highly
variable levels of LD were discovered in the wild ancestors, landraces and the elite
cultivars. In G. sofa. LD extends over 100 kb. But LD coverage is highly variable in the
landrace and elite cultivars. expanding from 90 to 574 kb because of the domestication
and increased self-fertilization (Hyten et al.. 2007a).

2) Population structure in association mapping

A population is said to be structured if individuals or families deviate from
Hardy-Weinburg equilibrium due to domestication. natural or artificial selection. and
admixture of populations. Association mapping investigate the association between
genetic diversity and phenotypic variation. But the association between population
structure and trait variation can complicate the identification of accurate correlations in

association mapping because of the indirect association between neutral polymorphism

l9

and phenotypic variation. Moreover. the trait of interest determines the magnitude of
variation could be explained by population structure in wheat, 4-6% for kernel
composition. or up to 51% for flowing time (Veyrieras et al., 2007).

3) Statistics in association mapping

Association mapping investigates the association between genetic diversity and
phenotypic variation. This approach has higher possibilities of type I and type II errors,
which means increased false positives and reduced power in association mapping
compared to biparental linkage analysis (Breseghello and Sorrells, 2006). SO many
statistical challenges cumber the dissection of phenotypic variation of complex traits of
interest.

The false positive rate (Type I error) genome-widely is caused by the multiple
hypotheses testing during the whole genome association mapping. whereby some random
or indirect associations occur in large collection of molecular markers. Type I error rate
for multiple testing can be controlled genome widely with the Bonferroni correction or
false discovery rate (FDR) (Benjamini and Yekutieli. 2005). Generally, the Bonferroni
correction is more conservative and reduces the power of detecting real association
between the polymorphism and the traits. The FDR controls the expected proportion of
false positives in the whole set of positive results from the multiple testing. So it is a
more flexible procedure with greater statistical power. In the FDR approach. the q-value

of a test measures the proportion of false positives incurred when a particular test is

20

called significant. The Q-value package estimates the q-values from the list of p-value of
the multiple testing. estimates a cutoff for a particular FDR, and estimates an FDR for a
particular cutoff (Chen and Storey, 2006).

Type 11 error. or false negative is attribute to population structure and familiar
relatedness among or within different subpopulations. Thus, association mapping has
limited application in detecting rare variant or genes existed within subpopulations. but
inconsistently fixed among populations (Zhang et al.. 2004; Veyrieras et al., 2007). So it
is necessary to discover the hidden effect of population structure before the investigation
ofcandidate polymorphism contribution to phenotypic variation in a collection of diverse
materials. Recently. some software or methodologies were developed to understand
population structure by analyzing molecular data. such as STRUCTURE (Hubisz et al.,
2009), principal component analysis (PC A). and Ward.

Nested association mapping in plants

Nested association mapping was developed for dissecting the genetic inheritance of
complex quantitative traits in maize. which combines the advantages of linkage analysis
and association mapping in a single population (Yu et al.. 2008). This NAM population
was developed by crossing the common parent 873 with 25 different founder parents.
Each individual obtained from 25 F3 populations were self-pollinated four generations.
producing a total of 5000 recombinant inbred lines (RIL). This RIL population can be

used for cursory QTL detection by linkage mapping with low-resolution markers,

21

followed by high-resolution association mapping with high-density maker, such as SNPs.
As a permanent and stable genetic mapping resource. the NAM population can be
evaluated for many quantitative traits in multiple environments. producing accurate
estimates of significant allelic effects. epistasis, pleiotropy and genotype-environment
interaction. This NAM methodology has been applied in the dissection of quantitative
traits in crop plants, such as northern leaf blight resistance and flowering time in maize

(Poland et al., 2009; Buckler et al., 2009).

MOLECULAR BASES FOR RESISTANCE (R) GENES

Five different classes of R-genes have been identified in plants based on the
combination of structural motifs. Class I contains a serine-threonine kinase (STK)
catalytic domain, such as [’10 conferring resistance to the bacterial pathogen
Pseudomonas syringae in tomato (Tang et al., 1999). As the largest two classes, the
second and third class both are composed ofa putative nucleotide binding site (NBS),
leucine-rich repeats (LRRs), but different in the presence ofeither a coiled-coil (CC)
domain or a Toll/Interleukin-l cytoplasmic receptor (TIR) at the amino terminus. Being
intracellular proteins, no transmembrane (TM) domain exists in Class I through 3 R-gene
families. Mi-l belongs to class 2 R-gene family. conferring resistance to root-knot
nematodes and potato aphids in tomato (Vos et al.. 1999). Class 4 R-genes have a TM

and an extracellular LRR without NBS. The coded protein in the fifth R-gene class

22

possesses an extracellular LRR. a TM and a cytoplasmic STK domain. For example the
rice R-gene Xa21 confers resistance against the bacterial pathogen Xanthomonas oryzae
pv. oryzae (Khush et al.. 1990).

Hundreds of NBS-LRR resistance gene analog (RGA)s were isolated and
sequenced. and genetically mapped to 8 of the 26 linkage groups of the soybean genetic
map. including groups C2. D.. H. J. L, M. N and P (Kanazin et al., 1996', Yu et al., 1996;
Graham et al.. 2000). The R-genes often possess similar sequence and are physically
clustered within a close distance on the same linkage group. Five classes of RGAs were
co-localized as a large cluster on LG I (Kanazin et al.. 1996). LG F is another important
region for disease resistance genes in soybean. where several RGAs related to virus,
bacteria fungus and nematode resistance are mapped (Jeong et al.. 2001). All this
information matched well with the available locations of aphid resistance genes in
soybean.

The NBS-LRR gene family is organized and evolves through a process of
birth-and-death. supercluster formation. and adaptive selection. That means the
superclusters are initiated from deleterious mutation. repeated gene duplication.
intra/inter-cluster recombination. transposition. or genome rearrangement. Then the
cluster/superculster are maintained intact or lost from the cluster/supercluster in natural
or artificial selection. For example. the conditional resistance to the soybean mosaic virus
(SMV; Potyvirus) resistance involves multiple-allelic gene on Rsv, which is tightly linked

23

to a NBS/LRR gene cluster on the LG F. The study showed that recombination within the

cluster can condition the specific resistance for all SMV strains in soybean (Hayes et al.,

2004)

MOLECULAR BREEDING PRACTICE

Marker-assisted selection (MAS) is a process widely used to accelerate plant
breeding through early generation selection. whereby molecular markers are used for
indirect selection of traits of interest. The first step of MAS is to map the gene or QTL by
genetic mapping. and then the closely linked flanking markers in the candidate gene
region are used for MAS. MAS is based on linkage analysis. which has been developed
for most crop plants to locate gene/QTL regions where molecular markers co-segregate
with traits of interest. Linkage analysis based on MAS is most likely to be used for
within-family selection in a limited number of elite families. The limitations of this
approach include the low resolution of MAS, inconsistent QTL detection in different
genetic background and environments. These properties prevent the development of
universal markers for marker-assisted. selection at multiple-population or germplasm
level. So gene-assisted selection (GAS) emerged to exploit the direct association between
gene(s) and trait based on association mapping. High resolution of marker-trait

associations is detected in association mapping due to the advances in high-throughput

sequencing and SNP genotyping platform. The closely linked markers (or genes) are
transferrable across multiple families, natural population and even species.

Progress of using MAS already existed in the area of quantitative disease resistance.
For an instance. the dominant gene Xa21 was located on chromosome 1 l, conferring
broad spectrum resistance to most isolates ofXanthomonas oryzae pv. oryzae (Ikeda et
al.. 1990. Khush et al., 1990). As a crucial gene for bacteria resistance improvement in
rice, Xa21 has been used in marker-assisted breeding by tagging with molecular markers
(Ronald et al.. 1992; Sharma et al.. 2001. Singh et al., 2001. Sridhar et al., 2001). In
current soybean aphid resistance studies. the close linkage SSR markers were used to

breed new productive resistant soybean cultivar (Kim and Diers, 2009).

Table 1.1 List of soybean aphid resistance sources available as of 2009.

 

 

 

 

 

 

 

 

Resistance Mechanism Reference
Antibiotic resistance Antixenotic resistance
‘Dowling’ Pl 71506 Hill et al., 2004
‘Jackson’
Pl 200538
Pl 5675988 Pl 567543C Mensah et al., 2005
PI 5675418 Pl 567597C
K1639 Diaz-Montano et al., 2006
Pioneer 95897
Pl 230977 Pl 595099 Hesler et al., 2007
PI 548664 PI 436684 Hesler and Dashiell, 2007
‘Perrin’
“Tracy-M”
Pl 243540 Pl 5673018 Mian et al.. 2008a
Pl 567324

 

26

Table 1.2 List of aphid resistance genes identified in soybean accessions available as of

 

 

2009.
Source Gene Linkage Flanking % variation Reference
group Markers explained

"Jackson’ Rag M Satt435~Satt463 - Li et al., 2007
’Dowling’ Rag] M Satt435~Satt463 - Li et al., 2007
PI 243540 Rug2 F Satt334~Sct_033 Mian et al., 2008b
Pl 5675418 - F Satt299~Satt435 50.3% Zhang et al., 2009

- M Satt649~Satt343 29.5% Zhang et al., 2009
P1 200538 - F Satt334~--Sct_033 - Hill et al., 2009

 

27

 

 

Ch

Cli-

C're'

Diaz

Diehl

References:

Associated Press. 2003. Aphids whittling soybean farmers’ profits. [Online] Available at:
www.cnn.com/2003/US/I\/Iidwest/l1/25/soybean.aphids.ap (posted 25 November
2003; verified 24 Mar. 2006)

Benjamini, Y., and D. Yekutieli. 2005. Quantitative trait loci analysis using the false
discovery rate. Genetics 171: 783—790 '

Bemardo, R., and J. Yu. 2007. Prospects for genomewide selection for quantitative traits
in maize. Crop Sci. 47:1082—1090.

Breseghello, F., and ME. Sorrells. 2006. Association mapping of kernel size and milling
quality in wheat (Triticum aestivum L.) cultivars. Genetics 172: 1165-1177.

Camus-Kulandaivelu, L., J.B. Veyrieras, D. Madur, V. Combes, M. Fourmann, S.
Barraud, P..Dubreuil, B. Gouesnard, D. Manicacci, and A. Charcosset. 2006.
Maize adaptation to temperate climate: Relationship between population structure
and polymorphism in the Dwart8 gene. Genetics 172:2449—2463.

Chen, L., and JD. Storey. 2006. Relaxed significance criteria for linkage analysis.
Genetics 173:2371—2381

Choi, I.Y., D.L. Hyten, L.K. Matukumalli, Q. Song ,J.M. C haky, C.V. Quigley, K. Chase,
K. Gordon Lark. R.S. Reiter. M.S. Yoon, E.Y. Hwang. S.I. Yi, N.D. Young, R.C.
Shoemaker, C.P. van Tassell, J .E. Specht, and PB. Cregan. 2007. A Soybean
Transcript Map: Gene Distribution, Haplotype and Single-Nucleotide
Polymorphism Analysis. Genetics 176:685—696.

Crepieux, S., C. Lebreton, B. Servin, and G. Charrnet. 2004. Quantitative trait loci (QTL)
detection in multicross inbred designs: recovering QTL identical-by-descent status
information from marker data. Genetics 168: 1 73 7-—1748.

Diaz-Montano, J., J.C. Reese, W.T. Schapaugh, and LR. Campbell. 2006.
Characterization of Antibiosis and Antixenosis to the Soybean Aphid (Hemiptera:
Aphididae) in Several Soybean Genotypes. J. Econ. Entomol. 99: 1884-1889.

Diehl and Bush. 1984. An evolutionary and applied perspective ofinsect biotype. Ann.
Rev. Entomol. 29:471-504.

28

Ii m

 

 

DiFonzo. C. 2004. Determining Optimum spray timing for the soybean sphid. [Online]
Available at: http://fieldcrop.msu.edu/documents/GRO4-006.pdf

Ersoz, 83., J. Yu, E.S. Buckler, R. Varshney, and R. Tuberosa. 2008. Applications of
linkage disequilibrium and association mapping in crop plants. p. 97—120. In
Varshney R., Tuberosa R.. (ed.) Genomic assisted crop improvement: Vol. I:
Genomics approaches and platforms. Springer Verlag, Germany.

Fox, T.B., D.A. Landis, F. F. Cardoso, and GD. DiFonzo. 2004. Predators suppress
Aphis glycines Matsumura population growth in soybean. Environmental
Entomology 33:608-618.

Gallun. R.L. 1972. Genetic Interrelationships between Host Plants and Insects. J Environ
Qual. 12259-265.

Graham, M.A., L.F. Marek, D. Lohnes, P. C regan, and RC. Shoemaker. 2000.
Expression and genome organization of resistance ene analogs in soybean. Genome
43:86——93.

Hartman, G.L., L.L. Domier, L.M. Wax. C.G. Helm, D.W. Onstad, J.T. Shaw, L.F. Solter,
D.J. Voegtlin, C.J. D‘Arcy, M.E. Gray. K.L. Steffey, S.A. Isard, and PL. Orwick.
2001. Occurrence and distribution oprhis glycines on soybeans in Illinois in 2000
and its potential control. Plant Health Progress. [Online] Available at:
http://www.plantmanagementnetwork.org/sub/php/brief/aphisglycines/

Hayes, A.J., S. C. Jeong, M.A. Gore, Y.G. Yu, G.R. Buss, S.A. Tolin, and MA. Saghai
Maroof. 2004. Recombination within a Nucleotide—Binding-Site
/Leucine—Rich-Repeat Gene Cluster Produces New Variants Conditioning
Resistance to Soybean Mosaic Virus in Soybeans. Genetics 166:493-503.

Hesler, S.L., K.E. Dashiell, and JG. Lundgren. 2007. Characterization of resistance to
Aphis glycines in soybean accessions. Euphytica 154:91—94.

Hesler, S.L., Dashiell, KB. 2008. Identification and characterization of new sources of
resistance to Aphis glycines Matsumura (Hemiptera: Aphididae) in soybean lines.
Journal of Applied Entomology and Zoology. 43:197-206.

Hill. C. 8., Y. Li and G. L. Hartman. 2004. Resistance to the soybean aphid in soybean
germplasm. Crop Sci. 44298-106.

Hill, C.B., Y. Li, and G.L. Hartman. 2006a. Identification of a single dominant gene for

resistance to the soybean aphid in the soybean cultivar Dowling. Crop Sci.
46:1601—1605.

Hill, C.B., Y. Li, and G.L. Hartman. 2006b. Soybean aphid resistance in soybean Jackson
is controlled by a single dominant gene. Crop Sci. 46:1606—1608.

Hill, C.B., K.S. Kim. L. Crulla, B.W. Diers and G.L. Hartman. 2009. Inheritance of
Resistance to the Soybean Aphid in Soybean PI 20053. Crop Sci 49:1193-1200

Hymowitz, T., and Harlan J. R. 1983. Introduction of soybean to North America by
Samuel Bowen in 1765. Economic Botany 37: 371-379.

Hyten, D., L.I. Choi, Q. Song, RC. Shoemaker, R.L. Nelson, J.M. Costa, J.E. Specht, and
RB. Cregan. 2007a. Highly Variable Patterns of Linkage Disequilibrium in
Multiple Soybean Populations. Genetics 175: 1937—1944.

Hyten, D.L., I.-Y. Choi, M.-S. Yoon, Q.-J. Song, J.E. Specht, R.L. Nelson, L. Chase, N.D.
Young, K.G. Lark, R.C. Shoemaker, and PB. Cregan. 2007b An Assessment of
Genome-wide Linkage Disequilibrium in Soybean. Plant & Animal Genome XV,
San Diego, CA.

Hubisz, M.J., D. Falush. M. Stephens, and J.K. Pritchard. 2009. Inferring weak
population structure with the assistance of sample group information. Molecular
Ecology Resources. 911322-1332.

Ikeda, R., G.S. Khush and RE. Tabien. 1990. A new resistance gene to bacterial blight
derived from 0. longistaminata. Jpn. J. Breed. 40: 280-281.

Jeong, S.C., A.J. Hayes, R.M. Biyashev, and MA. Saghai-Maroof. 2001. Diversity and
evolution of a non-TIR-NBS sequence family that clusters to a chromosomal
hotspot for disease resistance genes in soybean. Theor. Appl. Genet. 103:406—414.

30

Kanazin, V., L.F. Marek, and RC. Shoemaker, 1996: Resistance gene analogs are
conserved and clustered in soybean. Proc. Natl. Acad. Sci. 93:11746—1 1750.

Kang, S.T, M.A.R. Mian, and RB. Hammond. 2008. Soybean Aphid Resistance in P1
243540 Is Controlled by a Single Dominant Gene. CrOp Sci. 48:1744-1748

Kao, C.H., Z. Zeng, and R.D.Teasdale. 1999. Multiple interval mapping for quantitative
trait loci. Genetics 152: 1203—1216.

Kim. K.S., C.B. Hill, G.L. Hartmanb, M.A.R. Mian, and B.W. Diers. 2008. Discovery of
soybean aphid biotypes. Crop Sci. 48:923-928.

Kim, KS, and B.W. Diers. 2009. The Associated Effects Of the Soybean Aphid
Resistance Locus Rag] on Soybean Yield and Other Agronomic Traits. Crop Sci.
49:1726-1732;

Kudagamage, C ., J.E. Foster, P.L. Taylor, and EH. Chen. 1990. Biotypes of the Hessian
By (Diptera: Cecidomyiidae) identifed in the southeastern United States. J. Entomol.
Sci. 25: 575-580.

Khush, G.S.. E. Bacalangco and T. Ogawa. 1990. A new gene for resistance to bacterial
blight from 0. longistaminata. RGN 7: 121-122.

Lander, E.S. amd D. Botstein. 1986. Strategies for studying heterogeneous genetic traits

in humans by using a linkage map of restriction fragment length polymorphisms.
Proc. Natl Acad Sci. 83:7353—7357.

Landis, D., M. Brewer, and G. Heimpel. 2003. Soybean aphid parasitoid questionnaire
2003 [Online]. Available at www.ncera125.ent.msu.edu/StateRpts2003MI.htm
(verified 23 Mar. 2006) Michigan State Univ., East Lansing

Li, Y., C.B. Hill, S. Carlson. B.W. Diers, and G.L. Hartman. 2007. Soybean aphid
resistance genes in the soybean cultivars Dowling and Jackson map to linkage
group. M. Mol. Breed. 19:25—34

Li, Z., L. Qiu, J. A. Thompson, M.M. Welsh, and R.L. Nelson. 2001. Molecular genetic
analysis of US. and Chinese soybean ancestral lines. Crop Sci. 41:1330-1336.

Liu. X.M., C.M. Smith, and BS. Gill. 2002. Identification of microsatellite markers
linked to Russian wheat aphid resistance genes Dn4 and Dn6. Theor Appl Genet.
104:1042—1048.

Mensah, C., C. DiFonzo, R.L. Nelson, and D. Wang. 2005. Resistance to Soybean Aphid
in Early Maturing Soybean Germplasm. Crop Sci. 45:2228-2233.

Mensah, C., C. DiFonzo, and D. Wang. 2007. A case for the presence of soybean aphid
biotypes in Michigan. In 2007 annual meeting abstracts [CD-ROM]. ASA, CSSA,
and SSSA, Madison, WI.

Mensah, C., C. DiFonzo, and D. Wang. 2008. Inheritance of Soybean Aphid
Resistance in P1 5675418 and PI 5675988. Crop Sci. 48:1759-1763.

Mian, M.A.R., R.B. Hammond, and SK. St. Martin. 2008a. New plant introductions with
resistance to the soybean aphid. Crop Sci. 48:1055—1061.

Mian, M.A.R., S.T. Kang, S.E. Beil, and RB. Hammond. 2008b. Genetic linkage
mapping of the soybean aphid resistance gene in P1 243540. Theor. Appl. Genet.
117:955—962.

Momhinweg, D.W., D.R. Porter, and J .A. Webster. 2002. Inheritance of Russian Wheat
Aphid Resistance in Spring Barley Germplasm Line STARS-9577B. Crop Sci. 42:
1891 - 1893.

Murray, S. C., W.L. Rooney. M.T. Hamblin. S.E. Mitchell and S. Kresovich. 2009.
Sweet Sorghum Genetic Diversity and Association Mapping for Brix and Height.
The Plant Genome 2:48-62.

Newell, C.A., and T. Hymowitz. 1983. Hybridization in the genus Glycine subgenus
Glycine wild. (Leguminosae, Papilionoideae). American Journal of Botany 70:
334-348.

Painter. RH, 1951. Insect Resistance in Crop Plants. Macmillan. New York.

Pascal, T., F. Pfeiffer, J. Kervella, J.P. Lacroze, M.H. Sauge. 2002. Inheritance of green
peach aphid resistance in the peach cultivar ‘Rubira‘. Plant Breeding. 121:459-461.

Parisseaux, 8., and R. Bernardo. 2004. In silico mapping ofquantitativetrait loci in maize.
Theor Appl Genet. 109:508—5 14.

Patterson. F.L.. F.B. Maas. J.E. 111 Foster, RH. Ratcliffe. S. Cambron, G. Safranski, P.L.
Taylor, and H.W. Ohm. 1994. Registration of eight Hessian-fly resistant common
winter wheat germplasm lines (Carol, Erin, F lynn, Iris. Joy, Karen, Lola, and
Molly). Crop Sci. 34: 315—316.

Peng. J.H., Y. Bai, S.D. Haley , N.L. Lapitan. 2009. Microsatellite-based molecular
diversity of bread wheat germplasm and association mapping Of wheat resistance to
the Russian wheat aphid. Genetica 135:95-122.

Poland. J.A.. P.J. Balint-Kurti. R.J. Wisser. R.C. Pratt. and R.J. Nelson. 2008 Shades of
gray: the world of quantitative disease resistance. Trends in Plant Science
14:21 -29.

Poland, J.A., The Maize Diversity Project, Rebecca J. Nelson. 2009. Nested Association

Mapping of Northern Leaf Blight Resistance in Maize, Plant and Animal Genome,
Jan. 2009.

Puterka. G.J.. D.C. Peters. D.L. Kerns. J.E. Slosser. L. Bush. D.W. Worrall, and R.W.
McNew. 1988. Designation of two new greenbug (Homoptera: Aphididae)
biotypes G and H. J. Econ. Entomol. 81:1754—1759.

Ragsdalea. D.W., D. J. Voegtlinb. and R.J. O‘Neilc. 2004. Soybean aphid biology in
North America. Annals of the Entomological Society of America 97:204-208.

Ratcliffe, R.H., S.E. Cambron, K.L. Flanders. N.A. Bosque-Perez, S.L. Clement, and
H.W. Ohm. 2000. Biotype composition of Hessian Fly (Diptera: Cecidomyiidae)
populations from the southeastern. midwestem. and northwestern United States and

virulence to resistance genes in wheat. Journal of Economic Entomology
93:1319-1328.

Ronald, P.C., Albano B. Tabien R, Abenes L. Wu KS, McCouch S, Tanksley SD. 1992.
Genetic and physical analysis of the rice bacterial blight disease resistance locus.
Xa21. Mol. Gen. Genet. 236:113-120.

Ruth, D. 2007. A stingless wasp From China to Battle Soybean Aphids. [Online]
Available at: http://www.medicalnewstoday.com/articles/77810.php University of
Minnesota

Sharma, N.P., R.M. Sundaram and RV. Sonti, 2001. DNA markers in genetic purity
testing and marker assisted selection technology in rice improvement. In: Pages
32-38, Proc. 8‘“ Natl. Rice Biotechnol. Network Meeting, Oct 21-25, 2001.
Aurangabad, India.

Singh, K., Y. Vakil, J.S. Sidhu, D.S. Brar and HS. Shaliwal, 2001. Rice cultivar PR 106.
pyramided with three bacterial blight resistance genes, using marker assisted

selection. reaches plant-breeding trials. In: Pages 68a-71, Proc. 8th Natl. Rice
Biotechnol. Network Meeting, Oct 21-25, 2001, Aurangabad, India.

Singh, R. J., R.L. Nelson. and G. Chung. 2007. In Genetic Resources, Chromosome
Engineering. and Crop Improvement Series: Oilseed crops, Vol. 4. Edited by R.J.
Singh. CRC Press. Inc.. Boca Raton. Fla. pp. 15.

Sridhar, R. 2002. Use of molecular tools for understanding bacterial blight pathogen
populations of rice and usefulness ofdisease resistance genes. Indian Phytopathol.
55: 417-429.

Sneller, CH, DE. Mather and S. Crepieux. 2009. Analytical Approaches and Population

Types for Finding and Utilizing QTL in Complex Plant Populations. Crop Sci.
49:363-380.

Soy Stats. 2008. [Online] Available at
Http://www.soystats.com/2008/Default-frames.htm

Steffey, K.L. 2004. Soybean aphids in Illinois. [Online] Available at:
www.ipm.uiuc.edu/fieldcrops/insects/soybean_aphids/workshop/SoybeanAphids
Inlllinois_Steffey.ppt (verified 18 Mar. 2006). Univ. ofIllinois at
Urbana-Champaign.

Soroka, J.J., P.A. Mackay. 1991. Antibiosis and antixenosis to pea aphid (Homoptera:
Aphididae) in cultivars of field peas. Joumal of economic entomology
84:1951-1956.

Storey, JD. 2002. A direct approach to false discovery rates. I R Stat Soc Series B
64(3):479—498.

Tang, X., M. Xie, Y.J. Kim. J. Zhou. D.F. Klessig, G.B. Martin. 1999. Overexpression of
Pto activates Defense responses and confers broad resistance. Plant Cell 11:15—29.

Thomsberry, J.M., M.M. Goodman, J. Doebley, S. Kresovich. D. Nielsen, and ES.
Buckler, IV. 2001. Dwarf8 polymorphisms associate with variation in flowering
time. Nature Genetics 28: 286-289.

Tommasini, L.T., Schnurbusch. D. Fossati. F. Mascher and 8. Keller. 2007. Asociation
mapping of Stagonospora nodorum blotch resistance in modern European winter
wheat varieties. Theor. Appl. Genet.115: 697-708

Venette, RC, and D.W. Ragsdale. 2004. Assessing the invasion by soybean aphid
(Homoptera: Aphididae): Where will it end? Ann. Entomol. Soc. Am. 97:219—226.

Veyrieras, J. 8.. L. Camus-Kulandaivelu. B. Gouesnard, D. Manicacci, and A. Charcosset.
2007. Bridging genomics and genetic diversity: linkage disequilibrium structure
and association mapping in maize and other cereals. Crop Sci. 47: S60—S71.

Vos, R. G. Simons, T. Jesse. J. Wijbrandi. L. Heinen. R. Hogers, A. Frijters, J.
Groenendijk. P. Diergaarde. M. Reijans, J. Fierens-Onstenk, M. de Both, J.
Peleman, T. Liharska, J. Hontelez. and M. Zabeau. 1998. The tomato Mi-I gene
confers resistance to both root-knot nematodes and potato aphids, Nature

Biotechnology 16:1365 — 1369.

Workman, D. 2007. Top soybean countries America. Brazil & Argentina Lead Exporters
to largest importer China [Online] Available at:
http://intemationaltrade.suite101.com/article.cfm/top_soybean_countries#ixzzOL9b
u9UN7&D

Wu, Z., D. Schenk-Hamlinb, W. Zhan, D.W. Ragsdalea. and GE. Heimpelac. 2004
The soybean aphid in China: a historical review. Annals of the Entomological
Society of America 97:209-218.

35

 

 

 

Yu. Y.G.. G.R. Buss, and MA. Saghai Maroof. 1996: Isolation of a superfamily of
candidate disease-resistance genes in soybean based on a conserved
nucleotide-binding site. Proc. Natl. Acad. Sci. 93:1 1751—11756.

Yu, J., J.B. Holland, M.D. McMullen, and ES. Buckler. 2008. Genetic design and
statistical power of nested association mapping in maize. Genetics 178:539-551.

Zeng, Z. 1994. Precision mapping of quantitative trait loci. Genetics 136:1457-1468.

Zhang. G., C. Gu, and D. Wang. 2009. Molecular mapping of soybean aphid resistance .
genes in P1 5675418. Theor. Appl. Genet. 118:473—482.

Zhang, W., C. Andrew, J. Gibson. W.J. Tapper, S. Hunt. P. Deloukas, D.R. Bentley, N.E.
Morton. 2004. Impact of population structure, effective bottleneck time, and allele

frequency on linkage disequilibrium maps. Proc. Natl. Acad. Sci.
101:18075-18080.

Zhu. C., M. Gore. E.S. Buckler, and J. Yu. 2008. Status and prospects of association
mapping in plants. Plant Genome 1: 5-20.

CHAPTER 2
INHERITANCE OF RESISTANCE TO
THE SOYBEAN APHID IN SOYBEAN Pl 567585A
Abstract: The soybean aphid (Aphis glycines Matsumura) is an important insect pest of
soybean [Glycine max (L.) Merr.]. Soybean plant introduction (PI) 567585A has shown
strong resistance to soybean aphids. The objective Of the study was to determine the
inheritance of aphid resistance in P1 567585A. This resistant soybean PI 567585A was
crossed with two susceptible soybean cultivars. ‘Skylla‘ and IA2070. The parents, F1, F2
plants and F 2; 3 families were evaluated for aphid resistance in the field during the summer
of 2008 and 2009. All Fl plants exhibited phenotype intermediate between the resistant
and susceptible parents. The observed segregation ratios in the two F2 populations
(070082-1 and 07001 6-1) fitted a segregation ratio 1:2:1 (Resistant: Intermediate:
Susceptible). The F233 families also fitted the segregation ratio of 1:2:1 (Resistant:
Segregating: Susceptible). These data indicated that the aphid resistance in PI 567585A

was controlled by one major co-dominant gene.

Abbreviations: LG: linkage group: P1: plant introduction:

Key word: soybean aphid resistance. cO-dominant. PI 567585A. segregation ratio

37

The soybean aphid (Aphis glycines Matsumura) was first found in the United State in
2000 (Hartman et al.. 2000). Since then. it has been reported in 21 states in the United
States and three provinces in Cananda (Venette and Ragsdale, 2000; Ragsdale et al.,
2004). In Michigan. an estimation of 740.000 hectares was affected and cost $9 million
(DiFonzo. 2004). This insect sucks sap from plants. excrete honeydew on plants. The
sooty mold also develops on the honeydew which the aphids secrete, inhibiting the
photosynthesis process. Soybean aphids were reported transmitting viruses found in
soybean, potato. dry bean and vine crops (Mian et al.. 2008a). It caused severe yield
reduction in soybean in several north central states in 2003 (Zhang et al.. 2009).

Soybean aphids are generally controlled by application of foliar insecticides, though
it is costly and not environmentally friendly. A better alternative will be the use of aphid
resistance cultivars. However, no commercial aphid resistant soybean cultivar is currently
available in North America. Several aphid resistance sources were discovered in early
and late maturity germplasm. such as ‘Dowling’, ‘Jackson‘. PI 5675988, PI 5675418, PI
243550, and PI 200538 (Hill et al., 2004: Mensah et al., 2005; Mian et al., 2008a; Hill et
al., 2009). Recently we identified a new source of aphid resistance, PI 567585A (Dechun
Wang. unpublished data). PI 567585A is a maturity group II germplasm accession
originated from Shandong, China (Hill et al.. 2005). It showed resistance against the

soybean aphid in both choice and non-choice tests.

38

For some of existing aphid resistance sources. the inheritance patterns and the
location of resistance genes have been investigated since 2006. The aphid resistance in
‘Dowling’ and ‘Jackson’ is controlled by a single dominant gene. This gene is located on
linkage group (LG) M and named as Rag] and Rag, respectively (Hill et al., 2006a; Hill
et al., 2006b; Li et al., 2007). Resistance in both PI 5675988 and PI 5675418 accessions
is conditioned by two recessive genes (Mensah et al.. 2008). The resistance genes of PI
5675418 were recently mapped on LGs M and F (Zhang et al.. 2009). The antibiosis
resistance in P1 243550 and PI 200538 accessions was also controlled by a single
dominance gene designated Rag2. which is located on LG F (Kang et al., 2008; Mian et
al., 2008b; Hill et al.. 2009). The inheritance of aphid resistance in P1 567585A is
unknown. The objective of this study is to determine the inheritance of aphid resistance

in PI 567585A.

Materials and Methods
Population construction and aphid resistance evaluation

PI 567585A was crossed with ‘Skylla’ and IA2070. PI 567585A is resistant to
soybean aphids. and both ‘Skylla’ and IA2070 are susceptible cultivars. F 1 plants and F2
populations were developed from the crosses Skylla x PI 567585A and IA2070 x PI
567585A (070082-1 and 070016-l). The parental lines, F. plants. and F2 populations

were evaluated during the summer Of 2008 in the field on the Agronomy Farm of

39

Michigan State University (MSU). The field evaluation of soybean aphid resistance was
carried out in a 12.2- x 18.3-m aphid- and predator- proof cage. Fourteen days after
planting. each plant was inoculated with two Wingless aphids (Mensah et al., 2005) at the
V2 stage (Fehr and Caviness. 1977). All aphids used in inoculation were collected from
nearby naturally infested soybean fields. Three parents, PI 567585A, Skylla, and IA 2070
were planted 6.0 cm apart at row width of 38 cm with three replications. F 1 and F2 plants
were planted 5.0 cm apart at row width of 38 cm with no replications. Each parental, F1
and F2 plant was rated for aphid resistance 21 days after inoculation using the modified
half step scale ranging from 0 to 4 as described by Mensah et al. (2008).

Seeds from individual F2 plants in populations 070082-1 and 070016-1 were
harvested individually during the fall of 2008. Depending on seed availability, F2 plants
with a minimum of 1 1 progenies. resistant, susceptible or intermediate to soybean aphids,
were chosen for further aphid resistance study during the summer 012009. One hundred
and fifty eight F23 families were obtained from the 070082-1 F2 population, and 58 F23
families were collected from the 070016-1 F2 population. F23 plants were planted 3.0 cm
apart with no replication in the field cage during the summer of 2009. The soybean aphid

resistance was scored for each F23 family on a row basis as described before.

Statistical Analysis

40

C hi-square tests were performed to test the goodness of fit of observed segregation
ratios for F2 populations and F 23 families with the expected genetic ratios. In order to
analyze the segregation in F2 populations. each individual plant was classified as resistant
if it had a rating equal to or lower than the resistant parent, or as susceptible if it had a
rating equal to or higher than the susceptible parents. or as intermediate if the observed
phenotype Of an individual plant was between the resistant and susceptible parents.
Segregation among F 23 progeny was analyzed by classifying each family into three
groups: homozygous resistant (all observed ratings ofone family were equal to or lower
than resistant parent), segregating (all observed ratings of one family were segregating
for resistant. heterozygous and susceptible phenotypes). and homozygous susceptible (all

observed ratings of one family were equal to or higher than susceptible parents).

Results and discussion
Aphid resistance evaluation for F1 plants

The aphid resistance ratings for PI 567585A plants were 0.5 or 1.0., and 3 or 3.5 for
Skylla and IA 2070 plants. All F 1 plants from the two crosses demonstrated a phenotype
intermediate between the resistant and susceptible parents ranging from 1.5 to 2.5 (Table
2.1). Thus, an individual plant in F2 populations with a rating of 1.0 or less was classified
as resistant, a plant with a rating larger than 3.0 was regarded as susceptible, and a plant

with a rating ranging from 1.5 to 2.5 was considered intermediate.

41

Segregation analysis for aphid resistance in F2 populations

The frequency distributions of the aphid resistance ratings in the two F2 segregating
populations are shown in Figure 1. For the F2 population of 070082-1, the segregation
was 61 resistant plants. 1 15 intermediate plants and 45 susceptible plants. The
segregation ratio fits a 1:221 ratio (P:0.2614) (Table 2.2). For 070016-1 F2 population,
the segregation of resistant. heterozygous, and susceptible plants was 21:51:14, fitting a
1:221 ratio (P=0.1277) (Table 2.3). Thus. resistance to the soybean aphid showed a
qualitative character in two F2 populations. Moreover. both segregation ratios of F2
populations fit the expected 1:2:1 ratio. indicating a major co-dominant gene controlling

the aphid resistance in P1 567585A.

Segregation analysis for aphid resistance in F23 families

A total of 158 “Skylla” x PI 567585A F23 families were collected for the progeny
tests based on the requirement that they had produced a minimum of 1 1 seeds to allow
adequate statistical analaysi. The ratio of41:84:33 fits a 122:]
resistant/intermediate/susceptible ratio (P=0.4860). fully representing the 070082-1 F2
population. The F23 progeny test of 070082-1 F2 plants showed a ratio of 38:75:45
homozygous resistant/segregatirig/homozygous susceptible. significantly fitting the
ratio] 22:1 (P=0.5989) expected for a monogenic co-dominant gene (Table 2.4). For the

070016-1 F2 population, 58 F 23 families were collected for the progeny test. The ratio of

42

 

 

resistant/intermediate/susceptible. which was found to be 17:26:15, fits the expected
1:2:1 ratio (P=0.6843), fully representing the 070016-I F2 population. The results
showed a ratio of 16:28:14 homozygous resistant/segregating/homozygous susceptible,
which significantly fits the ratiol :2:1 (P=0.9017) expected for a monogenic co-dominant
gene (Table 2.3).
Dominant, recessive and co-dominant nature of aphid resistance

Both dominant and recessive genes have been identified for the aphid resistance in
plants. In wheat germplasm accessions. one recessive gene and eight dominant genes
have been identified for Russian wheat aphid resistance, and are generally qualitatively
inherited (Liu et al., 2001). In alfalfa and sweet clover. a single dominant gene controls
resistance to the pea aphid. A. pisum (Glover and Stanford, 1966), and the sweet clover
aphid, Therioaphis riehmi (Manglitz and Gorz. 1968). Spotted alfalfa aphid resistance in
alfalfa is controlled by several genes. suggesting the quantitative inheritance of resistance
(Glover and Melton. 1966). In some Solanum species. resistance to the green peach aphid,
Myzus persicae exhibits a partially dominant inheritance (Sams et al., 1976). Moreover,
in the soybean cultivar ‘Dowling‘, ‘Jackson’, PI 200538 and PI 243550. resistance to
soybean aphid is controlled by a single dominant gene (Kang et al.. 2008, Hill et al.,
2009). However, some insect resistance is conferred by co-dominant or recessive gene(s).
For example. the resistance genes underlying rhgl in soybean were found to be dominant,

recessive and co-dominant in the study of the Hg type resistance to the soybean cyst

43

nematode (Heterodera glycines) (Meksem et al., 2001; Afzal et al., 2009). A monogenic
co-dominant gene controlled resistance to the cyst nematode (Heteroa’era sacchari) in
African rice. ()ryza glaherrima (Lorieux et al., 2003). And two recessive genes control
the aphid resistance in soybean accessions Pl 5675988 and PI 5675418 (Mensah et al.,
2008).

In this study, aphid resistance in P1 567585A was shown to be controlled by a single
co-dominant gene controlling the aphid resistance. The inheritance pattern is different to
other host-plant aphid resistance sources. Louriex (2003) mentioned that different
phenotyping methods were applied to identify the possible co-dominant inheritance as
dominant in numerous pathogen resistance studies. In previous inheritance studies of
‘Dowling', ‘Jackson‘, PI 200538 and PI 243540. researchers used the 1-4 nonparametric
ordinal rating or 1-5 modified scoring (Hill et al., 2006a; Hill et al., 2006b; Mian et al.,
2008; Hill et al., 2009) because the segregating populations only showed two distinct
parental resistance phenotypes without any intermediate characteristics. In our study, the
F2 segregation populations showed intermediate phenotypes between two distinctive
parents. exhibiting discontinuous normal distributions. The 0-4 half-step rating scale can
effectively separate the variable aphid damage on individual plants in the field, allowing
for the clarification of plants into different scales. Hill et al. (2006) also pointed that there
would be more variability in aphid colonization on plants in the field compared with tests

carried out in the greenhouse. This inheritance study was completed in the field,

44

compared to the previous utilized inheritance studies in greenhouses. Thus, the 0-4
half-step rating scale was necessarily recruited in the field to evaluate variable aphid
damaging on individual plants.

In summary. a single co-dominant gene was discovered for soybean aphid resistance
in soybean accession PI 5675 85A. As a monogenic resistance source, it can be easily
introgressed into elite lines through backcross or pedigree breeding. However, it likely
will be overcome during a period of time due to evolution of insect biotype. Pyramiding
resistant genes is an effective strategy against the target insect/pathogen population,
which has been studied in soybean and rice (lIittalmani et al.. 2000; Maroofa et al., 2008).
Our next step is to map the resistance gene in P1 567585A. and discover whether it
co-localize with the current resistance gene(s) located on LG F, J or M, or it is a new
gene located elsewhere on the soybean genome. Ultimately. the genetic mapping of the
resistance gene in P1 567585A will hasten the selection of aphid resistance in breeding

program through use of marker assisted selection methods.

45

Table 2.1 Observed soybean aphid resistance rating for parental and F1 plants 21 days

after aphid inoculation in field in 2008 summer.

 

 

Genotype Total number of Number of Mean
plants tested plants rating
Pl 567585A 8 8 0.8
Skylla 10 0 3.5
IA2070 1 1 O 3.4
(Skylla x Pl 567585A) Fl 5 5 2.4
(IA2070 x PI 567585A) F1 5 --* --*

 

*2 The data were unavailable for the F1 plants from 1A 2070 x P1 567585A due to weakness and death in
field.

46

Table 2.2 Segregation of soybean aphid resistance in F2 populations derived from

different crosses.

 

 

Population Susceptible Resistant Total no. Observed X“ [:2] P 1.2.]
[D Parent Parent of plants R I S

070082-1 Skylla P1 567585A 61 1 16 45 2.683 0.2614
070016-1 1A207O P1567585A 21 51 14 4.116 0.1277

 

R: resistant with score 0.5 or 1.0

1: intermediate between resistant and susceptible with score ranging 1.5-2.5

S: susceptible with score 3.0, 3.5 or 4

47

 

 

0700 l

\
RR: I’IOr

RI: 55211
llc‘lertm1

IT: holllll

 

 

Table 2.3 Segregation of F23 progenies from PI 567585A x Skylla and IA2070 F2 plants

for aphid resistance rating.

 

 

Population NO. F2 plant F2 plant No. of F213 X2 [2;] P 12:!
phenotype genotype families
070082-1 Resistant RR 17
RT 20
rr 4
Intermediate RR 19
RT 44
rr 21
Susceptible RR 2
Rr 1 1
rr 20
Total 1.025 0.5989
070016-l Resistant RR 7
RT 10
rr 0
Intermediate RR
Rr 15
rr 2
Susceptible RR 0
Rr 3
rr 12
Total 0.207 0.9017

 

RR: homozygous resistant (all F23 plants in an individual family are resistant)
Rr: segregating progenies (all F23 plants in an individual family are a segregating resistant,
heterozygous and susceptible)

rr: homozygous susceptible (all F23 plants in an individual family are susceptible)

48

 

070082-1 and 070016-1 F2

 

I 070082-1 F2 population
El 070016-1 F2 population

 

 

 

 

0.5 1 1.5 2 2.5 3 3.5 4
Soybean Aphid Damage Rating

 

 

 

Figure 2.1 Frequency distribution Of soybean aphid resistance rating scores for F2

populations 070082-1 and 070016-l. respectively.

49

 

 

RCIt
Afz:l

DIFG

Fehr.

Glove

Glovei

Hill. C]

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r.

Hill, J.L

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(3

 

Hill. (1;

 

References:

Afzal A.J., N. Saini, A. Srour, and DA. Lightfoot. 2009. The multigeneic rth locus
complete sequence identifies alleles of three genes underlying root development,
nematode resistance and recombination suppression. Nature Genet (in review)

DiFonzo. C. 2004. Determining optimum spray timing for the soybean sphid. [Online]
Available at: http://fieldcrop.msu.edu/documents/GR04-006.pdf

Fehr, W.R., and GE. Caviness. 1977. Stages of soybean development. Iowa Coop. Ext.
Service, Iowa Agric. Home. Exp. Stn. Spec. Rep. 80. Iowa State Univ., Ames, IA.

Glover, E. and B. Melton. 1966. Inheritance patterns of spotted alfalfa aphid resistance in
Zea plants. New Mexico Agriculture Experimentation Station Research Report
127: 1 -40.

Glover, D.V., and EH. Stanford. 1966. Tetrasomic inheritance of resistance in alfalfa to
the Pea Aphid. Crop Sci 1966 6: 161-165.

Hartman G.L., L.L. Domier, L.M. Wax, C .G. Helm, D.W. Onstad, J.T. Shaw, L.F. Solter,
D.J. Voegtlin, C.J. D’Arcy, M.E. Gray, K.L. Steffey, S.A. Isard, and PL. Orwick.
2001. Occurrence and distribution oprhis glycines on soybeans in Illinois in 2000
and its potential control. Plant Health Progress. [Online] Available at:
httpzllwww.plantmanagementnetwork.org/sub/php/brief/aphisglycines/

Hill, C.B., Y. Li and G.L. Hartman. 2004. Resistance to the soybean aphid in soybean
germplasm. Crop Sci. 44298-106.

Hill, J.L., E.K. Peregrine, G.L. Sprau, C.R. Cremeens, R.L. Nelson, J.H. Orf, and DA.
Thomas. 2005. Evaluation Of the USDA Soybean Gerrnplasm Collection: Maturity
Groups 000-IV (PI 507670-PI 574486). [Online] Available at:
http://www.ars.usda.gov/is/np/SoybeanGermplasm/SoyGerm2005.pdf

Hill, C.B., Y. Li, and G.L. Hartman. 2006a. A single dominant gene for resistance to the
soybean aphid in the soybean cultivar Dowling. Crop Sci. 46:1601-1605.

Hill, C.B., Y. Li and G.L. Hartman. 2006b. Soybean aphid resistance in soybean Jackson
is controlled by a single dominant gene. Crop Sci. 46: 1 606-1608.

50

Hill, C.B., K. Kim, L. Crull, B.W. Diers, and G.L. Hartman. 2009. Inheritance of
resistance to the soybean aphid in soybean PI 200538. Crop Sci. 1 193-1200.

Hittalmani, S., A. Parco, T.V. Mew, R.S. Zeigler. 2000. N. Huang Fine mapping and

DNA marker-assisted pyramiding of the three major genes for blast resistance in
rice. Theor. Appl. Genet. 100:1121—1128.

Kang, S., M.A.R. Mian. and RB. Hammond. 2008. Soybean aphid resistance in P1 23540
is controlled by a single dominant gene. Crop Sci. 48:1744-1748.

Kogan, M. 1982. Plant resistance in pest management. In Metcalf, R.L. and Luckmann.
W.H. (Eds). Introduction to Insect Pest Management, 2nd Edition. New York,
USA: John Wiley & Sons, 93-134.

Li, Y., C.B. Hill, S. Carlson, B.W. Diers. and G.L. Hartman. 2007. Soybean aphid
resistance genes in the soybean cultivars Dowling and Jackson map to linkage
group M. MOI. Breed. 19:25—34.

Liu, X.M., C.M. Smith, B.S. Gill, and V. Tolmay. 2001. Microsatellite markers linked to
six Russian wheat aphid resistance genes in wheat. Theor. Appl. Genet.
102:504—510.

Lorieux, M,, G. Reversat, S.X. Garcia Diaz, C. Denance, N. Jouvenet, Y. Orieux, N.
Bourger, A. Pando-Bahuon, A. Ghesquiere. 2003.Linkage mapping of Hsa-1(Og),
a resistance gene of African rice to the cyst nematode, Heterodera sacchari. Theor.
Appl. Genet. 1074:691-696.

Manglitz, GR. and Gorz, HI. 1968. Inheritance of resistance in sweetclover to the
sweetclover aphid. Jounal of Economic Entomology 61 190-94.

Meksem, K., P. Pantazopoulos, V.N. Njiti. L.D. Hyten, P.R. Arelli, and DA. Lightfoot.
2001. 'Forrest' resistance to the soybean cyst nematode is bigenic: saturation
mapping of the Rhgl and Rhg4 loci. Theor. Appl. Genet. 103:710-717.

Mensah. C., C.DiFonzo. and D. Wang. 2008. Inheritance of soybean aphid resistance in
P1 5675418 an PI 5675988. Crop Sci. 48:1759-1763.

51

 

khan.

Khan.

hkuo

Venen

Zhang,

I'M

 

Mian, M.A.R., R.B. Hammond. and SK. St. Martin. 2008a. New plant introductions with
resistance to the soybean aphid. Crop Sci. 48:1055—1061.

Mian, M.A.R., S.T. Kang. S.E. Beil, and RB. Hammond. 2008b. Genetic linkage
mapping of the soybean aphid resistance gene in P1 243540. Theor. Appl. Genet.
1 1.7:955—962.

Maroofa, M.A.S, SC. Jeong, I. Gunduzc, D.M. Tuckera, G.R. Bussa and SA. Tolind.
2008. Pyramiding of Soybean Mosaic Virus Resistance Genes by Marker-Assisted
Selection. Crop Sci 48:517-526.

Ragsdale, D.W., DJ. Voegtlinb. and R.J. O’Neilc. 2004. Soybean aphid biology in
North America. Annals of the Entomological Society of America 97(2):204-208.

Sams, D.W., F.I. Lauer, and E.B.Radcliffe, 1976. Breeding behavior of resistance to
green peach aphid in tuber-bearing solanum germplasm. American Potato Journal
53:23-29.

Venette RC. and D.W. Ragsdale. 2004. Assessing the invasion by soybean aphid
(Homoptera: Aphididae): Where will it end? Ann. Entomol. Soc. Am. 97:219—226.

Zhang, G., C. Gu. and D. Wang. 2009. Molecular mapping of soybean aphid resistance
genes in P1 5675418. Theor. Appl. Genet. 118:473—482.

 

1&8

sov

V1 35

The

“as '

ands

CHAPTER 3

GENETIC LINKAGE MAPPING OF
THE SOYBEAN APHID RESISTANCE GENE IN PIS67585A

ABSTRACT

The soybean aphid (Aphis glycines Matsumura) is an important insect pest of
soybean [Glycine max (L.) Merr] in North America. In our previous study. PI 567585A
was shown to possess soybean aphid resistance controlled by a single co-dominant gene.
The objective of this study was to determine the genetic basis of aphid resistance in P1
567585A. A mapping population of 158 F435 recombinant inbred lines (RILs) derived
from the cross between PI 567585A (resistant to soybean aphid) and ‘Skylla’ (cultivar
susceptible to soybean aphid) was evaluated for aphid resistance in both the greenhouse
and field in 2009. Broad-sense heritability estimate of aphid resistance in the field trial
was 95.5%. The single aphid resistance gene was mapped in an interval between Satt674
and Sct_065, simple sequence repeat (SSR) markers on chromosome 16 (linkage group J)
using the composite interval mapping method. The locus explained 93.1% of the
phenotypic variation in the field trial. and is located in the same genomic region as Rag3.
This single aphid resistance gene in P1 567585A was confirmed in another F24 RIL
population derived from a cross between P1 5675 85A and a susceptible parent IA2070.

The SSR markers linked to aphid resistance in P1 567585A discovered in this study,

53

 

ahsng

used‘

INTF

2001 1
densn
20093
honeyt

additic

along with other independent aphid resistance genes from diverse germplams, could be

used to pyramid multiple genes into a soybean cultivar for more durable aphid resistance.

INTRODUCTION

Soybean aphids (Aphis glycines Matsumura) are native to eastern and southeastern
Asia. The insects were first reported in North America in July 2000 (Hartman et al.,
2001). Since it was detected. this new pest has rapidly increased to very high population
densities and spread to 21 states in the US. and three provinces in Canada (Zhang et al.,
2009a). High populations of soybean aphids suck sap from soybean plants, secret
honeydew on plants during the early reproductive stages, causing reduced pod set. In
addition, soybean aphids can transmit soybean viruses (DiFonzo and Hines, 2002).

Soybean growers typically control soybean aphid by applying foliar insecticides,
which increase the production costs and are released in the environment. The best
alternative control strategy would be the utilization of aphid resistant cultivars; yet, there
are no commercial aphid resistant soybean cultivars available in North America. To
date. several aphid resistance sources have been found in soybean accession. In the
genetic inheritance pattern of resistance has been determined in seven genotypes,
including ‘Dowling’, ‘Jackson’, PI 243550, PI 200538, PI 5675988, PI 5675418, and PI
567543C. In six genotypes (with the exception on of PI 5675988), the location of the

resistance gene(s) has been mapped. Single dominant genes Rag] and Rag on

54

 

et al.

Pl 5i

et a1

Clas
may
gent
N at
al..

Clus
n€m
\t'jjh

M 1r.

chromosome 7 (LG M) control aphid resistance in ‘Dowling‘ and ‘Jackson‘, respectively
(Hill et al.. 2004; Hill et al.. 2006a; Hill et al.. 2006b; Li et al., 2007). Aphid resistance in
P1 243550 and PI 200538 is also controlled by a single dominant gene designated Rag2,
which is located on chromosome 13 (LG F) (Kang et al.. 2008: Mian et al., 2008a: Mian
et al.. 2008b: Hill et al., 2009). Two recessive genes are involved in the resistance of both
PI 5675988 and PI 5675418 (Mensah et al.. 2008). The two resistance genes of PI
5675418 have been recently mapped on LGs M and F. namely ragl_C and rag-l (Zhang
et al.. 2009a). rag1_(,' was mapped to the same genome region as Rag], while rag-l was
distantly located from Rag2. Most recently, a major gene. Rag3 on chromosome 16 (LG
J), was identified in the aphid resistance in P1 567543C (Zhang et al., 2009b).

Clustering and sequence similarity of different resistance gene analog (RGA)
classes are known from other disease and insect resistance studies in plants. Genetic
mapping of nine classes of RGA located them on eight linkage groups of the soybean
genetic map. dispersing singly or in clusters along several LG, such as C2, D]. H. J, L. M,
N and P. Five classes of RGAs were co-localized as a large cluster on LG J (Kanazin et
al., 1996). LG F is another known important region where disease resistance genes are
clustered in soybean. where several RGAs related to virus. bacteria. fungus, and
nematode resistance are mapped (Jeong et al.. 2001). All ofthis information matches well
with the available locations of aphid resistance genes in soybean. Therefore, LG F, J and

M in soybean genome were given priority for mapping new aphid resistance locus.

55

 

gemir
resist:
Wang
contrt
Howe
object

with I

Used

3006i

Elal.:

CQndUC

A new source of aphid resistance was identified in P1 567585A, a maturity group II
germplasm accession originated from Shandong, China (Hill et al., 2005). It showed
resistance against the soybean aphid in both choice and non-choice tests (Dr. Dechun
Wang, unpublished data). In a previous study, one co-dominant gene was determined to
control the inheritance of aphid resistance in P1 567585A (Liu et al., unpublished data).
However, the location of the aphid resistance gene in P1 567585A is unknown. Thus, the
objective of this study was to map and validate the aphid resistance gene in PI 567585A

with linked SSR markers.

Materials and Methods
Plant materials and aphid resistance evaluation

A mapping population of 158 F45 lines (070082) was developed from the cross of PI
567585A x ‘Skylla’ by single seed descent. PI 567585A possesses antibiosis resistance to
the soybean aphid (Dechun Wang. unpublished data). The Chinese cultivar name of PI
567585A is ‘Ri Zhao Huang’. The morphological and agronomic traits of PI 567585A are
listed by Hill et al. (2005). ‘Skylla’ is an aphid-susceptible soybean variety (Wang et al.,
2006).

Based on the heritability of aphid resistance shown in previous experiments (Zhang
et al., 2009a). a single trial was carried out in the greenhouse and two replications were

conducted in field. The greenhouse trial was initiated in the Plant Science Greenhouse at

56

Michigan State University (MSU) in East Lansing. Michigan. Eight seeds per line or
parent were planted in a plastic pot. which is 210 mm in diameter and 125mm deep. In a
completely randomized design (C RD), two parents and the mapping population were set
on the bench without replication. The temperature was maintained at 26/15°C day/night
with 14-h supplemental lighting provided by sodium vapor lamps. In the summer of 2009,
the field evaluation of soybean aphid resistance was carried out in a 12.2 x 18.3m aphid-
and predator- proof cage (Redwood Empire Awning C 0.. Santa Risa, CA) on the
Agronomy Farm of MSU. The parental plants were planted randomly in the field, 5.1 cm
apart, with two replications. Depending on the seed availability. 4 to 16 seeds per line
were planted in a single row plot. 60cm long with a row spacing of 60cm. The average
number of plants per recombinant inbred line was around nine with most plots having at
least eight plants. Similarly. C RD was used to arrange the whole F45 population and its
parents in the field plots with two replications.

In both greenhouse and field trials. each plant was inoculated at the V2 stage with
two Wingless aphids. A single aphid clone was collected from a naturally infested field at
the MSU Agronomy Farm in summer 2008, and maintained in an isolation chamber in
the greenhouse for the inoculation of plants in the greenhouse trial in 2009 spring. The
soybean aphids used for inoculation in the field trial were collected from a naturally
infested field on the MSU Agronomy Farm in 2009 summer. The F45 mapping population

and parental plants were evaluated for aphid damage 3 wk after inoculation using a

57

 

modific-
resistan
replicat'
resistant
DI it as I

each can

DH ext

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hne1F45;
DNA extr
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modified 0-4 half step rating scale described by Mensah et al. (2008). The aphid
resistance score was detemrined as the mean of the rated plants in each line for each
replication. An aphid damage index (DI) for each line was used as an indicator of aphid
resistance, ranging from 0 (no damage) to 100 (most severe damage (Mensah et al., 2005).
D1 was calculated based on the following formula: D1 = 2 (scale value x no. ofplants in

each category) / (4 x total no. of plants) x 100 (Zhang et al.. 2009a).

DNA extraction and SSR marker genotyping

In the field trial of 2009. the unopened trifoliate from each individual plant of each
line (F435 mapping population) and their parents were bulk harvested for the genomic
DNA extraction. The C TAB (Hexadecyltrimethyl ammonium bromide) described by
Kisha et al.. (1997) was used to extract the genomic DNA. The concentration was
determined with a ND-1000 Spectrophotometer (NanoDrop Technologies. Inc..
Wilmington Delaware).

The genomic DNA from each RIL line and parent was amplified by PCR protocol
with SSR markers described by C regan and Quigley (1997) on a MJ TetradTM thermal
cycler (MJ Research Waltham. MA). The sequence information of SSR primers was
provided by Dr. Perry Cregan (USDA-ARS. Beltsville. Maryland). A total of 1056 SSR
primers were used to screen for the polymorphism between PI 5675 85A and ‘Skylla’.

Bulked segregant analysis (Michelmore et al.. 1991) was used to accelerate the

58

ide:

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on If

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DASC
Wang

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Present

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identification of the aphid resistance locus. Ten resistant lines with the lowest DI scores
were selected and bulked into a resistance pool for analysis. The resistant bulk and
parental DNA samples were genotyped with polymorphic markers. Priority was placed
on the polymorphic SSRs on chromosomes 7, 13, and 16 (LG M, F, and J) with coverage
of a marker at every 10 cM because these LGs were linked to aphid resistance in other
soybean accessions.

The PC R products were separated on 6% non-denaturing polyacrylamide gels with a
DASG-400-50 electrophoresis unit (C.B.S. Scientific Co.. Del Mar, CA) as described by
Wang et al.. (2003). The ethidium bromide stained gels were visualized and
photographed under UV light. For polymorphic SSR markers. the PCR products of each
line in the mapping population were scored as ‘a‘ (only the band of the resistant parent
present). ‘b’ (only band of susceptible parent present) or ‘h‘ (bands from both parents

present).

Statistical and QTL analysis

The DI data from the field trial was analyzed by the analysis of variance (ANOVA)
with the GLM procedure of SAS V9.1. The broad-sense heritability of D1 was estimated
according to the method described by Fehr (1987). The SSR genotyping data and the
aphid resistance phenotyping data of F415 RIL lines were analyzed to construct a linkage

map with Join-Map 3.0 by using the Kosarnbi function and a LOD score of 3.0 (Van

59

 

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expt

map

back
other
was c

LOD

belittle
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Its Patel
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Ooijen and Voorrips. 2001). At each locus of potential aphid resistance, the segregation
ratio of alleles was determined by X2 goodness of fit to detect ifthe locus met the
expected 7:2:7 ratio with a significance threshold ofP = 0.05. Composite interval
mapping (C 1M) was performed to detect aphid resistance loci by using QTL C artographer
V2. with a standard model Zmapqtl 6 (Wang et al.. 2008'). In order to control the genetic
background. the forward and backward regression method was applied to select markers
other than the interval being tested as cofactors (Zeng, 1994). A window size of 10 CM
was chosen and the target markers interval distance was at 2 cM for CIM. The empirical
LOD at 5% probability level was determined by a 1.000- permutation test (Churchill and
Doerge. 1994). The linkage map and the aphid resistance loci were visualized by

MapChart (Voorrips. 2002).

Resistance locus validation

A validation population of 162 F314 lines (070016) was derived from the cross
between PI 567585A and IA 2070 by single seed descent. IA 2070 is an
aphid-susceptible soybean cultivar. In the summer of 2009. the validation population and
its parents were evaluated for aphid resistance in a field trial similar to the mapping
population with two replicates. Ninety four RILs were randomly selected as a subset
population from the validation population. The genomic DNA of these 94 RILs was

extracted by method described above. Polymorphic markers within the potential regions

60

containing the aphid resistance locus were genotyped for the validation population.
Linkage map construction and genetic mapping analysis were carried out in the same way

as for the mapping population.

RESULTS
Phenotypic data analysis for mapping and validation populations

The phenotypic data for aphid damage index of mapping and validation populations,
and parents in the field trial were shown in Table 3.1. Resistant parent PI 567585A had
significantly (P<0.05) lower DI than susceptible parents ‘Skylla’ and IA2070, which
were heavily infested by soybean aphids. The broad-sense heritabilities for aphid
resistance were 0.96 and 0.89 for population 070082 and 070016 in the field trial,
respectively (Table 3.1). This indicates that substantial variation exists among RILs
within both mapping and validation populations. The DI for the two populations showed
discontinuous variation and approximate bimodal distribution with a ratio of 1:1,
confirming that aphid resistance is controlled by one single gene (Figure 3.1) (Liu et al.,

unpublished data).

Genetic mapping of aphid resistance
A total of 313 SSR markers were polymorphic between PI 567585A and ‘Skylla’.

Analy Sis of the bulked resistant lines from the 070082-2 population indicated that the

61

SSR markers Satt622 and Satt215 on chromosome 16 (LG J) were associated with aphid
resistance. These two SSR markers were genotyped for the entire 070082 RIL population,
and their associations with aphid resistance were confirmed. Five other polymorphic SSR
markers within i 30 cM of Satt622 and Satt215 were genotyped for the entire mapping
population. The segregation of all markers except Satt674 fit a 7:2:7 (homozygous SSR
allele of the resistant parent: heterozygous SSR alleles from both resistant and susceptible
parents: homozygous SSR allele of the susceptible parent) segregation ratio (P>0.05) at
F4 generation (Table 3.2).

A linkage group was constructed by analyzing these seven markers with Join-Map.
The marker order was highly consistent with the consensus map (Song et al., 2004),
although the spanning distance was 70.2 cM. about 7.3 cM larger than the corresponding
map distance of 62.9 cM (Figure 3.2). The aphid resistance gene was identified in the
interval between Satt674 and Sct_065 in both greenhouse and field trials (Figure 3.2 and
Table 3.3). The major phenotypic variation of aphid resistance contributed by the PI
567585A gene in the greenhouse and field trials was 93. 1% and 90.1%, respectively. The
additive effect of this resistance gene was also determined for the mapping population in
both trials. The PI 567585A resistance allele decreased the soybean aphid DI value by
32.35 and 30.50 in the greenhouse and field trials. respectively (Table 3.3). In addition,
the average DI value was calculated for each genotype class of Sct_065, and analyzed by

ANOVA. The heterozygous class showed an intermediate level of resistance to soybean

62

aphid between resistant and susceptible classes. which was not significantly different
from the average of the two homozygous classes (Table 3.4). These results indicated that
aphid resistance in P1 567585A is controlled by a single co-dominant gene with additive
effect, which is consistent with a previous genetic inheritance study of PI 567585A (Liu

et al., unpublished data).

Validation of aphid resistance gene

Seven SSR markers linked to the resistance locus on LG J in the mapping population
were genotyped for the validation population (070016). The segregation ratio of each
marker fit the 7:2:7 ratio (Table 3.2). These seven markers were used to construct a
linkage map, which was similar to the consensus map, except for the inverted order of
Satt654 and Sct_065 (Figure 3.2). A single aphid resistance gene was identified in the
interval between Satt654 and Sct_065 in the QTL analysis using the CIM analysis. This
gene is located at a position of 8 CM above Sct_065, which is the same as the QTL
detected in the mapping population. Moreover. the resistance gene identified in the
validation population explained 85.6% of the phenotypic variation in the field trial (Table
3.3). Thus, analysis ofthe validation population confirmed the location of the aphid

resistance locus identified in the mapping population.

DISCUSSION

63

The increased genetic distance in two mapping populations for LG J was due to
three possible reasons, when compared to the consensus map: 1) the parents used for our
SSR map are more distantly related and expected to have a lower recombination rate; 2)
the average distance is larger among the markers. which are closely linked to the
resistance gene; 3) Join-Map utilizes two-point detection unlike the MapMaker that uses
three-point detection approach. However. MapMaker cannot be set to accept data from
F4-derived families. The aphid resistance in P1 567585A was controlled by a single
co-dominant gene with additive effect. that mapped between Satt674 and Sct_065 on
Chromosome 16 (LG J). In a previous study. the resistance gene in P1 567543C was also
found to be a single co-dominant gene possessing additive effects. This gene was located
in the same general region as the aphid resistance gene Rag} in P1 567543C (Zhang et al.,
2009b). Further fine mapping of Rag3 and Rag3-1 with more SNP markers will be
considered to identify the relationship between them. either closely-linked or multiple
allelism within the same locus.

The discovery ofa common mapping location for two aphid resistance genes was
not unexpected because the two PIs were both collected from Shandong province, China
(Chen et al., 2007). This kind of genetic allelism also exists in other soybean aphid
resistant accessions. For example. Rag] in ‘Dowling’ and Rag in ‘Jackson’ were mapped
to the same position on LG M (Li et al.. 2007). The resistance gene in P1 200538 was

mapped to the same region as the aphid resistance gene Rag2 in P1 234550 on soybean

64

LG F (Mian et al., 2008; Hill et al., 2009). Moreover. a QTL conferring resistance to
brown stem (caused by Phialophora gregata) was mapped to the same region in five
different PIs. which all originated from central China (Klos et al., 2005). So data
suggested that the resistance gene (Rag3_1) in P1 567585A is not a new aphid resistance
gene, but may be a resistance source in addition to Rag3 ofPI 567543C.

The mapping of soybean aphid resistance to LG J is interesting because several
resistance genes. such as powdery mildew resistance locus (Rmd). corn earworm (CEW)
resistance genes (C EW6-2 and C EW 7-4). brown stem rot (BSR) resistance gene,
Phytophlhora resistance Rps2. soybean cyst nematode (SCN) race-2 and race-3 resistance
genes, sudden death syndrome resistance genes (SDS). and soybean rust resistance genes
(RppZ) (Grant et al., 2009). has been localized to the LG. In addition, five classes of
disease resistance gene analogs (RGAs) were extensively clustered on chromosome 16
(LG J), including RGAI. RGA2. RGA3. RGA5. and RGA6 (Kanazin et al., 1996). Some
previous studies showed the presence of genes in the same region conferring resistance to
several diseases may explain the correlation between a variety of disease resistance. For
example, the similar gene location for BSR and powdery mildew resistance (Rma’) were
suggested to explain the positive association between these resistance traits (Lewers et al.,
1999). Among these resistance loci. the Rag3-l region only overlapped with the two
CEW resistance QTLs and SDS resistance locus (,Sanitchon et al., 2004). Previous studies

on CEW and aphid resistance showed that the two traits were inherited separately. SO it is

65

suggested that aphid resistance gene Rag3 or Rag3-1 and the CEW QTLs may not occupy
the same locus on chromosome 16. However. it is unknown if the association of aphid
resistance and SDS resistance exists. whether the underlying resistance genes for both
traits are separated. close-linked or pleiotrophic.

At least three soybean aphid biotypes have been discovered: the Illinois, Ohio and
Michigan biotypes (Kim et al.. 2008; Dr. Dechun Wang, unpublished data). The aphid
resistance genes Rag] and Rag2 did not provide resistance in plants that were infested
with the aphid collected from Michigan in 2008 and 2009 (Dr. Dechun Wang,
unpublished data). However. Rag3 in P1 567543C conferred a broad resistance to aphid
isolates from Ohio and Michigan (Zhang et al., 2009a). The gene in PI 567585A provided
an additional source ofRag3. resistance to Michigan aphid isolates in the field trial in
2008 and 2009. This aphid resistance gene locus and the linked molecular markers will

be useful for developing new aphid-resistant soybean cultivar.

66

Table 3.1 Damage index of soybean aphid in the field in the summer of 2009 for the
parents: PI 567585A. Skylla, and IA2070; 158 F45 RILs derived from 070082-2
validation population (PI 567585A x ‘Skylla’); and 162 F24 RILs derived from 070016-2

mapping population (PI 567585A x IA2070).

 

 

 

Population ID Parents RILs population

P1 567585A 1A 2070 ‘Skylla MeaniSE“ Range H2“:
70082 16.7a - 87.5b 58.1i8.55 12.5-87.5 95.50%
70016 12.5a 73.2b - 39.7:t12.55 8.3875 88.70%

 

Means followed by different letters within the same row are significantly different at P<0.05
DI+=Z (scale value x no. of plants in each category) l (4 x total no. of plants) x 100.
SE*= standard error

7 . . .
H‘**=broad sense herItabIIIty

67

Table 3.2 X2 test of segregation ratio for the aphid resistance gene (Rag3-l) and seven
SSR markers among 158 F25 RILs from the PI 567585A x ‘Skylla’ mapping population

and 94 F34 RILS from the PI 567585A x IA 2070 validation population.

 

Number of F34 RILs in

 

 

Population Locus each category X2 727 P

[D 3* b* h* 2*

070082 Satt674 77 7 74 0 9.472 0.0088
Sct_065 75 22 62 0 1.474 0.4785
Satt406 70 17 69 1 0.374 0.8296
Satt654 75 18 62 3 1 358 0.5072
Satt622 74 27 57 0 5.132 0.0768
Satt215 68 26 64 0 2.376 0.3048
Satt431 76 12 70 0 3.736 0.1544
Rag3-l 70 19 69 0 0.040 0.9803

070016 Satt674 36 6 52 0 6.328 0.0423
Sct_065 40 15 39 0 1.040 0.5947
Satt406 51 2 41 0 10.462 0.0053
Satt654 40 1 1 43 0 0.164 0.9212
Satt622 46 9 39 0 1.331 0.5139
Satt215 34 13 47 0 2 207 0.3318
Satt431 44 7 43 0 2.207 0.3318
Rag3-l 40 12 42 0 0.055 0.9730

 

a*=homozygous SSR allele ofthe resistant parent, P1 5675988

b*=homozygous SSR allele ofthe susceptible parent. ‘Skylla' or IA 2070

h*=heterozygous SSR alleles from both resistant and susceptible parents

a:

=missing band for SSR alleles

68

Table 3.3 Summary for aphid resistance loci detected in the mapping population and the

validation population with aphid DI data using the C [M method

 

 

Population LG/ Peak
Trial Chr* Pos.(cM)* * Flanking markers+ LOD R2++ at
070082 Greenhouse Js’ 1 6 15.5 Satt674-Sct_065 21.66 93.1 32.35
Field Cage Ii] 6 16.0 Satt674~Sct_065 15.66 90.1 30.50
070016 Field Cage 1,116 20.0 Satt654-«Sct_065 28.17 85.6 26.25

 

LG/Chr*=linkage group/chromsome

Peak Pos.(cM)**=QTL peak position is expressed in cM

Flanking markers+=Markers flanking the peak position or the marker at the peak position
R2++=Percentage of phenotypic variation explained by a QTL

aI=Additive effect. The positive value implies that the PI 5675988 allele decreases the D1

69

Table 3.4 Average aphid DI for different genotypes of marker Sct_065 in the field trial

for mapping and validation populations

 

 

Population PI 567585A Heterozygous Skylla / IA 2070 Average of P1 567585A
Type (a') Type (b2) Type (113) Skylla / IA 2070 type

070082 39.75a 53.75b 81 .25c 60.50b

070016 22.50a 42.50b 67.50c 45.00b

 

al=homozygous SSR allele ofthe resistant parent. PI 5675988
b2=homozygous SSR allele ofthe susceptible parent, ‘Skylla‘ or IA 2070

3 . .
h =heterozygous SSR alleles from both rCSIstant and susceptible parents

70

 

a) 070082

60-
50*

40
30 I 070082

RILS
20 ll
10-
0 . . .-.[. .
0 0.5 1 15 2 2

. 5 3 3. 5 4
Soybean aphid damage rating

I

 

I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a)070016

80 r

70 -

60 -

50 r

40 F .070016
30 _ RILS
20 _

10 - I j

0 I I I!1 - l l

0 0.5 1 1 5 2 2 5 3 3.5 4

Soybean aphid damage rating

 

 

 

Figure 3.1 Distribution of D1 scores in RIL populations: a) 070082 F25 RILs validation

population; b) 070016 F334 RILs mapping population.

71

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0 Satt674 15.9 Satts74 0.0 Satt674
m
g 8
a» ‘33 8
.5 A CO
‘71 O ‘r’
in fig 32.1 Sct_065 ,1:
_ TI
§ 3 36.7 \L/ Satt406 ‘92 5311654 ii
23.2 Sct_065 ‘3 g 38.2 /~\ Sattes4 22.7 Sct_065 g
I! 42.4 Saf1622 ‘g
44.8 Sa11215 V
33.3 Satt406 33.9 Satt406
35.7 SattBS4
41.1 Satt622 426 SattGZZ
47.5 Satt215 48.7 Satt215
78.8 Satt431
70.8 Satt431
78.8 Satt431

Figure 3.2 Linkage maps showing the locations of the soybean aphid resistance genes
from P1 567585A that were mapped on soybean linkage group J. (a) Aphid resistance
gene position in the mapping population for greenhouse and field trials. (b) The

relevant segment ofthe soybean LG according to the integrated soybean map of Song

et al., (2004). (c) Aphid resistance gene position in the validation population.

References:

Chen C.Y., C. Gu. C. Mensah. R.L. Nelson. D. Wang. 2007. SSR marker diversity of
soybean aphid resistance sources in North America. Genome 50:1104—1 l l 1.

Churchill G.A., R.W. Doerge. 1994. Empirical threshold values for quantitative trait
mapping. Genetics 138:963-971

C regan P8, Quigley CV. 1997. Simple sequence repeat DNA marker analysis. In
C aetano-Anolles G. Gresshoff PM (eds) DNA markers: Protocols, applications and
overviews. John Wiley & Sons, New York, pp. 173-185

DiFonzo, CD, and R. Hines. 2002. Soybean aphid in Michigaanpdate from the 2001
season. Michigan State Univ. Ext.8ull. E-2748. Michigan State Univ., East
Lansing.

Fehr, W.R. 1987. Principles of cultivar development: theory and technique. MacMillan
Publishing Co, New York

Grant, D., M.I. Imsande, R.C. Shoemaker. 2009. SoyBase, The USDA-ARS soybean
genome database. http://soybase.agron.iastateedu, Accessed on 24 Feb 2009

Hartman G.L., L.L. Domier, L.M. Wax. C.G. Helm, D.W. Onstad, J.T. Shaw, L.F. Solter,
D.J. Voegtlin, C.J. D’Arcy, ME. Gray. K.L. Steffey, S.A. Isard, and PL. Orwick.
2001. Occurrence and distribution Oprhis glycines on soybeans in Illinois in 2000
and its potential control. Plant Health Progress. [Online] Available at:
http://www.plantmanagementnetwork.org/sub/php/brief/aphisglycines/

Hill, C. 8., Y. Li and G. L. Hartman. 2004. Resistance to the soybean aphid in soybean
germplasm. Crop Sci. 44:98-106.

Hill, J.L., E.K. Peregrine, G.L. Sprau, C.R. Cremeens.R.L. Nelson, J.H. Orf, and DA.
Thomas. 2005. Evaluation of the USDA Soybean Gennplasm Collection: Maturity
Groups 000-IV(PI 507670-PI 574486). United States Department of Agriculture,

Agricultural Research Service Technical Bulletin Number 1914

73

Hill, C.B., Y. Li, and G.L. Hartman. 2006a. Identification of a single dominant gene for
resistance to the soybean aphid in the soybean cultivar Dowling. Crop Sci.
46:1601—1605.

Hill. C.B., Y. Li, and G.L. Hartman. 2006b. Soybean aphid resistance in soybean Jackson
is controlled by a single dominant gene. Crop Sci. 46:1606—1608.

Hill, C.B. K.S. Kim, L. Crulla, B.W. Diers and G.L. Hartman. 2009. Inheritance Of
Resistance to the Soybean Aphid in Soybean PI 20053. Crop Sci 49:1193-1200

Jeong, S.C., A.J. Hayes, R.M. Biyashev, and MA. Saghai-Maroof. 2001. Diversity and
evolution of a non-TIR-NBS sequence family that clusters to a chromosomal
hotspot for disease resistance genes in soybean. Theor. Appl. Genet. 103:406—414.

Kanazin, V., L.F. Marek, R.C..Shoemaker. 1996. Resistance gene analogs are conserved
and clustered in soybean. Proc Natl Acad Sci 93:1 1746-11750.

Kang. S.T, M.A.R. Mian, and RB. Hammond. 2008. Soybean Aphid Resistance in P1
243540 Is Controlled by a Single Dominant Gene Crop Sci 48:1744-1748.

Kim, K., C.B. Hill, G.L.Hartman, Mian M.R., B.W. Diersm. 2008. Discovery of soybean
aphid biotypes. Crop Sci 48:923—928

Kisha, T., C.H. Sneller, B.W. Diers. 1997. Relationship between genetic distance among
parents and genetic variance in populations of soybean. Crop Sci. 37:1317-1325.

Klos, K.L.E., M.M. Paz, L.Fredrick Marek, P.B. Cregan and RC. Shoemaker. 2000.
Molecular Markers Useful for Detecting Resistance to Brown Stem Rot in Soybean.
Crop Sci. 40:1445-1452.

Lewers, K.S., E.H. Crane, C.R. Bronson, J.M. Schupp. P. Keim and RC. Shoemaker.
1999. Detection of linked QTL for soybean brown stern rot resistance in ‘BSR 101’
as expressed in a growth chamber environment. Molecular Breeding 5: 33—42,
1999.

74

Li, Y., C.B. Hill, S. Carlson, B.W. Diers. and G.L. Hartman. 2007. Soybean aphid
resistance genes in the soybean cultivars Dowling and Jackson map to linkage
group M. MOI. Breed. 19:25—34.

Mensah, C., C. DiFonzo, and D. Wang. 2008. Inheritance of Soybean Aphid
Resistance in P1 5675418 and PI 5675988. Crop Sci 48:1759-1763.

Mian, M.A.R., R.B. Hammond, and SK. St. Martin. 2008a. New plant introductions with
resistance to the soybean aphid. Crop Sci. 48:1055—1061.

Mian, M.A.R., S.T. Kang. S.E. Beil, and RB. Hammond. 2008b. Genetic linkage
mapping of the soybean aphid resistance gene in P1 243540. Theor. Appl. Genet.
117:955—962.

Michelmore, R.W., J. Paran. R.V. Kesseli. 1991. Identification of markers linked to
disease resistance genes by bulked segregant analysis: a rapid method to detect
markers in specific genomic regions by using segregating populations. Proc. Natl.
Acad. Sci. 88:9828-9832.

Sanitchon, J. A. Vanavichit. S. C hanprame. T. Toojinda. K. Triwitayakom, V.N. Njitid
and P. Srinivesa. 2004. Identification of Simple Sequence Repeat Markers Linked
to Sudden Death Syndrome Resistance in Soybean. Science Asia 30: 205-209.

Van Ooijen, J.W., R.E. Voorrips. 2001. JoinMap 3.0. Software for the calculation of
genetic linkage maps in experimental populations. Kyazma, BV, Wageningen

Voorrips, RE. 2002. MapChart: Software for the graphical presentation of linkage maps
and QTLs. J Heredity 93:77-78

Wang. D., J. Boyse. B.W. Diers. 2006. Registration of ‘Skylla' soybean. Crop Sci.
46:974-975

Wang, D., J. Shi, S.R. Carlson, P.B. Cregan. R.W. Ward. B.W. Diers. 2003. A low-cost
and high-throughput system for high-resolution genotyping with microsatellite
DNA markers. Crop Sci. 43:1828-1832.

75

Wang, 8., CI Basten. Z.B. Zeng. 2008. Windows QTL Cartographer 2.5. Dept of Stat,
North Carolina State University, Raleigh. http://statgen.
ncsu.edu/qtlcart/WQTLCart.htm. Cited 21 Apr 2008

Zeng. 2.8. 1994. Precision mapping ofquantitative trait loci. Genetics 136:1457-1468.

Zhang. G.. C. Gu, and D. Wang. 2009a. Molecular mapping of soybean aphid resistance
genes in PI 5675418. Theor. Appl. Genet. 1 18:473—482.

Zhang, G., D. Wang. 2009b. Detection and verification of an aphid resistance locus in
Soybean PI 567543C. In 2009 annual meeting abstracts '[CD-ROM]. ASA, CSSA,

and SSSA, Pittsburg. PA.

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CHAPTER 4

IDENTIFICATION OF APHID RESISTANCE GENES IN SOYBEAN
USING MODIFIED NESTED ASSOCIATION MAPPING (MNAM)

ABSTRACT

The soybean aphid (Aphis glycines) has become an important pest of soybean
[Glycine max (L.) Merr] in the US. since 2000. PI 5675988 was found to possess
antibiosis resistance to the soybean aphid. In this study. a modified nested association
mapping (MNAM) approach was used to locate resistance genes in P1 5675988 on the
integrated soybean linkage map. PI 5675988 was crossed with 10 different susceptible
cultivars to construct 10 recombinant inbred lines (RILS) populations, where only
resistant progenies were selected in each population for MNAM. We expected that the
genomic regions containing the aphid resistant genes from P15675988 were present in the
most resistant progenies. C hi-square test was used to discover the significant association
between aphid resistance and single sequence repeat (SSR) markers. False discovery rate
and Bonferroni correction were applied to control the type I error rate. Genomic regions
on linkage groups F, G, J and M were found associated with soybean aphid resistance in
MNAM. Linkage analysis ofa population of 94 BC1F4;5 RILS derived from P1 5675988
and a F45 RIL population derived from E06902 (elite advance breeding line developed
from P15675988) were used to confirm the MNAM results. The results of linkage

analysis showed that genomic regions on the linkage groups F, J, and N were associated

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with aphid resistance. Ultimately. we showed that MNAM was efficient for the discovery

of aphid resistance genes in soybean breeding and germplasm improvement.

INTRODUCTION

Soybean [Glycine max (L.) Merrill] is a leading crop worldwide. providing an
important source ofoil and protein. In 2008. the soybean production area was 30.19
million hectares in the US, and the production represented 38% (80 million metric tons)
of the world‘s total soybean production (USDA-National Agricultural Statistics Service,
2008). Insect pests, including the soybean aphid (Aphis glycines Matsumura), are the
major constraints to the production and economic yield of soybean. Soybean aphids
feed directly on soybean aboveground biomass and transmit several viruses, causing
8-25% yield loss in Michigan (DiFonzo and Hines, 2002). Native to Asia, the soybean
aphid was detected in the US. in July 2000. Since then. it has spread to at least 21 states
in the US and 3 provinces in Canada. becoming one of the most destructive pests of
soybean in North America (Chen et al., 2007).

Insecticides are currently the only effective method to control soybean aphid.
However, utilization of host resistance could provide a more practical method of pest
control without releasing pesticides into the environment and increasing production costs
(Sun et al., 2000). Host resistance is classified as tolerance, antixenosis. or antibiosis

(Painter 1951). Tolerance is defined as host plant resistance that restricts an infestation

78

L /-'___. _

without yield loss. Antixenosis is present when insect pests show non—preference for a
specific host plant. Antibiosis reduces insect abundance by disrupting the life cycle Of the
insect during feeding. decreasing plant damage. Antibiosis and antixenosis have been
studied extensively in soybean aphid resistance research. Choice and nonchoice tests
have been used to distinguish between these two resistance mechanisms. Recently,
certain soybean plant introduction lines were found to possess antibiosis resistance in
soybean germplasm: ‘Dowling’. ‘Jackson’ (Hill et al.. 2004). PI 5675418. PI 5675988
(Mensah et al., 2005): ‘K1639‘. ‘Pioneer 95897‘. PI 230977 (Diaz-Montano et al., 2006;
Hesler et al.. 2007): PI 59099 (Hesler et al., 2007): PI 243540 (Kang et al., 2008), PI
567543C (Zhang et al., 2009b). and PI 567585A (Dr. Dechun Wang, unpublished paper).
Classic genetic studies indicate that aphid resistance in ‘Dowling‘, ‘Jackson', PI 200538,
and PI 243540 is controlled by a single dominant gene (Hill et al., 2006a, 2006b; Mian et
al., 2008a; Hill et al.. 2009). and that the aphid resistance in P1 5675418 and PI 5675988
is controlled by two recessive genes (Mensah et al., 2008; Zhang et al.. 2009a). Recently.
one major co-dominant gene was shown to control the aphid resistance in P1 567585A
was identified (Liu et al., unpublished data).

In complex agricultural traits such as aphid resistance. many quantitative trait loci
(QTL) contribute to phenotypic variation. each with a small effect and influenced by

environmental factors. Thus. QTL mapping was developed to identify QTLs associated

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with a desirable phenotype. Linkage analysis and association mapping are the two widely
used approaches for exploring QTLs underlying quantitative traits (Zhu et al., 2008).

The linkage analysis was widely used in mapping soybean aphid resistance. This
genetic mapping can detect marker allele-trait associations within a structured pedigree,
such as an F2, a backcross or a F25 RIL population. Six aphid resistance genes have been
located on F, J and M linkage groups (LG) by linkage analysis: Rag] in ‘Dowling’, Rag
in ‘Jackson’ (Li et al., 2007). Rag2 in P1 243540 and PI 200538 (Mian et al., 2008b; Hill
et al., 2009), Rag3 in P1 567543C (Zhang et al.. 2009b). and rag1_3 and rag-'3t in P1
5675418 (Zhang et al.. 2009a). However, the limitation of linkage analysis is
low-resolution mapping in a specific population, leading to inconsistent results occurring
among different populations. Therefore, association mapping is regarded as an essential
supplement to linkage analysis because it allows high—resolution and genome-wide QTL
scanning (Zhu et al., 2008). Nested association mapping (NAM) was developed for maize
(Zea mays L.) to dissect the genetic inheritance of complex quantitative traits by
combining the benefits of linkage analysis and association mapping (Yu et al., 2008). As
the common parent. inbred line 873 was crossed with other 25 founder parents. A total of
5000 RILs derived from these 25 founder populations were genotyped by common parent
specific (CPS) markers. By using computer simulation. NAM detected the historical
recombination in populations and identified the relatively narrow gene regions underlying

complex traits (Yu et al., 2008). However. discovering resistance genes by applying

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NAM directly is impractical. First, during artificial selection, susceptible lines will be
discarded after each generation. It is also not necessary to retain all RIL populations for
genotyping. Second. plant breeders have to consider the conflicts between rapid cultivar
replacement and the time spent to create numerous founder populations. The cost of
genotyping is another restriction for most plant breeding programs.

In this study. we developed a modified nested association mapping (MNAM) design
which is more suitable for detecting the aphid resistance genes in soybean breeding and
germplasm improvement. PI 5675988 serves as a common aphid resistance parent, which
, was crossed with 10 founder susceptible parents. Ten F2 populations were advanced to F4
generation through single seed descent (SSD). Unlike NAM, we only selected and
analyzed resistant progenies in each F25 RIL founder populations. Because of the high
Type I error in association mapping, MNAM also uses Bonferroni and false discovery
rate (FDR) controlling procedures to adjust the genome-wide false positives (Yu et al.,
2008). In summary. the objectives of this study were to: 1) identify the genes responsible
for aphid resistance in soybean PI 5675988 by using MNAM; 2) confirm the resistance

genes by using traditional linkage analysis.

MATERIALS AND METHODS

Primary and secondary populations for MNAM

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The eleven founder soybean accessions were PI 5675988, Titan, ADO-711003,
AGO-711020, A02-381 100. E00003. IA2064. IA2070. IA2072. SDAOOR-039-42. and
Skylla. The common resistant parent. PI 5675988, was crossed with the other 10 founder
accessions, followed by selfing. to generate 10 segregating F2 populations. Out of each
population. RILs were derived through single seed descent. Individual RILs of each F45
population were evaluated for soybean aphid resistance in greenhouse and field during
2006. A total of 85 resistant RILs were obtained (Table 4.1). Among them, 41 F435
resistant RILS were derived from the cross of PI 5675988 and Titan. which was called
the primary population. In the secondary population, 44 resistant RILs were developed
from the cross of PI 5675988 with the other nine founder parents (Dr. Dechun Wang,

unpublished data).

DNA extraction, PCR and gel system

For primary, secondary, and confimiation populations. the unexpanded trifoliates of
each line were harvested and pooled for genomic DNA isolation. The DNA was isolated
by the CTAB method (Kisha et al., 1997) and the DNA concentration was detected by a
ND-1000 Spectrophotometer (NanoDrop Technologies. Inc; Wilmington, DE). SSR
markers (Song et al.. 2004) were used to amplify the genomic DNA according to the PCR
TM

protocol described by Cregan and Quigley (1997) using a MJ Tetrad thermal cycler

(MJ Research, Waltham. MA). PC R products were detected on 6% non-denaturing

82

polyacrylamide gels by using a DASG-400-50 electrophoresis system (C.B.S. Scientific

Co.. Del Mar, CA) as described by Wang et al.. (2003). Gels stained with ethidium

bromide were photographed and scored under UV light.

Identification of QTLs by MNAM

MNAM included the following steps:

1)

2)

3)

4)

5)

Screen the polymorphism of 1056 SSR markers in parents PI 5675988 and Titan in
the primary population along the whole genome. The SSR primer sequences were
obtained from Dr. Perry Cregan at USDA-ARS, Beltsville, MD.

Genotype 41 RILs in the primary population using the polymorphic SSR markers of
approximate 20 cM interval coverage in each linkage group.

Use the Chi-square test to detect SSR markers significantly associated with soybean
aphid resistance in the primary population. and apply Bonferroni and FDR methods to
control for false positive results.

Select SSRs that were consecutively and significantly associated with resistance over
an approximate distance of 30 cM. The selected SSRs were assigned to several
potential regions that potentially contribute to the aphid resistance in the primary
population.

Genotype 1 1 parents of the primary and secondary populations using saturated

polymorphic SSR markers within the potential aphid resistance regions. The SSRs

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with rare specific banding pattern for PI 5675988 were called
common-parent-specific (CPS) SSRs. The selection criterion for these PI
5675988-rare SSRs were set to be segregating in >8 populations.

6) Genotype CPS SSRs in the secondary populations. Those CPS SSRs significantly
associated with resistance in both primary and secondary populations were defined as

consistcnt-common-parent-specific ( CC PS) SSRs.

Statistical analysis
1) Chi-square test

For the inheritance of each SSR marker locus. the segregation of the banding
patterns in the primary and secondary populations in MN AM were tested to fit the
expected ratio of 1:1 (resistant parent: susceptible parent) by C hi-square tests. The
amplified SSR bands were scored and classified: homozygous for the banding pattern of
PI 5675988 (a): heterozygous for the banding patterns of PI 5675988 and susceptible
parent (h); or homozygous for the banding patterns of the susceptible parent (b). The
Observed number of resistant RILs (m) that inherited the banding pattern from P1
5675988 was calculated as the sum ofa and h. The p-values of Chi-square tests were
calculated for 1 degree of freedom. In each individual Chi-square test, the SSR marker
locus was considered significantly associated with aphid resistance when p S 0.01 and the

banding patterns were skewed toward the resistant parent.

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2) Bonferroni corrections and FDR-controlling procedures

Because MNAM in the primary population was a procedure of multiple hypotheses
tests. p-values obtained for each marker locus by the C hi-square test were subjected to
Bonferroni and FDR correction procedures (Benjamini and Yekutieli. 2005). in order to
reduce false discoveries. In the Bonferroni correction. or S 0.01 was fixed for entire set of
N C hi-square tests. For each C hi-square test. the SSR was regarded as significantly
associated with aphid resistance if the p S a/N. In the F DR-controlling procedure, the
QVALUE software was used to estimate the q-values for the list ofp-value resulting
from the multiple Chi-square tests (Storey. 2002). Q-value was controlled at the level of

0.01.

Validation of QTLs by linkage mapping
1) Confirmation populations

The following two populations were constructed to map aphid resistance genes in P1
5675988. and test the efficiency and accuracy of MNAM.

First. a BCF4,5 population was composed of 94 RILs. which were derived from a
cross between PI 5675988 and Titan, that was backcrossed with Titan. Genotyping was
carried out on this population with the CCPS SSRs discovered in MNAM. Considering

the monomorphism resulting from backcrossing, polymorphic markers between two

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parents were genotyped within the potential aphid resistance gene regions around CC PS
SSRs.

Second. an F25 RIL population (070063) was developed from the cross between
E06902 and IA2070. E06902 is an elite advanced breeding line derived from the original
PI 5675988 resistance source that possessed resistance levels similar to PI 5675988 in
field evaluations during 2006 (Dr. Dechun Wang. unpublished data). E06902 was crossed
to the susceptible soybean accession IA 2070 in 2007. followed by selling. to generate
segregating F2 population. SSD was applied to reach F4 generation. A total of 1 18 F435
RILS were obtained. Considering the monomorphism due to inbreeding practice,
polymorphic markers between two parents were added within the potential aphid

resistance gene regions around CCPS SSRs.

2) Evaluation of soybean aphid resistance

For the BCF435 population. aphid resistance was evaluated in the greenhouse in spring
2008 without any replication and in the field in 2008 summer with two replicates.
Phenotypic data were collected by evaluating aphid resistance in the field in the summer
of 2009 without any replication in the F415 RIL population (070063). The greenhouse trial
was conducted in the Plant Science Greenhouse at Michigan State University (MSU) in
East Lansing Michigan. Eight seeds per line or parent were planted in a plastic pot, which

is 210m in diameter and 125mm deep. The two parents and the mapping population

86

were set on the bench without replication in a completely randomized design (CRD). The
temperature was maintained at 26/15°C day/night with l4-h supplemental lighting
provided by sodium vapor lamps. The field evaluation of soybean aphid resistance was
carried out in a 12.2 x 18.3m aphid- and predator- proof cage (Redwood Empire Awning
Co., Santa Risa. CA) on the Agronomy Farm of MSU. The parental plants were planted
randomly in the field. 5.1 cm apart. Each RIL was planted in a single row plot, 60cm long
with a row spacing of 60cm. The average number of plants per line was around 10 with
most plots having at least 12 plants. A similar C RD was used to arrange each population
and its parents in the field plots.

In both greenhouse and field trials. each plant was inoculated at the V2 stage with two
Wingless aphids. The BC 1F“ population and parental plants were evaluated for aphid
damage without replication in greenhouse in 2008 spring. with two replications in field in
2008 summer. A single aphid clone was collected from a naturally infested field at the
MSU Agronomy Farm in summer 2007. and maintained in an isolation chamber in the
greenhouse for the inoculation of plants in the greenhouse trial in 2008 spring. The
soybean aphids used for inoculation in the field trial were collected from a naturally
infested field on the MSU Agronomy Farm during 2008 summer. The BC [F45 population
and parental plants were evaluated for aphid damage the 3rd and 4lh week after
inoculation using a modified 0-4 half step rating scale described by Mensah et al.. (2008).

The F45 RIL population (070063) and parental plants were evaluated for aphid damage

87

without replication in field in 2009 summer. Same aphid infestation and damage rating
methods were used as described before.

The aphid resistance score was determined as the mean of the rated plants in each line
for each replication. An aphid damage index (DI) for each line was used as an indicator
of aphid resistance. ranging from 0 (no damage) to 100 (most severe damage (Mensah et
al.. 2005). D1 was calculated based on the following fonnula: D1 = 2 (scale value x no. of

plants in each category) / (4 x total no. ofplants) x 100 (Zhang et al., 2009a).

3) Statistics for linkage analysis

Linkage analysis was performed for these two populations with Map Manager
QTXb20 (Manly et al.. 2001). The linkage maps were constructed using JoinMap and
MapC hart (Van Ooijen, 2001: Voorrips. 2002). Then linkage groups were assigned to
specific chromosomes according to the soybean consensus map (Song et al., 2004).
Simple interval mapping (SIM) and composite interval mapping (C IM) were applied to
locate QTLs for aphid resistance with the use of QTL C artographer V2.5 (Zeng. 1994;
Wang et al.. 2008). The QTL results for the confirmation populations obtained by linkage
analysis were compared to the results from MNAM to test whether these QTLs are

co-located at the same positions along chromosomes.

RESULTS

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1) Identification of QTL regions in the primary population

Among 1056 SSR markers. 31 1 polymorphisms were identified between the parents
in the primary population. In order to accelerate the identification of potential regions
associated with aphid resistance. 1 12 polymorphic SSR markers at an average distance of
22 cM were used to genotype 41 resistant RILs in the primary population. The amplified
SSR bands were scored. and the m was defined as the observed number of resistant RILs
that inherited the banding pattern from P1 5675988 in the primary population. Figure 4.1
shows an example ofa banding pattern amplified by primer satt406 in the primary
population. A total of38 resistant RILs showed a banding pattern inherited from P1
5675988. P-value was 4.6E-08 based on the Chi-square test. indicating that satt406 was
significantly associated with aphid.

For the Bonferroni correction, the alpha value ofthe entire set of 1 12 comparisons
was 0.01. For each comparison. the alpha value equaled to 8.9E-05 (0.01/112). In the
FDR controlling procedure. the q-values were calculated according to the p-values of
C hi-square tests. A total of 18 consecutive SSR markers were significantly associated
with aphid resistance over an approximate distance of 30 cM. These 18 SSR markers
were distributed on five linkage groups: 82. F, G, J and M (Table 4.2). Five potential
regions were defined as associated with aphid resistance in the primary population: from

66 to 95 cM on B2 linkage group (Region 82): from 16 to 130 cM on F linkage group

89

(Region F); from 22 to 59 cM on G linkage group (Region G); from 15 to 52 CM on J

linkage group (Region J); and from 38 to 67 cM on M linkage group (Region M).

2) Identification of CPS SSRs

Eleven parents of the primary and secondary population were genotyped using
saturated polymorphic SSR markers within the five potential regions. PI 5675988
showed specific banding patterns at 18 SSR marker loci. unlike the ten susceptible
parents. These SSR marker loci. whose banding patterns differed from those of the
susceptible parents were defined as CPS SSRs (Figure 4.2 and Table 4.3). distributing
over four linkage groups: F. G. J. and M. No CPS SSRs were located in the potential
regions on LG 82.
Identification of CCPS SSRs

The eighteen CPS SSRs were tested for the association with aphid resistance in the
primary and secondary population. The p-values were calculated for each CPS SSR locus
in each population (Table 4.3). Fourteen CPS SSRs were identified as CCPS SSRs. which
meant they were consistently and significantly associated with aphid resistance in both
populations. The CC PS SSRs were located on three linkage groups: F , G. and J.
Validation of QTLs in two confirmation populations

Seven polymorphic SSRs within the potential aphid resistance regions on LG J were

genotyped for 94 lines in the BC IF4;5 Population. The QTL was located between Satt285

90

and Satt380, which were mapped in the interval of 9.5 and 27.1 cM on the soybean
consensus mapping. The QTL explained 69.2%~91.1% of the phenotypic variance for
both 3rd and 4h week screening in the greenhouse and field trials (Table 4.4). The
soybean aphid resistance QTL identified in MNAM and linkage mapping was located at
the similar position along the linkage group I (Figure 4.3). The marker Sat_304 on LG N
was found to be linked with aphid resistance gene. explaining 4. 1%~8. 1% of phenotypic
variation for 3rd week in either greenhouse or field trials.

Six polymorphic SSRs within the potential aphid resistance regions on linkage
group J were genotyped on 1 12 lines in a F435 RIL population (070063). The soybean
aphid resistance QTL identified in MNAM and linkage mapping was located at the
similar position along the linkage group J (Figure 4.3). The QTL was located between
Sct_065 and Satt596. which are mapped in the interval of 16.1 and 23.8 CM on the
soybean consensus mapping. The identified QTL explained 53.4% of the phenotypic
variation for 3rd week in the field trial. The markers Satt334 (LG F) and Sat_304 (LG N)
identified as linked with aphid resistance loci. explaining 1 1% and 18% of phenotypic
variation for 3rd week in field trial (Table 4.4).

Band pattern analysis of markers linked to QTLs for aphid resistance

Five aphid resistance germplasm, accession PI 567543C, P15675418. PI 5675988,

PI 567585A. and ‘Dowling‘. together with two susceptible accessions were genotyped

using markers Satt030 (LG F). Satt522 (LG F). Satt622 (LG J), Satt529 (LG J), Satt463

91

(LG M). Sat_304 (LG N). which were tightly linked with the potential QTLs identified in
this study. On LG J. the band patterns of the PC R products from P1 5675988 were as
same as PI 567585A for both of the two markers. but different from Dowling. The PI
5675988 and PI 5675418 accessions share the same band pattern for marker Satt46 on

LG M, whereas PI 567543C and PI 567585A share a different band pattern.

DISCISSION
1) Modified nested association mapping

The resistant genotypes present a pattern of gene identity by descent (IBD), which is
underlying the patterns of observed phenotypes. Genotypes of progenies or relatives are
similar because they share genes that are 18D, inherited from a common ancestor within
the defined pedigree. Several studies have been carried out to infer the IBD information
of QTL by using flanking markers. including nested association mapping (C harlier et al.,
1996). NAM was first proposed and implemented in maize: in order to dissect complex
traits at the gene level by using designed multiple mapping populations from linkage
analysis. The genetic architecture of quantitative traits. for flowering time and northern
leaf blight (NLB) has been studied using NAM (Buckler et al.. 2009; Chung et al., 2008;
Poland et al.. 2009).

In the MNAM experiment. PI 5675988 is the common parent. whose resistance

gene(s) can be followed in the progenies through flanking CPS SSRs. The CPS SSR

92

allowed the prediction of inheritance of chromosome segments in progenies among
populations. rather than tested ofbi-allelic contrasts across each bi-parental population
(Yu et al., 2008). However. all RIL in each population were retained and analyzed for the
genetic dissection ofquantitative traits in NAM. In this study, unlike these quantitative
traits analyzed in NAM, soybean aphid resistance has been discovered to be controlled by
oligogenes in a number of resistant PIS. either as one or two dominant/recessive genes. So
selection was used to increase the efficiency in MNAM, where only resistant RILS

derived from founder populations were selected and genotyped in MNAM.

2) Resolution of MNAM

Association mapping exploit the historical recombination events at the population
level. The resolution of association mapping relies on the density of molecular markers,
and the linkage disequilibrium (LD) between an array of linked markers and the
functional mutations responsible for trait variation. Generally. LD decays at a much
greater distance in self-pollinated crops than in cross-pollinated species. For example, the
LD decayed within 04-10 kb in maize depending on the gene length (F lint-Garcia et al.,
2003). In cultivated soybean, LD extended from 90 to 574 kb because ofincreased
self-fertilization during domestication (Hyten et al.. 2007). Thus. if LD decays within a
long distance, the mapping resolution will be low. and a relatively small number of

markers are required in soybean association mapping. The goal of MNAM is to quickly

93

the aphid resistance gene location/s on the soybean consensus map. In consideration Of
the cost and necessity of genotyping. relatively low-resolUtion SSR markers were used to
localize the QTL regions. SNP markers would be added to the potential regions to

pinpoint the genetic variation at the gene level in future fine mapping study.

3) Association mapping and linkage mapping

Comparison of locations of reported QTL showed that more significant QTLs
identified using association mapping were located within the previously reported QTL
regions. In association mapping of yellow pigment in durum wheat germplasm, 48% Of
the significant markers identified in AM were associated with QTLs found through
linkage analysis (Reimer et al., 2008). Due to the combined infonnation across all
families. the NAM analysis identified nearly twice as many significant QTLs compared
with individual family linkage analysis. A total of 29 QTLs were identified that explained
64% of the ASI (anthesis-silking interval) variation. meanwhile 36 and 39 QTLs
contributed to the 89% of the total variance for DA (days to anthesis) and DS (days to
silking). The QTLs identified in NAM were concordantly located within six major QTL
regions previously mapped in meta-analysis of maize flowering date (Buckler et al.,,
2009). As another quantitative disease resistance trait. NLB resistance was investigated
for genetic dissection by NAM. A total of 21 QTLs were detected in this study. but new

QTLs were also detected. Most of these QTLs co-localized with previous identified

94

disease resistance loci for NLB. Furthermore, th8. 06 (th for quantitative resistance to
Exserohilum turcicum) was consistently identified as the largest-effect QTL across all
populations. and one QTL on chromosome 8 significantly contributing to resistance
(Chung et al., 2008).

In this study. the aphid resistance gene region on LG J (chromosome 16) was
detected in MNAM. and then confirmed by linkage analysis. This QTL region explained
53.4~91.1% of aphid resistance variation either in greenhouse and field trials. In MNAM,
additional two resistance regions were found on LG F and N (chromosome 13 and 03).
The QTL on LG F was confimied only in population 070063, not in the BCIF4;5
population. which may be due to different genetic background of these two populations.
Similar aphid resistance regions on LG F were identified in other soybean aphid
resistance sources, PI 200538 and PI 243540. The QTL explaining 4.1%~18% of
phenotypic variation on LG N was identified in both populations for phenotypic data
evaluated in 3rd week. The Sat_304 marker was significantly associated with aphid
resistance in the first step of MNAM. but no consecutive SSR markers significantly
associated with aphid resistance around Sat_304 were detected. Thus. the QTL region on

LG N was not studied further and regarded as potential aphid resistance QTL region in

MNAM.

4) Prediction of molecular function of aphid resistance QTLs in P1 5675988

95

In the past decade. resistance to insects has been identified in various plant species.

A series of R genes have been mapped. and molecular markers linked to these loci have
been identified. These identified genes confer resistance to Russian wheat aphid, Hessian
fly. and Mayetiola destructor (Kaloshian. 2004) in wheat. In this study, the resistance
genes on LG J and N of soybean genome were identified together mainly in the 3rd week.
The genome sequence search showed that a cluster of leucine-rich-repeat (LRR), toll and
interleukin-1 receptor (TIR). and nucleotide binding site (NBS) genes were located
within the region between Satt285 and Satt3 80. It is not surprising that an array of R
genes exist within a resistance locus conferring disease resistance. For example. three
candidate genes were identified within the NLB resistance locus qNLB8.06Dt-883.
including two tandem protein kinase(PK)-like genes and one protein
phosphatase(PP)-Iike gene (Chung et al.. 2008). Thus. fine mapping and cloning of
candidate genes is required to identify their real functional roles of individual members.

Moreover, several ERF. MYB. WRKY transcription factors (TF) were located on LG
N closely linked to Sat_304. These results are reasonable because the induction of plant
defense by insect feeding is regulated by several signaling pathways. including salicylic
acid (SA)-. jasmonic acid (JA)- regulation pathways. Li et al. (2008) demonstrated that
aphid feeding on soybean induced expression ofNBS-LRR. Myb family TF, and genes
associated with both SA and JA mediated response pathways. In their study.

Gm-r1070-4664, a potential MYB family transcription factor, is one of the top five

96

constitutively higher expressed genes in aphid resistance in cv. Dowling. By genome
sequence search. the closely linked marker to Gm-r1070-4664 was Sat_304 located on
LG N (Chromosome 03). near the aphid resistance locus found in this study. The TFs
could be potentially involved in the early basal resistance stage. before the initiation of
numerous expression ofR genes. Thus. it may be reasonable to collect phenotypic data in

the 3rd week to capture/detect more QTL regions conferring aphid resistance in soybean.

5) The relationship among different soybean aphid resistant sources

Understanding the allelic relationship among different resistance sources can be
used to determine the breeding strategy to control the most effective alleles R gene
pyramid. PI 5675418. PI 567543C. PI 5675988. and PI 567585A all originated from
Shandong province. China. In this study. the major QTL of PI 5675988 was coincidently
located in a similar genomic region on LG J as the resistance genes in P1 567543C and PI
567585A in both MNAM and linkage analysis. PI 5675988 share the same SSR
amplification bands as PI 567585A. but different from P1 567543C. On LG F, the band
patterns did not show similarity between PI 5675988 and PI 5675418 for Satt030,
suggesting that there is no resistance locus on the upper region of LG F in P1 5675988.
Also, unpublished data showed that the segregation ratio of F2 progenies derived from a
cross between PI 5675988 and PI 5675418 was 3:1, indicating that resistance genes on

LG F and I both contribute to the resistance. All above information indicated that the

97

resistance loci in P1 5675988, PI 567585A and PI 567543C are either allelic at the same
locus or tightly linked genes on LG J.

P1 5675988 and PI 5675418 shared the same banding patterns for Satt463 on LG M,
and this resistance locus was detected in MNAM (Figure 4.4). However, this locus was
not detected in the confimration population using linkage analysis. Moreover. the
segregation ratio suggested the locus on LG M not involving in the resistance in P1
5675988. It is possible that the resistance loci in these two PIs are located within the
small region. where no recombination event occurs. or maybe the resistance loci on LG
M. such as Rag], were already overcome due to the evolution of resistant aphid biotypes
in Michigan (Dr. Dechun W ang, unpublished data). As mentioned above, the resistance is
generally determined by one or few members in the R genes cluster. Several populations
derived from crosses among these resistance sources will be used to determine whether
the aphid resistance is conferred by same R gene member or different members in

individual PI accessions.

6) MNAM and breeding practice

In this study. MNAM was developed to investigate the genetic basis of traits
controlled by oligogenes that could offer several advantages for the plant breeding and
genetic research community. F irst. the susceptible lines are generally discarded in each

generation each year. Facing the rapid cultivar replacement in market place, the critical

98

point in marker-assisted selection or molecular plant breeding is the timely application of
molecular markers. Thus only aphid resistant RILs in each population were used in
MNAM, which means the population built for breeding purposes can also be used in
MNAM for genetic studies. without the intent to construct bi-parental population for
QTL mapping. Eventually. plant breeders conducting selection to achieve breeding goals,
can generate genetic research at the same time. The cost of maintaining and genotyping
whole RIL population in each founder population restricts many breeding research labs
using NAM for genetic studies. In MNAM. partial founder populations are retained and
genotypes, which decrease the labor time and expense. However, MNAM is not suitable
for quantitative traits. whose phenotypic data show a normal distribution, as selection
from extreme tails will be problematic. Thus MNAM favors qualitative traits or traits

controlled by oligogenes.

99

Table 4-1 List of primary and secondary populations subjected to MNAM

 

Population name Female Male Number of F45
(Susceptible parent) (Common resistant parent) resistant RILS

 

 

Primary population Titan PI 5675988 41
A00-71 1003 PI 5675988 5
AGO-711020 PI 5675988 7
A02-381100 PI 5675988 2
E00003 PI 5675988 3

Secondary 1A2064 PI 5675988 3

Pepulation IA2070 PI 5675988 5
1A2072 PI 5675988 2
SDAOOR-039-42 PI 567598B 8
Skylla PI 5675988 9

 

100

Table 4-2 Consecutive SSR markers significantly associated with aphid resistance on five

linkage groups: 82. F. G. J and M.

 

 

SSR marker _ p-value q-value
name Linkage group Linkage Group Position m
Sat_3 5 5 B2 66.24 36 1.29E-06 1.26E-05
Satt474 B2 75.35 35 5.93E-06 4.75E-06
Satt06 3 B2 93 .49 34 2.48 E-05 1.69E-05
Satt2 52 F 16.08 35 5.93E-06 4.75E-06
Satt663 F 56.17 36 1.29E-06 1.26E-06
Sat_ 1 20 F 75.97 36 1.29E-06 1.26E-06
Satt490 F 97.97 36 1.29E-06 1.26E-06
SattS 22 F 119.19 35 5.93E-06 4.75E-06
Sat__090 F 130.64 35 5.93 E-06 4.75E-06
Satt23 5 G 21.89 36 1.29E-06 1.26E-06
Satt34O G 48.54 35 5.93E-06 4.75E-06
SattS 94 G 52.94 35 5.93 E-06 4.75 E-06
Satt2 87 J 15.69 34 2.48E-05 1.69E-05
Satt285 J 25.51 32 3.28E-04 1.99E-04
Satt406 1 38.19 38 4.6E-08 2.48E-05
Sat_3 66 I 52.84 32 3.28E-04 1.99E-04
Satt43 5 M 38.94 36 1.29E-06 1.26E-06
M M 65.79 35 5.93E-06 4.75 E-06

 

m I calcaulated as the Observed number of resistant RILs that inherited the banding pattern from P1
5675988 in the primary population.

101

Table 4.3 C C PS SSR markers significantly associated with aphid resistance on three

linkage groups: F, G, and M.

 

 

 

Observed number of RILS inherited p-value
the banding pattern from the
Linkage common parent PI 5675988 (m)
S S R Linkage Group Primary Secondary Primary Secondary
marker group Position population (41) population (44) population population
satt2 52 F 16.08 35 27 5.93E-06 0.132
satt423 F 20.56 34 18 1.26E-05 0.228
sat_240 F 25.58 35 27 5.93E-06 0.132
satt663 F 56.17 35 30 5.93E-06 0.016
Satt334 F 76.41 34 38 1.26E-05 1.41 E-06
satt522 F 1 19.19 34 33 1.26E-05 6.30E-04
sat_3 08 G 43.09 35 30 5.93E-06 0.016
satt l I 5 G 43.78 33 27 9.45E-05 0.132
sattS 94 G 5294 35 34 5938-06 1908-04
satt406 J 38.19 38 39 4.60E-08 2.96E-07
satt596 I 39.64 37 41 2.55E-07 1.0lE-08
sat_ I 51 I 41.35 33 33 9458-05 6308-04
satt529 J 41.90 38 39 4.60E-08 2.96E-07
satt622 I 42.25 38 36 4.60E-08 2.43E-05
satt3 80 J 43.01 33 38 9.45E-05 1.41E-06
sat‘255 J 43.85 38 36 4.60E-08 2.43 E-05
Satt2 l 5 1 44.08 35 34 5.93E-06 1.90E-04
WI J 44.68 36 38 1.29E-06 1.41E-06

 

102

Table 4.4 Summary for aphid resistance locus detected in mapping population and the

validation population with aphid DI data using the CIM/SIM method

 

 

Population Rep/ Peak
week Pos.(cM)b
lConsensus Flanking
Trial LG/Chra Pos.c markersd Looc R2f ag
BC 1 F45 Greenhouse 1/3 J/16 25.41 Satt285~Satt406 10.17 69.2% 0.86
Greenhouse lt’3 N/‘3 77.10 Sat_304 - 8.0% 0.20
Greenhouse 134 1:" l 6 22.41 Satt285~Satt406 8.98 89.2% 0.81
Field Cage 13 J” 16 25.91 Satt285-Satt406 13.39 79.1% 0.88
Field Cage 153 N13 77.10 Sat__304 - 4.2% 0.15
Field Cage 2:3 1:16 24.91 Satt285-Satt406 16.36 78.6% 0.89
Field Cage 22’3 N13 77.10 Sat_304 - 8.1% 0.26
Field Cage 14"4 17"16 25.91 Satt406~Satt3 80 12.87 84.6% 1.09
Field Cage 2.1-"‘4 1:16 25.91 Satt285-«Satt406 22.87 91.1% 1.09
070063 Field Cage 13 1116 7.14 Satt4 l4~Satt280 6.31 53.4% 1.02
Field Cage 13 F»"13 78.06 Satt334 - l 1% 0.40
Field Cage 1’3 Nt’3 77.10 Sat_304 - 18% 0.52

 

 

DJ

LG/Chr= linkage group/chromsome

CT

QTL peak position is expressed in cM

C
consensus position is expressed in CM

d
Markers flanking the peak position or the marker at the peak position
C
‘The LOD threshold is 6.8 and 4.6 for BC] F45 and 070063populations, respectively

t
Percentage of phenotypic variation explained by a QTL

g . .
AddItrve effect. The positive value implies that the PI 5675988 allele decreases the phenotypic value

Satt406

 

Figure 4.1 SSR amplification banding patterns Of 41 resistant RILS in the primary
population using primer satt406. Upper and lower bands were the amplification banding

pattern for Titan and PI 5675988 using SSR primer satt406, respectively.

104

Satt522

 

Sat_308

 

Satt622

 

Satt463

 

Figure 4.2 Banding patterns of PCR products Of the 1 1 parents in the primary and
secondary populations using SSR marker Satt522 (LG F). Sat_308 (LG G), Satt622 (LG
J), and Satt463 (M). The order of the 11 parents is: A00-711003(1), A00-711020(2),
A02-381100(3), EOOOO3(4), IA2064(5), RR Titan(6), PI 5675988(7), IA2070(8),

IA2072(9), SDXOOR-039-42(10), Skylla(l 1).

105

 

 

 

 

 

 

0.0 sct_065
5.1 satt414
9.6 satt280
13.5 sat1596
14.8 ~~- sat1529
15.9 ~4~+~ 5311622
19.3 satt215

Figure 4.3 Locations of soybean aphid resistance locus on LG J using composite
interval mapping method. a map shows the identified resistance locus in BCIF435
Population for 3-week and 4-week rating in either greenhouse or field trials: greenhouse
3-week (GH3 W). greenhouse 4-week (GH-l ll"). replication 1 for 3-week rating in field
trial (FREP13 ll"). replication 1 for 4-week rating in field trial (FREPI-IW), replication 2
for 3-week rating in field trial (F REP23 ll). replication 2 for 4-week rating in field trial
(FREP24W). b map is the soybean consensus map for linkage group J. In c map, the
filled black bar represents the locus for the 3-week rating in the field cage trial (FL3 W)

for F45 RIL population (070063). In a and c map. the LOD threshold is 6.8 and 4.6 for

 

ME‘H

”L.

>01

91

 

 

 

 

 

 

 

 

 

 

 

0.0 $811674

95 8811285
16 1 Sct_065
20 7 \,_,/‘ $811406
21 8 \,_./ 5811414
22 3 /’_‘\ Satt280
23.8 “'f—‘t‘ 8811596
252 ~~<r Satt529
26.4 “HP- Satt622
27 1 /7\ Satt380
28.8 880215
36.7 Sat_66

BC1F4;5 and 070063populations, respectively.

106

 

 

+ FL3W

 

 

 

Figure 4.3 (Cont’d)

(10

'14

324)

3815
4013

5513

Ci

 

 

 

 

 

 

 

 

sa0674

san285

san406

san380
sau215

sat_366

AHMHS

Afl}fi)
AAVAiSEkI AAEhiSEkl

l———l
l—_—l

lMEZdEHd

IMvadSHd

~9l

~02

 

 

 

 

 

-O-.FL3VV
-—.—-(HH3VV

"a."
‘iFFM3313VV
'IWREFW4VV
"ffl?EFQ3VV
'FTWSP24VV

66+?

GH44VV

 

 

 

107

 

 

Satt622

 

Satt529

 

Satt285

 

Figure 4.4 PCR products amplified by SSR markers Satt622 (LG J), and Satt529 (LG J),
for PI 567543C (1), PI 5675418 (2), PI 5675988 (3), PI 567585A (4), Skylla (5), Titan

(6), Dowling (7).

108

References:

Benjamini, Y., and D. Yekutieli. 2005. Quantitative trait loci analysis using the false
discovery rate. Genetics 171: 783—790

Buckler. E.S., J.B. Holland. P.J. Bradbury. C.B. Acharya, P.J. Brown, C. Browne, E.
Ersoz, S. F lint-Garcia, A. Garcia, J.C. Glaubitz, M.M. Goodman, C. Harjes, K.
Guill, D.E. Kroon, S. Larsson, N. K. Lepak, H. Li, S.E. Mitchell, G. Pressoir, J. A.
Peiffer, M. O.Rosas, T.R. Rocheford, M.C. Romay, S. Romero, S. Salvo, H.S.
Villeda, H.S. da Silva, Q. Sun. F. Tian, N.Upadyayula, D. Ware, H. Yates, J. Yu, Z.
Zhang, S. Kresovich. M.D. McMullen. 2009. The Genetic Architecture of Maize
Flowering Time. Science 3252714 - 718.

C harlier. C., F. Famir, P. Berzi. P. Vanmanshoven, B. Brouwers. 1996. IBD mapping of
recessive traits in livestock: application to map the bovine syndacty locus to
chromosome 15. Genome Res. 62580—589.

Chen C.Y., C. Gu, C. Mensah, R.L. Nelson. D. Wang. 2007. SSR marker diversity of
soybean aphid resistance sources in North America. Genome 50:1104—1111.

Chung, G.L., J. Poland, R.Wisser, J. Kolkman, The Maize Diversity Project, and Nelson,
R. 2008. Analysis of th8.06, a major QTL for resistance to northern leaf blight in

maize. Annual Research Meeting of Generation Challenge Programme, Bangkok,
Thailand.

Cregan P.B.. C.V. Quigley. 1997. Simple sequence repeat DNA marker analysis. In
CaetanO-Anolles G, Gresshoff PM (eds) DNA markers: Protocols, applications and
overviews. John Wiley & Sons, New York, 173-185.

I)iaz-Montano, J., J.C. Reese, W.T. Schapaugh, and LR. Campbell. 2006.
Characterization of antibiosis and antixenosis to the soybean aphid (Hemiptera:
Aphididae) in several soybean genotypes. J. Econ. Entomol. 99:1884—1889.

DiFonzo, CD, and R. Hines. 2002. Soybean aphid in Michigan: Update from the 2001
season. Michigan State Univ. Ext. Bull. E-2748. Michigan State Univ., East
Lansing.

Flint-Garcia, S. A., J. M. Thornsberry and E. S. Bucker, IV, 2003. Structure of linkage
disequilibrium in plants. Annu. Rev. Plant. Biol. 54: 357—374.

109

Hesler. S.L., K.E. Dashiell, and JG. Lundgren. 2007. Characterization of resistance to
Aphis glycines in soybean accessions. Euphytica 154291—94.

Hill, C.B., Y. Li and G.L. Hartman. 2004. Resistance to the soybean aphid in soybean
germplasm. Crop Sci. 44:98-106.

Hill. C.B., Y. Li, and G.L. Hartman. 2006a. Identification ofa single dominant gene for

resistance to the soybean aphid in the soybean cultivar Dowling. Crop Sci.
46:1601—1605.

Hill. C .B., Y. Li, and G.L. Hartman. 2006b. Soybean aphid resistance in soybean Jackson
is controlled by a single dominant gene. Crop Sci. 46:1606—1608.

Hill, C.B. K.S. Kim, L. Crulla. B.W. Diers and G.L. Hartman. 2009. Inheritance of
Resistance to the Soybean Aphid in Soybean PI 20053. Crop Sci 49:1 193-1200

Hyten, D.L., I.-Y. Choi, Q. Song, R.C. Shoemaker, R.L. Nelson et al.,, 2007. Highly
variable patterns of linkage disequilibrium in multiple soybean populations.
Genetics 175:1937—1944.

Kaloshian, I. 2004. Gene-for-gene disease resistance: bridging insect pest and pathogen
defense. J. Chemical Ecology 30:2421-2440.

Kang, S.T, M.A.R. Mian, and RB. Hammond. 2008. Soybean aphid Resistance in P1
243540 Is Controlled by a Single Dominant Gene. Crop Sci. 48:1744-1748.

Kisha T, C.H.Sneller, B.W. Diers. 1997. Relationship between genetic distance among
parents and genetic variance in populations of soybean. Crop Sci 37:1317-1325.

Li, Y., C.B. Hill, S. Carlson. B.W. Diers, and G.L. Hartman. 2007. Soybean aphid
resistance genes in the soybean cultivars Dowling and Jackson map to linkage
group M. MOI. Breed. 19:25—34.

Li, Y., J. Zou, M. Li, D.D. Bilgin, L.O. Vodkin, G.L. Hartman, and SJ. Clough. 2008.

Soybean defense responses to the soybean aphid. New Phytologist 79:185-195.

110

Manly K.F., RH. Cudmore, J.M. Meer. 2001. Map Manager QTX. cross-platform
software for genetic mapping. Mamm Genome 122930—932

Mensah C, C. Difonzo, R.L. Nelson. D. Wang. 2005. Resistance to soybean aphid in
early maturing soybean germplasm. Crop Sci 45:2228—2233.

Mensah. C., C. DiFonzo. and D. Wang. 2008. Inheritance of Soybean Aphid
Resistance in P1 5675418 and PI 5675988. Crop Sci. 48:1759-1763.

Mian. M.A.R., RB. Ilammond, and SK. St. Martin. 2008a. New plant introductions with
resistance to the soybean aphid. Crop Sci. 48: 1055—1061.

Mian. M.A.R., S.T. Kang. SE. Beil, and RB. Hammond. 2008b. Genetic linkage

mapping of the soybean aphid resistance gene in P1 243540. Theor. Appl. Genet.
117:955—962.

Painter. RH. 1951. Insect resistance in crop plants. Macmillan. New York.

Poland, J.A., The Maize Diversity Project. R.J. Nelson.2009. Nested Association

Mapping of Northern Leaf Blight Resistance in Maize, Plant and Animal Genome,
Jan.2009

Reimer, S., C.J. Pozniak, F.R. Clarke, J.M. Clarke, D.J. Somers, R.E. Knox, and AK.
Singh. 2008. Association mapping of yellow pigment in an elite collection of
durum wheat cultivars and breeding lines. Genome 51:1016-1025.

Song Q.J., L.F. Marek, R.C. Shoemaker, K.G. Lark, V.C. Concibido, X. Delannay, J.E.
Specht, P.B. Cregan. 2004. A new integrated genetic linkage map of the soybean.
Theor. Appl. Genet. 109:122—128.

Storey JD. 2002. A direct approach to false discovery rates. Journal of the Royal
Statistical Society, Series B. 64:479-498.

S un B., S.B.Liang. W.X. Zhao. 2000. Outbreak ofthe soybean aphid in Suihua prefecture
in 1998 and its control methods. Soybean Bull 1:5

111

USDA-National Agricultural Statistics Service. 2008. USDA Report Assesses Corn and
Soybean Acreage USDA-NASS. Washington, DC.

Van Ooijen, J.W., RE. Voorrips. 2001. JoinMap 3.0, Software for the calculation of
genetic linkage maps in experimental populations. Kyazma, BV, Wageningen

Voorrips RE. 2002 MapC hart: Software for the graphical presentation of linkage maps
and QTLs. J Heredity 93:77-78.

Wang D., J. Shi, SR. Carlson, P.B. Cregan, R.W. Ward, B.W. Diers. 2003. A low-cost
and high-throughput system for high-resolution genotyping with microsatellite
DNA markers. Crop Sci. 43:1828-1832.

Wang S.. C.J. Basten. Z.B. Zeng. 2008. Windows QTL Cartographer 2.5. Dept of Stat,
North Carolina State University. Raleigh.
http://statgen.ncsu.edu/qtlcart/WQTLCart.htm. Cited 21 Apr 2008

Yu. J., J.B. Holland, M.D. McMullen. and ES. Buckler. 2008. Genetic design and
statistical power of nested association mapping in maize. Genetics 138:539-551.

Zeng Z.B. 1994. Precision mapping of quantitative trait loci. Genetics 136:1457-1468.

Zhang. G.. C. Gu. and D. Wang. 2009. Molecular mapping Of soybean aphid resistance
genes in P1 5675418. Theor. Appl. Genet. 1 18:473—482.

Zhang. G., D. Wang. 2009b. Detection and verification of an aphid resistance locus in
Soybean PI 567543C. In 2009 annual meeting abstracts [CD—ROM]. ASA, CSSA,
and SSSA, Pittsburg. PA.

Zhu. C., M. Gore, E.S. Buckler, and J. Yu. 2008. Status and prospects of association
mapping in Plants. The Plant Genome 1:5-20.

APPENDIX

113

Table A1 0-4 Half step scale for soybean aphid damage (Mensah et al., 2008)

 

 

Half Step Scale Soybean Aphid Damage

0 No aphids. health and normal plants

0.5 Less than 10 aphids. no colony formed. health and normal plants
I 1 1-100 aphids/plant. still normal and health plants

1.5 101-150 aphids/"plant. mostly located on the young leaves

2 151-300 aphids/plant. located both on the young leaves and stems

2.5 3111 -500 aphids/plant. aphids dispersed on leaves and stems

3 501-800 aphids/plant. curly and shiny leaves. young leaves and stems covered
by aphids

3.5 More than 800 aphidsfplant. stunted plants. curled and yellow leaves. but no
sooty mold and few cast skins

4 More than 800 aphids/plant. stunted plants. curled and yellow leaves. sooty

mold and cast skins

 

114

Table A2 The aphid resistance scores for Skylla, IA2070. PI 567585A, and F.

generations derived from 070082-1 and 070016-1 populations.

 

 

 

 

 

Genetic 11) Genetic Name Individual No. Rating
7P76 Pl 567585A I 1.0
7P76 Pl 567585A 2 0.5
7P76 1’1 567585A 3 1.0
7P76 PI 567585A 4 0.5
7P76 Pl 567585A 5 0.5
7P76 I’l 567585A 6 1.0
7P76 PI 567585A 7 0.5
7P76 Pl 567585A 8 1.0
7P02 Skylla 1 3.5
7P02 Skylla 2 3.5
7P02 Skylla 3 3.5
71’02 Skylla 4 3.5
7P02 Skylla 5 3.5
71’02 Skylla 6 3.5
7P02 Sky Ila 7 3.5
7P02 Skylla 8 3.5
7P02 Skylla 9 3.5
7P02 Skylla 10 3.5
7P38 IA2070 1 3.5
7P38 IA2070 2 3.5
7P38 IA2070 3 3.0
7P38 IA2070 4 3.5
7P38 IA2070 5 3.5
7P38 IA2070 6 3.5
7P38 IA2070 7 3.5
7P38 1A2070 8 3.0
7P38 IA2070 9 3.5
7P38 IA2070 10 3.5
7P38 IA2070 l 1 3.0

(Skylla x 1’1 567585A)F1 I 2.5

(Skylla x PI 567585A) F1

IQ
IO N
UI

 

(Skylla x P1 567585A) 151 3 5
(Skylla x P1 567585A)1-‘1 4 2.0
(Skylla x Pl 567585A1FI 5 2.5
(IA2070 x PI 567585A) Fl 1 -
(1A2070 x PI 567585A) Fl 2 -
(IA2070 x Pl 567585A) F] 3 -
(IA2070 x PI 567585A) Fl 4 -
(IA2070 x PI 567585A)171 5 -

 

TableA3 Aphid resistance scores for 070016-1 F2 population

 

 

 

070016-1 F2 population 070016-I F2 population 070016-l F2 population
F3 line No. Rating F2 line No. Rating 152 line No. Rating

1 2 31 2 61 3
2 2.5 32 2 62 3
3 2 33 2.5 63 2
4 1.5 34 3.5 64 3.5
5 3 35 2 65 2
6 3 36 1 66 l
7 2 37 2 5 67 0.5
8 1 38 3 68 l
9 2 39 I 5 69 2.5
10 2 40 1 70 1
II 2 41 1.5 71 2
12 2.5 42 2 72 2
13 0.5 43 1 73 3
14 2.5 44 2 74 2
15 l 45 1 5 75 -
16 2 46 1 76 I
17 l 47 2 77 I
18 2 48 2 78 1
l9 3 49 1.5 79 2
20 I 5 50 2 80 3
._l 1.5 51 l 5 81 1
22 1 52 1.5 82 2.5
23 l 53 1.5 83 2
24 2 5 54 l 5 84 3 5
25 2 55 1 5 85 3
26 2 56 2 86 2
27 I 57 l 87 2
28 2 58 3 88 3
29 2 59 2 5
30 2 60 l

 

116

Table A4 Aphid resistance scores for 070082-1 F2 population

 

 

 

070082-1 F2 population 070082-1 F2 population 070082-1 F2 population

F2 line No. Rating F2 line NO. Rating F2 line NO. Rating
1 3 31 1.5 61 3
2 1 32 2 62 0.5
3 1.5 33 1 63 2
4 1.5 34 1.5 64 1.5
5 2 35 0.5 65 1.5
6 2 36 1.5 66 3
7 1 37 l 67 3
8 l 38 1.5 68 3
9 1.5 39 5 69 2.5
10 3 40 0.5 70 1
1 1 - 41 5 71 3
12 2.5 42 0.5 72 1.5
13 1.5 43 1 73 3
l4 1 44 1.5 74 3
15 1.5 45 2.5 75 1.5
16 1.5 46 0.5 76 2
17 3 47 2 77
18 1 48 0.5 78
19 2 49 1 5 79
-0 l 50 0 5 80 2.5
21 2 51 0 5 81 1
22 1 5 52 1 5 82 3.5
23 1 53 0.5 83 2
24 2.5 54 2 84 0.5
25 1.5 55 2 85 1
26 1 5 56 2 5 86 1.5
27 1.5 57 1 5 87 2
28 .. 58 1 5 88 1
29 2 59 1 89 0.5
30 1.5 6() 1.5 90 1.5

 

117

Table A4 (Cont'd)

 

 

 

070082-1 F2 population 070082-I F2 population 070082-1 F2 population
F2 line No. Rating F2 line No. Rating F2 line No. Rating

91 1.5 121 0.5 151 1
92 1.5 122 1.5 152 1.5
93 2 123 2 153 2
94 1 124 2 154 1.5
95 1.5 125 2 155 2
96 1.5 126 2 156 1.5
97 1 127 1 157 2
98 1.5 128 I 158 I
99 1.5 129 0.5 159 1.5
100 1 130 1.5 160 0.5
1(11 1 131 2 161 1.5
102 0.5 132 1.5 162 2
103 1.5 133 1.5 163 1.5
104 0.5 134 I 164 2
105 1.5 135 0.5 165 1.5
106 1 136 2 166 3
107 2 137 I 167 2
108 2.5 138 1.5 168 1
109 1.5 139 2 169 3
110 2 140 2.5 170 1.5
111 0.5 141 3.5 171 3
112 0.5 142 3 172 0.5
1 l3 1 143 3 173 1.5
1 14 1.5 144 3 174 1.5
1 15 1.5 145 3 175 1.5
1 16 1 146 1 176 1.5
117 0.5 147 1.5 177 1.5
1 18 2 148 1.5 178 1.5
1 19 2 I49 3 179 I
120 1 150 3 180 -

 

118

Table A4 (Cont‘d)

 

 

 

()70082-1 F3 population 070082-l F2 population
F3 line No. Rating F3 line No, Rating

181 1.5 203 1
182 1.5 204 .
I83 2 205 3
184 l 206 3.5
185 3 207 2.5
186 I 208 3
187 1.5 209 1.5
188 0.5 210 1.5
189 2 21 1 2.5
190 1.5 212 I
191 0.5 213 3.5
192 1.5 214 2
193 3 215 3.5
194 3.5 216
195 3 217 3
196 3 218 3.5
197 1.5 219 1.5
198 1.5 220 1.5
199 3 221 3.5
200 1.5 222 3.5
201 3 223 3.5
202 3 224 3 5

 

119

Table A5 Progeny test for F3 families derived from 070016-1

 

F2 individual

F ~ r0 enies in 2009
tested in 2008 2.3 P 8

 

Line WK3 '1 2 3 4 5 6 7 8 9 10 1 1

 

l 2 3 l 5 3 1.5 3 2 2 2 3 5 2.5 2.5
2 3 3 2 3.5 3.5 2.5

3 2 1 5 1 1 5 1.5 1 1 5 1 5 1

4 2 3 5 1.5 l 5 3 5 3.5 1 5

5 2 3 5 2.5 3 5 3.5 3 5 5 3.5

6 3 1 2 2 1.5 2 2.5 1 5

7 2 1 1 5 1.5 1 5 1 1

8 l 2 5 2 2 5 l 1.5 2 3 5 1 5

9 2 3.5 3 5 3 3.5 3."

11 2 2 1 5 2 1.5 2 5 3 5 1 5 2

12 2.5 2 5 1 2 3.5 3 5 3 3.5 3 5 1 2
13 0.5 1 1 l 1.5 1 1.5 1

15 1 3.5 1 1.5 2 1.5 1.5 3 1.5 1

19 3 1 3 2 3 2.5 1.5 1.5 1

20 1.5 3.5 3.5 3.5 3.5

21 1.5 1.5 3 3.5 1.5 3.5

22 1 0.5 1 0.5 0.5 1 1 1

26 2 l l 3 1 3 1 5 2 5 3 5

27 l 3.5 3 3.5 1 5 1 5

30 2 3.5 3 5 3

31 2 1 l l 1 1

32 2 2 1 5 2 3 3 5 l 5 3 5 2 1

35 2 3.5 3.5 3.5 3.5 l 5 1 5

36 l 2 1 5 1.5 2.5 2 2 1.5 3 2 1
37 2.5 1.5 l 5 2.5 3 5 2 3.5 2 1.5 1.5 1.5 3.5
41 15 3.5 3 5 3.5 3 5 3.5 3 5 3 5 3.5 3.5

44 2 1 5 3 3 5 l 3 1.5 1 5 3 5 l 5 2
47 2 0.5 1 1 0 0.5 0.5 0.5 0.5 0.5

48 2 1 0.5 l 2 3.5 1 5 1

49 1 5 3 5 3 3.5 3 5 3 5 3 5 3 5

50 2 3 5 2 3.5 3.5 3 5 1 1 1.5

51 15 1 l 1 1.5 15 2 1 0.5 1.5 1.5
52 1 5 0.5 0 5 0.5 1.5 1.5 0.5 0.5 1 1 0.5 0.5
55 1.5 1 1 0.5 0 5 l 1 1 l 1

56 2 l 3 0.5 0 5 1 0.5 1

58 3 1 5 1.5 l 2 5 3.5 1.5 1 5 1

59 2 5 1 0.5 0 5 0 5 0.5 1 l 5

60 l 1 5 3.5 3 5 1.5 2 5

61 3 3 5 3.5 3 5 3.5

 

120

Table A5 (Cont‘d)

 

F2 individual

F— ' ' 2009
tested in 2008 2,3 progenies In

 

 

Line WK3 1 2 3 4 5 6 7
63 2 3 5 3.5 3.5 3.5 3 5

64 3.5 3 5 3 5 3.5 3 5 3 5 3 5

65 2 1 5 0 5 0.5 0 5 1 l 0.5
66 1 1 1 3.5 2 1 3.5 l .5
67 0.5 3.5 3 5 3 5 3 1 5

68 l 0.5 0 5 0.5 0.5

69 2.5 0 5 0.5 0 5 0.5 ()5 '

72 7 l 5 3.5 3 5 .. 3 5 0.5
73 2.5 0.5 3 5 0 5 0.5 1 5 l 5
78 1 2 0 5 l 3 3.5 1 5 1
79 2 3.5 3 5 3.5 3 5

80 3 0 5 3 3 3 3 3 1
81 1 1 0.5 0.5 5 0 5

82 2 5 5 0.5 0.5 O 5 5

83 2 1 1 1.5 1 5 2 1 1 5
84 3 5 - 5 3.5 3 5 ' 3 5 3 5

85 3 s 5 3.5 3 5 3 3 5

86 2 2 1 1 1 2 2 3.5
88 3 3.5 3.5 3.5 3.5

 

121

Table A6 Progeny test for F 33 families derived from 070082-l

 

F2 individual
tested in 2008

F23 progenies in 2009

 

 

Line WK3 1 2 3 4 5 6 7 8 9 10 11
1 3 3.5 3.5 3.5 3 5 3.5 3.5 3.5 3.5 3.5 3.5
2 1 2 2 l 2 2 1 1.5 3.5 1
3 1.5 3.5 3.5 3.5 3 5 3.5 2 2 3.5
4 1.5 3 3 2 1 1 2 3.5 2.5 1.5 l
6 2 3.5 3.5 ' 3.5 3 5 3.

10 3 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5

15 1.5 3.5 1 1 3 1 2.5 1 1 3

16 1.5 1.5 1 1.5 15 1.5 1.5 1.5 1 1.5

18 1 3.5 3.5 3.“ 3.5 3.5 3.5 3.5 3.5

19 2 3.5 3.5 3.5 3.5 3 5 3.5 3.5

20 l 3.5 2.5 2 2 2 2.5

21 2 3.5 3.5 3.5 3 5 2.5 3.5 1.5 1.5 1.5

22 l 1.5 1 1 1.5 1 l 1 1.5 1

24 2 2 2 2 3.5 3 5 3.5 3.5 3.5 3.5 3.5 3.5
25 1.5 3.5 3.5 1.5 3.5 1 1

26 1.5 1 l 1 3 5 3.5

27 1.5 1 1 1 l 2 1.5 1 1 1

28 2 1 1 1 1

30 1.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5

33 l 3.5 3.5 3.5 3.5 3 5 3.5

34 1.5 1.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5

35 0.5 3 3 1 3 1.5 1.5 l 1 1 1
36 1.5 2 3.5 3.5 3.5 3 5 3.5 3.5 3.5

37 1 1 1.5 3 3 l | 2.5 l l

38 1.5 1 1 1 1 l 1 1 1 1.5

40 0.5 1

42 0.5 1 1 1 1 1 1 1 1

43 1 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5

44 1.5 1 1 1 l 1

47 2 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5

48 0.5 1 1 1 l 1

51 0.5 1 3.5 3.5 1.5 1 l 3.5

53 0.5 1.5 3.5 2 1 l

55 2 3 3 2.5

56 2 l 1 1 1 1 1 l 1

59 1 1.5 1 1 l 1 1 1 1 l 1
60 1.5 2 2 2 1.5 2 1.5 2

61 3 2.5 2 1.5 2 2 3 2 2.5 1 1.5
63 2 1.5 1.5 1.5 2 1.5 3 2 1.5

 

122

Table A6 (Cont'd)

 

 

 

F2 individual tested in 2008 F33 progenies in 2009
Line WK3 l 2 3 4 5 6 7

64 1.5 l 1 1 3.5 3.5 3.5

66 3 1 l 2 2 1 2 1 2
69 2.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5

70 1 l 1 1 l 1 1 1

71 3 3.5 1 5 3

73 3 3.5 2 2 5 3 5

74 3 3.5 3.5 3 5 3.5 3 5 3 5

75 1 5 3 1.5 1 2 l 3 5

76 2 3 1 l 1 1 1 5 3 5

77 3 3 .5 3.5 3 5 3.5 3.5 3 5

78 3 2 3.5 3 5 3.5 3 5 1 5 2 .5
79 1 5 1 1 l 1 l 2 5 1 .5
81 1 3 5 1 2 1 1 5 l 1

82 3 5 2 3 3 2 2 3 S 3 5

83 2 1.5 1 I 1 1 1 2

84 0.5 1 1 1 1 5 l l 1

85 l 1 1 1 l 1 l 1

86 1 5 l 5 1.5 l 3.5 1 2 5 3

87 2 3 5 1 l 5 1 1 1 1

88 1 1 5 1 1 1 l 5 1 5 1

90 1.5 1.5 1.5 l 2 2 1.5 1

91 1.5 3.5 3.5 3 5 3.5 3.5 3 5

93 2 3.5 3 ' 3 3.5 3 5

94 1 1 1.5 1 l l l 1

95 1 5 1 I 2 5 1 3 l 5 2.5

96 1.5 1.5 2 1 5 1 5 2 2 2

97 l 3 5 2.5 1 5 3.5 3 5 l

98 l 5 l 1 2 1.5 2 3 5 3 5

100 1 3 5 1.5 1 5 1 5 2 3 5 2
101 1 1 2.5 l l 1 3 1 5

102 0 5 1.5 l 1 1 1 l 5 l .5
103 1.5 2 3 3 3 3 3 .5
105 1.5 3 1 1 1 2 3 1 5 1.5
106 l 1.5 1.5 3 3 2.5 2.5 3

107 2 1.5 2.5 3 1.5 1.5 3.5 3.5

108 2.5 3.5 3.5 3.5 3 5 3.5 3 5 3 5

110 2 1 l 1.5 1 1 5 1 1 .5
113 1 1.5 l 3 l 5 l 5 2 2.5

 

123

Table A6 (Cont‘d)

 

F2 individual tested in ‘ _ .
133 progenies in 2009

 

 

2008
Line WK3 1 2 3 4 5 6 7 8
114 1.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5
115 1.5 l 1 l 1 1 1 1
116 1 2 1 1 1 2 1 2.5
118 2 2 l 1 2 1 3 3.5
120 1 1 3 1 l 1 3 l 3.5 2
121 0.5 2 1 1 1 0 0.5 0.5
123 - 2 5 3 3 5 3 3 5 3 3 3
124 2 2 l 5 1 1 - 5 2 3 1
125 2 0.5 0.5 0.5 0.5
126 2 1 2 1 2 O l 1 5 2
127 1 0.5 0.5 0.5 0.5 0.5 0.5
129 0.5 0.5 1 0 3 3 1 1 1.5
130 1.5 2 l 1 1.5 3 2 1 5
131 2 1.5 1.5 1.5 3.5 3.5 35
133 1.5 1 3 3 1 1 1 3 1
I34 1 1 1 1 2 1 1 1 1
135 0.5 2 1.5 3 1.5 1.5 1 1
136 2 1 1 1 1 1.5 1 1.5 1.5
137 1 l 1 1 1 1 1 I
138 1.5 1.5 3 3 1 1 1.5 l
139 2 2 1.5 3 3 1 1 3 1 5
140 2.5 3 1 2 2 5 3 2 5 3 1.5
141 3.5 3.5 3.5 3 5 3 5 3.5 3 5 3 5
146 l 3 1.5 1.5 1 3.5 3.5 1
147 1.5 3.5 1 |
150 3 35 35 35 35 3.5 35 35 35
151 1 2 1.5 1 1 1 1 l
157 2 1 3 1.5 1.5 2 1.5
159 1.5 3.5 l 1.5 1 3 3 2 3.5
161 1.5 1.5 25 15 2 15 2 15
162 2 3.5 2 5 1 5 1 5 2.5 3 2 5
163 1 5 1 5 1 1.5 1 5 1 5 1 1
164 2 3.5 3.5 3.5 3.5 3 5 3.5 3 5
165 1.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
167 2 3.5 3.5 1 l 2 2.5 1 0.5
169 3 3.5 3 5 .5 3.5 3 5
170 1.5 0.5 0 5 0.5 0 5 0 5 0.5
171 3 3.5 3 5 3.5 3.5 3 5 3.5 3.5 3.5
172 0.5 1 1 0.5 1.5 2 0.5

 

124

Table A6 (C ont‘d)

 

 

 

F3 individual tested in 2008 F3 3 progenies in 2009

Line WK3 1 2 3 4 5 6 7 8 9 10 11
173 1.5 3 5 2 1 1 2 2

174 1.5 2 1 5 3 5 3.5 3 1 1 5 2
175 1.5 2 3 5 3 5 3.5 3 5 3 2 5

176 1.5 2 5 3 5 3.5 3 3 2 5

177 1.5 3 5 1 1.5 1 3 5 1 l 0.5
178 1.5 1 0.5 2 3.5 2 3.5 2 2
179 l l 0.5 0.5 0.5 3 3 0.5 0.5
181 1.5 2.5 1 1 1 7.5 1 2 2
182 1.5 1.5 0.5 1 1 5 1

183 2 1.5 3 5 3 5 3 1 5 l 5 3 5

184 1 1.5 l 1 0 0.5 ()5 1

185 3 3.5 3 5 3 5 3.5 2 5 2 5

186 1 2.5 2 1 2.5 1

187 1.5 1 1.5 3.5 2 1.5 2

189 2 0.5 0.5 0.5

190 1.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5
192 1.5 3.5 3.5 3.5 3.5 3.5 3.5

193 3 3.5 3.5 3.5 3.5 3.5 3.5 3.5

194 3.5 3.5 3.5 3 5 3.5 3 5 3.5 3 5

195 1 1.5 3 1.5 1 1 3

”196 3 3.5 3.5 3.5 3.5 3.5 3.5 3.5

198 1.5 3 5 1 1 3 3 3

199 3 1 2.5 1 3.5

200 1.5 2.5 3 3

201 3 3 5 3.5 3.5 3.5 3.5

202 3 0.5 0 5 0.5 0.5 0.5

203 1 2.5 1 5 1 1.5 2 1

204 3 2 2 2 2 2.5

205 3 3.5 a 5 3.5 3 5 3 5

206 3.5 3.5 3 5 3.5 3.5 3 5 3.5 3 5

207 3.5 3 5 35 3.5 35 35 5

208 3 1.5 1.5 3 5 3.5 3.5 3 5

209 1.5 1 1 1 1 1 1 1

215 3.5 1 1.5 1 3 3.5 2 5 3

217 3 1 2.5 3 5 3.5 2 1 3.5

218 35 35 3.5 35 3.5 3.5

219 1.5 1 1 1 5 2 3.5 3 5 3 5
220 1.5 0 5 0 5 0.5 ()5 O 5 0 5 0.5

221 3.5 3.5 3 5 3.5 3 5 -

 

125

Table A6 (Cont‘d)

 

F3 individual

F » ro enies in 2009
tested in 2008 3 3 p g

 

l J
b)
A
VI
0‘
\1
00
C
'5

Line WK3 1
3.5 3.5 3.5 3.5 3.5

223 3.5 0.5 0.5 l 1 1.5 1 3.5 3.5
224 3.5 3.5 3.5 3.5 3.5 3.5 3.5

77’) 35

Table A7 RIL information for MNAM

 

 

RIL Name Female Parent Male parent R11. Name Female Parent Male parent
M1,18 RR Titan P15675988 Ml.50 RR Titan P15675988
Mli20 RR Titan P15675988 ML51 RR Titan P15675981)
Ml.21 RR Titan P15675988 Ml.52 RR Titan P15675988
Ml.23 RR Titan P15675988 MLS3 RR Titan P15675983
Ml,24 RR Titan 13156759813 ML55 RR Titan P15675988
Ml.27 RR Titan 1’156759813 MLS6 RR Titan P15675988
M129 RR Titan P15675988 ML57 RR Titan P15675988
Ml,30 RR Titan P15675988 ML63 RR Titan P15675988
ML31 RR Titan P15675988 ML64 RR Titan P15675988
Ml.32 RR Titan 1’156759813 ML78 RR Titan P15675988
Ml.33 RR Titan P15675988 ML79 RR Titan P15675988
Ml.34 RR Titan P15675988 ML82 RR Titan P15675983
Ml.35 RR Titan P15675988 ML88 RR Titan P15675988
Ml.36 RR Titan P15675988 ML89 RR Titan P1567598B
Ml.37 RR Titan P15675988 ML91 RR Titan P15675988
M138 RR Titan P15675988 Ml,94 RR Titan P15675988
M1 .42 RR Titan P15675988 M L95 RR Titan P15675988
M1 ,43 RR Titan 13156759813 ML96 RR Titan P15675988
ML44 RR Titan P15675988 ML98 RR Titan P15675988
M1445 RR Titan P15675988 Ml.99 RR Titan P15675988
Ml,48 RR Titan P15675988

 

7

-1.

Table A7 (Cont'd)

 

 

RlL Name Female Parent Male parent R11, Name Female Parent Male parent
(70001 A00-71 1003 P1 56759813 (70132 1A2070 Pl 56759813
(70004 A00-71 1003 PI 5675983 (70133 1A2070 Pl 5675983
C0008 A00-71 1003 P1 56759813 C0374 1A2070 Pl 5675983
(70009 A00-7l 1003 P1 56759813 (70135 1A2072 PI 56759813
(‘0013 A00-7l 1003 P1 56759813 (‘0375 1A2072 Pl 56759813
(70255 A00-71 1020 PI 56759813 C0120 SDXOOR-O39-42 P15675983
C0257 A00-71 1020 P1 56759813 (70144 SDXOOR-O39-42 P15675983
(70260 A00-71 1020 P1 56759813 (70347 SDXOOR-039—42 P15675983
(70268 A00-71 1(120 Pl 56759813 C0348 SDXOOR-039-42 P15675983
C0270 A00-71 1020 P1 5675983 C0350 SDXOOR-039-42 P15675983
C0272 A00-7l 1020 P1 56759813 (70353 SDXOOR-039-42 P15675983
(70273 A00-71 1020 P1 56759813 (70359 SDXOOR-039-42 P15675983
C0295 A02-381 100 P1 5675983 C0386 SDXOOR-039-42 P15675983
(70297 A02-381 100 1’1 56759813 (‘0199 Skylla P15675983
(70041 1300003 P1 56759813 ("0201 Skylla P15675983
(70042 300003 P1 56759813 C0202 Skylla P15675983
C0301 1500003 Pl 56759813 (‘0203 Skylla 1315675983
C0123 1A2064 Pl 56759813 (70204 Skylla P15675983
(70125 1A2064 P1 56759813 (70207 Skylla P15675983
(70127 1A2064 Pl 5675983 (70208 Skylla P15675983
(70129 1.A207() Pl 56759813 (70210 Skylla Pl 5675983
(‘0130 IA2070 Pl 56759813 (‘021 | Skylla P1 56759813

 

128

Table A8 Genome-wide screening for marker trait association in primary population

 

 

1.0 group Position SSR marker m total MLS P-value q-value
1-1 0.5 satt666 15 26 8.58E-02 2.57E-02
H 59 sat—-334 5 36 1293-06 1.26E-06
11 106 satt434 18 23 4.353-01 1033-01

A; 14.65 sat1572 20 21 8763-01 1.72E-01
A. 32.68 satt155 1 1 30 3003-03 1.3213-03
A] 42.8 sat_356 21 20 8.7613-01 1.72E-()1
A I 64.74 satt385 17 24 2.7413-01 6.84E-02
A] 71.39 sattS45 16 25 1.601-i-01 44813-02
A] 93. 3 satt236 14 27 44233-02 1.34E-02
A3 28 satt480 12 29 7.93E-O3 2.8715-03
A3 54 salt 1 87 7 34 2.481305 1.69E-05
A3 77.7 satt341 21 20 87613-0] 1.72E-01
A3 90 satt377 19 22 63913-0] 1.34E-O1
A3 128 satt209 12 29 7.93 3-03 2.87E-03
A3 159.63 sat1538 22 19 6393-01 1.34F.-01
31 14.32 sat‘272 0 41 1523-10 NA

31 31 sat_41 1 12 29 7933-03 2873-03
31 80.89 satt332 13 28 1.91E-02 6511-2-03
31 96.36 satt665 18 23 4353-01 1.03 Fl-Ol
13] 125.74 sat_331 0 41 1523-10 NA

33 6.05 Satt5 77 16 25 1 6015-01 44817-02
33 27.63 satt126 1 1 30 30015-03 1.32E-03
33 55.2 sattl68 15 26 8.5813-02 2573-02
33 66.24 sat33 55 5 36 12913-06 1263-06
33 75.35 satt474 6 35 5933-06 4753-06
33 93 5311063 7 34 24813-05 1.69E-05
C, O sattS65 15 26 8583-02 2.5713-02
CI 32.1 sat_337 9 32 3 .2813-04 1.99E-O4
C; 65 sa11578 1 1 30 3.0013-03 1.32E-03
C] 73.39 satt 1 61 17 24 2.743-01 6.8413—02
C] 87 sat_207 12 29 7.93E-03 2.87E-03
C. 128 satt 1 80 10 3 1 1043-03 5.83 E-04
C3 41 satt281 17 24 2.74E-01 6.84E-02
C3 91 sat_246 12 29 7.9313-03 2.87E-03
C3 1 17.77 satt460 12 29 7.9313-03 2.87E-03
C3 127.67 satt316 19 22 6.39E-01 1.3413-01
C3 145.48 satt371 20 21 87613-01 NA

[)18 5 sat_332 l 1 30 3003-03 1.32E-03
D13 36.23 sat_353 14 27 423172-02 NA

1) I a 55.22 satt295 2 39 7543-09 9.95 E-09
D 1 a 77.46 satt077 7 34 2.483-05 1.6913-05

 

129

Table A8 (Cont‘d)

 

 

1.0 group Position SSR marker m total Ml.s P-value q-value
[)1a 106.69 Satt408 0 41 1523-10 NA
[)1 h 37 sat1157 16 25 1.60E-01 4483-02
1)”, 46.6 satt634 13 28 1.91 E-02 6.51 E-(l3
D”) 75.29 sat1005 1 l 30 3.00F.-03 1.3212-03
016 98.7 Satt703 1 l 30 3003-03 1.32E-03
Um I 18.6 satt459 14 27 4.2313-02 1.34E-02
1)”, 137.1 satt271 20 21 8.76E-01 1.7213-01
[)3 6 sat_296 1 1 30 3.0013-03 NA
D3 29 sat3277 18 23 43513-0] 1.03E-01
[)3 68 satt669 1 1 3(1 3.0013-03 1.3213-03
[)3 84.62 satt31 l 19 22 63913-01 1.34E-01
[)3 93.71 satt301 21 20 87613-0] 1.72Ii-01
[)3 128.95 set_l37 0 41 1.52E-10 NA

F. 21 satt720 20 21 8.7613-01 1.72E-01
[*1 41.68 satt602 0 41 1.52[i-10 NA

E 56.27 satt369 12 29 79313-03 2.87E-03
E 64.18 sat__381 24 17 2743-01 68413-02
E 67.92 sat1553 0 41 1.521i-10 NA

F 2.23 satt325 10 31 1.0415-03 58313-04
F 16 satt252 6 35 5.931i-06 4.75 E-06
F 26 sat_240 8 33 2.48F.-05 1.69E-05
F 56 satt663 5 36 1.29E-06 1.26E-06
F 75 sat_ 120 6 35 1.29E-06 1.26E-06
F 98 salt-190 5 36 1.2913-06 1.2615-06
F 130.46 sat_090 6 3 5 5 .93 F.-06 4.7513-06
0 0 satt 163 I9 22 63913-01 1.3413-01
0 21.89 satt235 5 36 1293-06 1.26E-06
0 48.54 satt340 8 33 5.93E-06 4.7513-06
0 59.33 satt594 6 35 5.93 E-06 4.75E-06
0 76.77 satt288 12 29 7.‘)31i-()3 28713-03
0 96.57 satt191 19 22 6393-01 1.34E-01
l 27.98 satt367 24 17 2.7413-01 6.8415-02
l 46.22 satt354 19 22 6.3915-01 1.3415-01
l 75 sat_l70 19 22 6.391‘7-01 1.34F.-01
l 82.78 satt292 1 1 30 3.00303 1.3215-03
1 98 sat_155 18 23 4353-01 1.0313-01
l 1 12.7 satt440 19 22 6.391i-01 1.34FL-01
J 15 satt287 14 27 2.4811-05 1.6915-05
J 25.51 sat1285 12 29 3.2813-04 1.9913-04
J 39 salt-406 3 38 4603-08 5.74177-08
J 52.71 Sat_366 9 32 3.2813-04 1.99F7-04

 

130

Table A8 (Cont‘d)

 

 

1.0 group Position SSR marker m total MLS P-value q-value
J 72 satt547 13 28 1.91 E-02 6.511i-03
R 30.28 salt 1 ()2 18 23 4.3513-01 10313-01
k 45 .74 satt 1 67 16 25 1.60F.-01 4.4815-02
k 54.97 satt559 19 22 6.39E-01 1.3415-01
k 87 sat_243 10 31 1043-03 5831-7-04
1. 0 salt-195 0 41 1 .5213-10 NA
1. 19.93 sat1238 l 1 30 3.00303 1.321i-03
1. 34.54 satt313 19 22 6.39L-‘-01 1.341i-01
l. 56 satt156 17 24 2.741-7-01 6.841.7-02
1. 87.42 sat 286 14 27 4.2315-02 1.34Fl-02
l. 107 satt3 73 17 24 2.74E-01 6.843-02
M 5 satt636 20 21 8763-01 1.7213-01
M 19 satt150 1 1 30 30013-133 1.32li-03
M 38.94 satt435 6 35 1293-06 1263-06
M 66 sat_226 6 35 5.93F.-06 4.753-06
M 80.9 satt728 17 24 2.74 E-0 1 6.841-7-02
M 95.45 sat1551 14 27 4231-7-02 1.3412-02
M 1 12.08 Satt210 13 28 1.91F.-02 6.5113-03
N 27 satt159 12 29 7.93 F.-03 2.8715-03
N 58 sat__0.33 0 41 1523-10 NA
N 77 sat3304 15 26 8583-02 2573-02
N 103.34 sat_125 12 29 7.93F.-03 28713-03
O 14.17 satt500 17 24 2.74E-01 6.84E-02
() 59.49 satt241 1 1 30 3.00E-03 1.3215-03
O 74.05 sat_242 12 29 7.9313-03 2.141i-10
() 86.86 satt123 25 16 16013-01 4.4813-02
() 93.37 satt331 22 19 6.393-01 1.343-01
() 118.14 satt153 17 24 2.74F.-01 68413-02
0 129.8 sat~190 1 1 30 30012-113 1.3213-03

 

131

 

fins—a >e 001m A0828 305 03338030 1. 0 man A A: A<AZ>A<A

 

 

:33 Ah >8- >8- >3- xx AVA mAvchAx-

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AEDAN A.. Aobx A A A A A A A A A.A A A
mmsAmA 1 NPAO A A A A A A A A A A A
.321qu 1 NAAA A A A A A A A A A A A
2:53 A” A0. A .\. A A A A A A A A A A A
mmzAAA 1 AAA A A A A A A.A A A A A A A
REAP». A.. A A c. A c A A A A A A A A A A A
SALSA -A AAbc A A A A A A A A A A A
8:. AA 0 AAAAA A A A A A A A A A A A
mmzACA C ANAAA A A A A A A A A A A A
AEAAAAA A AA. A c A A A A A A A A A A A
.4333. A Ac.oA A A A A A A A A A A A
AB! A AA A A A .AA A A A A A A A A A A A
mmzAmc A A A .c: A A A A A A A A A A A
$338 A ANNA A A A A A A A A A A A
AEAAAAA A AA: A A A A A A A A A A A
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$2.5. A A A AAbx A A A A A A A A A.A A A
maria: A A AA .3 A A A A A A A A A A A

 

132

Table A10 Aphid resistance scoring for BCIF45 population in CH and field

 

 

1.1 ncs G/3 w (1/4w REP1w3 R13P2w3 REP 1 W4 REP2w4
1 0.9 1.6 0.8 0.7 0.8 0.7
2 0.9 1.3 1.5 0.5 1.2 0.5
3 0.9 0.9 0.8 0.8 1.1 0.7
4 1.8 1 1.2 0.8 1.2 0.5
5 1.8 1.8 1.5 1.2 0.9 0.9
6 1.3 1.6 1.7 1.8 1.4 1.8
7 1.3 2.1 0.7 0.5 0.5 0.5
8 1.4 1.3 1.4 1.2 0.9 1.2
9 2.2 1.3 0.8 0.8 0.6 1
10 2.2 1.8 1.9 2.3 1.7 1.6
11 1.4 2.4 1.3 1.5 0.7 0.8
12 1.7 1.7 1.1 1.8 1.2 1.3
13 1.6 1.8 1.8 1.5 1.3 1.4
14 1.1 1.3 0.5 0.6 0.5 0.5
15 1.2 1.2 0.6 0.9 0.5 0.5
16 3.1 3.2 2.8 3.3 2.2 3.2
17 2 1 2.2 1.3 0.6 (1.6 0.6
18 1.5 1.1 1.5 1.2 0.8 0.5
19 1.2 0.9 1 1.5 0.5 0.8
20 1.8 2.3 1.8 1.5 1.4 1.5
21 1.8 2.8 1.2 2.1 1 1.4
22 2.1 2.8 0.8 2.8 0.8 1.7
23 1.4 2.4 1.1 1.5 1.3 0.8
24 1.5 2.8 2.5 3 1.7 2.2
25 2.4 3.1 1 2.3 1.3 3.5
26 2 6 3.5 O 7 1 7 0.5 1.4
27 2.5 3.5 2 3.1 1.7 1.8
28 2.7 3.1 2.9 2.5 2.4 1.5
29 2 4 2.3 1.4 1.3 0.8 1.1
30 2.1 3.7 2.7 2.3 2.8 2.4
31 1.4 2 0.7 0.7 0.8 0.8
32 2 1.4 1 5 1.6 0.9
33 1.6 2.2 0.5 1 0.5 0.5
34 2.9 3.5 3.2 3.5 3.3 3.8
35 2.9 3.7 3.2 3.3 3.3 3.7
36 3.3 3.6 3.3 3.3 3.3 3.8
37 3.4 3.6 3.4 3 2 3.1 3.3
38 2.5 3.3 3 2 1.4 2
39 1.6 1.9 1.6 1.4 0.9 1.2
40 2.3 3.6 3 1.6 1.3 1.6
41 2.5 1.8 1.5 1.3 0.9 1

 

133

Table A10 (Cont‘d)

 

 

Lines (1/3 w (}./4\\ R1i1’1w3 REP2W3 REP 1 W4 REP2w4
42 1.3 2 0.6 0.5 0.6 0.6
43 3.3 3.5 2.2 3.3 2.7 3.3
44 2.3 3.3 3 3.3 3.6 3.5
45 1.6 1.5 1.9 1.8 0.9 1.9
46 1.4 2.3 1.5 1 0.5 0.6
47 2.6 3.4 3.5 3.4 3.7 3.6
48 2.8 3.4 3.2 3.5 3.2 3.4
49 2.4 3.2 3.4 3.3 3.1 3.3
50 1.7 1.7 1.5 2.8 1.1 1.6
51 3.5 3.5 3.2 3.3 2.2 3.3
52 3.4 3.5 3.5 2.6 3.3 2.3
53 1.6 2.4 2 1.4 1.2 1
54 1.6 2.1 3.3 1.4 2.5 0.7
55 1.1 1.4 1.1 1.8 0.8 1.1
56 3.3 3.5 2.8 2.9 3.8 3.2
57 3.3 3.5 3.5 3 3.4 3
58 1.1 2.4 1.6 1.1 0.6 0.5
59 1.4 2 1.9 0.9 0.8 0.6
60 1.5 1.3 2.1 0.8 1.1 1.1
61 3.4 4 3.3 3.3 2.5 3.3
62 1.7 2.5 1.3 3.1 0.5 1.1
63 3.1 3.5 2.7 3.3 3.3 3.4
64 2.6 3.4 2.8 3.4 2.8 3.4
65 2 3.3 2.7 3.3 2.7 3.4
66 1.4 1.9 0.6 0.5 0.6 0.5
67 2.1 2.6 0.7 2.4 0.7 1.4
68 2 2.7 0.9 0.7 0.8 0.7
69 2.4 3.5 3.5 2.6 3.6 2.4
70 1.6 1.8 1.1 1.5 0.9 1.3
71 1.7 2.4 1.4 0.9 0.9 2.8
73 3.1 4 3.5 2.1 3.5 2.2
74 1.7 2.5 1.4 1.5 1.7 1.8
74 1.7 2.5 1.4 1.5 1.7 1.8
76 1.4 2.1 1 1.2 0.5 0.8
77 3.3 3.5 1.8 2.3 3 2.5
78 2.3 3 2.5 2.8 3.5 2.8
79 2.3 3.5 3.1 2 3.7 2.8
80 3.2 3.5 2.9 1.8 3.6 2.5
81 1.3 2.1 1.3 0.7 0.8 0.6
82 1.8 3.3 2.2 1.8 1.5 1.8
83 1.3 2.9 0.5 0.6 0.5 0.5

 

134

Table A10 (Cont'd)

 

 

Lines (1/3 W (1/4W REP 1 W3 R1iP2W3 REP 1 W4 R11P2W4
84 3 3.5 2.6 2.1 3.2 3.1
85 1.6 2.4 1.1 1.1 0.5 1.2
86 2.2 3 1.4 1.1 0.8 1
87 2 2.7 1.3 0.8 0.5 0.7
88 3.2 3.5 3 3.3 3.3 3.3
89 1.4 2.6 1 O 7 0.5 0.7
90 2.3 3.5 3 3.4 4 3.1
91 2.5 3.5 3 5 3 4 3.8 2.9
92 1.1 2.2 2.1 0.7 1.3 0.7
93 1.6 2.3 1.3 1.8 0.9 1
94 1.9 2.6 1 4 1.9 0.9 1
95 (1.9 1.7 1.2 1.6 0.7 1.6
96 0.8 1.8 1 1.4 0.5 0.6
97 2.7 3.5 3.3 3 3.3 3.2
98 1.2 2.8 17 1.5 2.1 1.1
99 2.9 3.4 2.9 3.3 2.4 3.2
100 2.1 3.4 2.5 2.8 2.7 3.3
101 1.1 1.8 0.6 0.8 0.9 0.7
102 3.4 3.6 3.3 2.9 2.5 2.7
103 1.9 3 1.6 2.3 1.1 1.3
104 3.2 3.5 3.1 2.6 3.4 2.7
105 3 3.4 3.4 2.4 3.4 3.1
106 1.1 3.4 1.6 0.9 1.1 0.9
107 2.8 3.3 2.5 1.9 2.6 1
108 1 3.3 2.2 1.5 1.7 1.2
109 1.8 3 1.1 0.6 0.6 0.7
110 2.2 2.7 1.6 1.2 1.1 1.1
111 2.4 3 3 2.4 3.5 3
112 2.1 2.4 3.2 2.3 2.4 2.5
113 0.5 2.1 1.3 0.8 0.8 0.8
114 2.9 3.5 2.9 2.3 3.6 2.5
115 3.3 3.5 2.8 2.5 3.6 2.6
116 0.5 1.3 0.6 0.5 0.5 0.5
117 0.5 1.7 0.8 0.5 0.5 0.5
118 2.3 2.8 3.2 2.8 3.1 3.1
119 1.4 1.5 0.7 1.6 0.5 1
120 1.6 1.8 2 1.5 O 9 1 1
121 1.3 2.7 1.6 1.3 0.6 1.1
122 2.8 3.1 2.5 1.9 3.3 2.6
123 0.8 2 0.9 0.5 0.7 0.8
124 1 9 2 2 1.6 0 7 0 6 0 8

 

135

 

Table A10 (Cont’d)

 

 

Lines (1/3 w (3/4 W Rli P 1 W3 R F. P2w3 RE P 1 W4 R1iP2w4
125 1.8 2.3 2 1.7 1.8 1.8
126 1.4 2.3 1.6 1.4 1.4 0.8
128 1.9 3 2.4 2.9 2.3 2.2
129 0.6 1.6 1.3 1 1 0.6
130 1.7 3 2.1 1.5 1.6 1.2
131 1.3 2.5 2.3 3.3 2 3.5
132 3.5 3.5 3.3 3.1 3.4 3.2
133 3.5 3.5 3.5 3.5 3.6 3.5
134 3.3 3.5 3.2 3 3 3.3
135 2.9 3.5 3.2 3.1 2.2 2.9
136 3.5 3.5 3.3 3.5 3.2 3.4
137 1.6 2.5 0.9 0.6 0.6 0.5
138 3.5 3.5 3.4 3.1 3.5 3.3
139 3.2 3.5 2 2.8 2.2 3.1
140 2 2.3 1 1.4 1
141 3.4 3.5 3.1 2.9 3.5 2.8
142 1.9 2.3 1.2 0.5 0.8 0.6
143 2.5 3.5 1.4 1 0.9 1
144 1.3 1.8 1.9 0.9 0.8 1.1
145 1 1.7 0.9 0.7 0.6 0.6
146 1.4 1.8 0.7 0.5 0.5 0.5
147 3.5 3.5 3.2 2.5 3 2.8
148 1.1 2.7 1.5 0.8 1.3 0.7
149 0.6 1.4 0.6 (1.6 0.5 0.5
150 1.1 1.3 0.7 0.5 0.5 0.5
151 2.1 3 1.4 0.5 0.8 0.5
152 3.4 3.5 3.3 2.7 3.8 2.8
153 2.3 3.4 2 1.9 0.9 1.1
154 1 1.8 1.4 1.4 0.6 0.9
155 3.7 3.5 3.2 3.4 3 3.3
156 3.4 3.7 2.9 2.6 2.2 3.5
157 1.1 2.2 0.5 0.6 0.7 0.8
158 3.4 3.5 3 2.8 3 3.3
159 1.5 2 2.8 1.3 2.5 1.5
160 3.7 3.8 2.6 2.3 3.1 2.3
161 1.6 2 1 0.8 1.8 0.5
162 1.4 1 3 0.6 0.5 0.5 0 5
163 3.4 3.5 2 6 2.9 18 3 3
164 1.5 1.5 1 6 1.5 1 1
165 3.4 3 5 3 2 3.1 2 6 2
166 3.4 3.5 3.1 2.8 3.7 ._

 

136

 

Table A10 (Cont‘d)

 

 

Lines 0/ 3 W (1/4W RIP 1 W3 RliP2W3 R15P1W4 REP2W4
167 1 2.2 1 1.4 2 0.5
168 2.5 3.5 3.1 2.1 3 1.8
169 1.7 1.7 0.8 0.6 0.7 0.7
170 1.5 2 | .3 0.6 1 0.6
171 1.7 2.5 1.8 1.1 1.6 1.1
172 1.9 3 2.6 3 3.3 2.7
173 1.9 3 2.3 3.2 2.6 2.7
174 3.5 3.5 3.2 2.3 3.2 2.3
175 3 3 0.5 0.7 1 1.5
176 2 3.3 2.8 1.7 3.2 2.4
177 0.9 1.5 1.6 1 1.3 1.1
178 1.1 1.8 (1.8 0.5 0.5 0.5
179 3.2 3.5 2.8 3.2 3.3 3.2
180 2.6 2.6 2.8 3 1.6 1.7
181 0.7 1.4 0.9 1.1 0.5 0.5
182 2.1 3.5 3.3 3 3 2.9
183 2 3.5 3.4 2.8 3.3 2.3
184 3.5 3.5 3.2 2.6 5 2.4
185 3 5 3.5 1.8 2.9 2.3
186 2.4 3 2.8 2.2 3.5 2.4
187 2 3 2.9 2.8 3.1 2.6
188 1.5 2.1 1.4 1.4 0.7 1.1
189 0.7 1.1 0.8 0.7 0.5 0.6

 

137

 

Table All Aphid resistance scoring for 070063 population in the field

 

R11. N0. 1 2 3 4 5 6 7 8

 

1 3 5 3 5 3.5 3 5 3 5

2 3 5 3 5 3.5 3 5

3 0 5 1 1.5 1 1 5 1

4 3.5 1 5

5 - 3 5 3.5 3 5

6 -

7 3.5 3.5

8 3.5

9 1 1.5 2 1.5 1.5 1 5

10 1.5 1.5 3.5 3.5

11 3 3.5

12 0.5 l

13 3 2 1 5

14 3.5 2.5 3.5 3.5 3.5

15 3.5 3.5 3.5 3.5

16 3.5 3.5 3.5 3 2 1.5
17 0.5 0.5 0.5 0.5 0.5 ' 0 5
18 1.5 0.5 1 0.5 0.5 5

19 3.5 3.5

20 3

21 3 5 3.5 3 5

22 3 3 3

-3 ()5 0.5 0 5

24 3 '

25 '

26 2. 2

27 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
28 1.5 1 2 1 5 0.5 2

29 1 0.5 1 0.5 1 1 0 5
30 3.5 3.5 3.5 3.5 3.5 3.5 3.5
31 2 2 1 1.5 1 1 5 2
32 0.5 0.5 0.5 0.5 0.5

33 2 3.5 3.5 3.5

34 1.5 1.5 2 1 5 2 1 1 5 1 5
35 3.5 3.5 3.5 3.5 3.5 3.5 3.5
36 3.5 3.5 3.5

37 2 2 2

38 3 5 3 5 3.5 3 5 3 5 3 5

39 1.5 2 1 1.5 2 1.5 1.5
40 1 1.5 1 1.5 1 1 1
41 3.5 3.5 3.5 3.5 3.5 3 5

 

 

Table All (Cont‘d)

 

 

RILNO. 1 2 3 4 5 6 7 8
42 3.5 3.5 3.5 3.5 3.5

43 3.5 3.5 3.5 3.5 3.5

44 1 1.5 1.5 1 1.5 1.5

45 3.5 3.5 3.5 3.5 3.5 3.5 3.5
46 2 1.5 2 1.5 1.5

47 1 0.5 1.5 0.5 0.5

48 0.5 (1.5 0.5 0.5 0.5 0.5

49 0.5 0.5 0.5 0.5 0.5 .5

50 0.5 0.5 0.5 0.5 0.5 0.5 1
51 3.5 3.5 3.5

52 3.5 3.5 3.5 3.5 3.5 3.5 3.5
53 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5
54 0.5

55 3.5 3.5 3.5 '

56 0.5 1 l ' 1.5 ()5 0.5 15
57 3 3 3.5 3.5 3

58 2 1.5 1.5 2 1.5 1

59 0.5 0.5 0.5 0.5 0.5 0.5 ()5
60 0.5 0.5 0.5 0.5 0.5 0.5 15
61 3.5 3.5 3.5 3.5 3.5

62 2 1.5 1.5 2 1.5 1.5

63 2 2 1.5 2 2

64 3.5 3.5 3.5 3.5

65 3.5 3.5 .5 3.5 3.5 3.5 .5
66 2.5 2 2.5 1.5 2 2.5
67 3.5 3.5 3.5

68 2.5 2 2.5 2 2.5

69 3.5 3.5 3.5 3.5 3.5 3.5 35
70 3.5 3.5 3.5

71 3.5 3.5 3.5

72 2 3 2 3 2 2 2
73 3.5 3.5 3.5 3.5 3.5

74 1.5 1.5 2 2 1 1.5

75 3 3 2 3. 3.5

76 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
77 3.5

78 3.5 3.5 3.5 3.5

79 0.5 0.5 0.5 0.5

80 0.5 0.5 0.5

81 2 3.5

82 0.5 0.5 0.5 0.5

 

139

 

Table All (Cont‘d)

 

 

R11. N0. 1 2 3 4 5 6
83 0.5 1
84 3.5 3.5 3.5
85 1 1
86 3.5
87 3.5 3.5 3.5
88 3.5 3.5
89 0.5 0.5 0.5 0.5 0.5 0.5
90 2 0.5 0.5 0.5
91 3.5 3. 3.5
92 0.5 0.5 0.5
93 3.5 3.5 3.5
94 3.5 3.5 1.5 1.5 2
95 3.5 3.5 3.5 3.5 3.5
96 3.5 3.5 3.5 3.5
97 3.5 3.5 3.5 3.5
98 3.5 3.5 3.5 3.5
99 2 2.5 2 2.5
100 2 1.5 2 1.5
101 3.5 3.5 3.5 3.5 3.5
102 0.5 0.5
103 1 0.5 0.5
104 3.5 3.5 3.5
105 3.5 3.5 3.5
106 3.5 3.5 3.5 3.5
107 0.5 0.5 0.5 0.5 0.5
108 3.5 3.5
109 0.5 0.5 0.5
110 1.5 1.5 1.5 2 2
111 3. 3.5 3.5 3.5 3.5
112 3.5 3.5 3.5 3.5
113 2 2.5 2 2.5 2.5
114 2 2
115 3.5 3.5 3.5 3.5
116 0.5 0.5 0.5 0.5
117 0.5 0.5 0.5 0.5
118 3.5

 

140

   

ml" 16111111111111 1111111111111.111111111111111“
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