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2008

LIBRARY
Michigan State
University

This is to certify that the
dissertation entitled

INHERITANCE OF APHID RESISTANCE IN PI 5675413 AND Pl
5675988, IDENTIFICATION OF APHID RESISTANCE QTL IN PI
5675988, AND A NEW APHID BIOTYPE IN MICHIGAN

presented by

Clarice Mensah

has been accepted towards fulﬁllment
of the requirements for the

Ph.D. degree in Plant Breeding and Genetics

 

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5/08 K:lProj/Acc&Pres/CIRC/DateDueindd

INHERITANCE OF APHID RESISTANCE IN PI 567541B AND PI 567598B,
IDENTIFICATION OF APHID RESISTANCE QTL IN PI 567598B, AND A NEW
APHID BIOTYPE IN MICHIGAN

By

Clarice Mensah

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Plant Breeding and Genetics

Department of Crop and Soil Sciences

2008

ABSTRACT

INHERITANCE OF APHID RESISTANCE IN PI 567541B AND PI 567598B,
IDENTIFICATION OF APHID RESISTANCE QTL IN PI 567598B, AND A NEW
APHID BIOTYPE IN MICHIGAN ‘

By

Clarice Mensah

The soybean aphid (Aphis glycines Matsumura) has become a very important pest
of soybean [Glycine max (L.) Merr.] in North America since it was ﬁrst reported in 2000.
In 2005, four new plant introductions (PI) with aphid resistance: PI 567543C, PI
567597C, PI 5675413 and PI 5675983 were identiﬁed. Since then, other sources of
aphid resistance have been identiﬁed, but only in two sources has genetic and molecular
characterization been conducted. The objectives of this research were to: 1) determine the
inheritance of antibiosis resistance in P1 567541B and PI 5675983, 2) determine if a
different soybean aphid biotype exist in Michigan and 3) identify and map quantitative
trait loci (QTL) underlying aphid resistance in PI 5675983. Field studies were conducted
to determine the inheritance of antibiosis resistance in PI 567541B and PI 567598B. The
two resistant PIs were crossed with one or two susceptible soybean lines and the F1 and
F2 plants and F23 families were evaluated for aphid resistance. All F1 plants were found
to be susceptible to soybean aphids. The plants in seven F2 populations segregated in a 15
susceptible to 1 resistant ratio, which is the expected ratio for a trait controlled by two

recessive genes. The segregation data shows that two recessive genes are involved in the

resistance in P1 567541B and PI 5675983. This information will be useful for breeders to
design efﬁcient breeding schemes for developing soybean cultivars with resistance to
aphids. To achieve our second objective, 188 F2 individuals ﬁom a cross between Titan
and PI 5675983 were genotyped with 109 polymorphic simple sequence repeats (SSR)
markers. Both single marker analysis (SMA) and composite interval mapping (CIM)
methods were used to determine locations of QTLs. SMA revealed 24 markers associated
with aphid resistance. QTL mapping by CIM identiﬁed a putative QTL on LG J. The
SSR markers ﬂanking these resistance genes can be used in marker-assisted selection for
aphid resistance in soybean breeding programs. With the testing of several soybean aphid
resistant genotypes, it was expected that resistant biotypes would evolve. In a ﬁeld study
in 2006, Dowling, a resistant check was found to be susceptible to the soybean aphid. A
greenhouse study was conducted to compare the effect of the aphids which overcame the
resistance in Dowling and aphids collected in the ﬁeld in 2006. Dowling was found to be
susceptible to both aphid colonies. In a follow up greenhouse study Dowling was found
to be resistant to aphid colonies which had been raised in a growth chamber and
greenhouse since 2002.These two studies indicate that, there is a difference in the feeding
behavior on Dowling by aphids collected in 2002 and 2006 and suggests that a new

soybean biotype may have evolved in Michigan.

Cowright by
CLARICE MENSAH
2008

Dedicated to the three Adovor’s in my life: Doe, Volta and Qwekqem

ACKNOWLEDGEMENTS

I would like to thank Dr. Dechun Wang, my major professor for his patience, guidance
and ﬁnancial support throughout my stay at MSU, and also for giving me the opportunity-
to work on such a wonderful project. To the members of guidance committee Drs. David
Douches, Christiana DiFonzo and Amy Iezzoni, thank you so much for all the
suggestions and the time you took to help me in my research and program of study. I
would like to thank Dr. James D. Kelly for critically reviewing my dissertation and for
agreeing to substitute on my committee. To Dr. Russell Freed, for all his help in the area
of career development and rich experience he shared during my time here, I am very
grateful. To all the friends I made, thank you for your support. This research was
supported by Project GREEEN (Generating Research and Extension to meet Economic
and Environmental Needs, the State of Michigan's plant agriculture initiative) and the
Michigan Soybean Promotion Committee. To the Graduate School and the Elmer
Rossman Endowment who co-funded my ﬁnal semester, I am indeed grateful. And to my
family who have always been there for me, Mama, Ama and Awo thanks for your love

and support.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
CHAPTER 1 ....................................................................................................................... 1
INTRODUCTION ...................................................................................................... 1
REFERENCES ......................................................................................................... 10
CHAPTER 2 ..................................................................................................................... 14
INTRODUCTION .................................................................................................... 1 5
MATERIALS AND METHODS .............................................................................. 17
RESULTS AND DISCUSSION ............................................................................... 18
REFERENCES ......................................................................................................... 26
CHAPTER 3 ..................................................................................................................... 29
ABSTRACT .............................................................................................................. 29
INTRODUCTION .................................................................................................... 30
MATERIALS AND METHODS .............................................................................. 33
RESULTS ................................................................................................................. 36
DISCUSSION ........................................................................................................... 37
REFERENCES ......................................................................................................... 40
CHAPTER 4 ..................................................................................................................... 45
ABSTRACT .............................................................................................................. 45
INTRODUCTION .................................................................................................... 46
MATERIALS AND METHODS .............................................................................. 47
RESULTS ................................................................................................................. 51
DISCUSSION ........................................................................................................... 64
REFERENCES ......................................................................................................... 67
APPENDIX ....................................................................................................................... 7O

vii

LIST OF TABLES

Table 2.1: Segregation of aphid resistance in F2 populations derived from
susceptible x resistant crosses ...................................................... 23

Table 2.2: F; and parental lines classiﬁed as resistant to soybean aphid ............. 24

Table 3.1: The average Damage Index (DI) based on three replications in Study] Fall
2006 4 weeks after inoculation in a No-choice test in the greenhouse. . . . . ...40

Table 3.2: Damage Index (DI) based on results obtained in Study 2-resistant sources
tested in the greenhouse, winter 2006 at 3 and 4 weeks after inoculation using
aphids from 2002. ....................................................................... 41

Table 4.1: Markers signiﬁcantly associated with soybean aphid resistance in
PI 567598B in single marker analysis in 2005, 2006 and 2007 at three weeks after
inoculation ................................................................................. 59
Table 4.2: Markers signiﬁcantly associated with soybean aphid resistance in

P1 567598B in single marker analysis in 2005, 2006 and 2007 at three weeks after
inoculation. ................................................................................ 60

Table 5.1: Visual rating scale used to establish the Damage Index (DI) of a plant ...... 71

Table 5.2: Phenotypic data for 188 individuals of mapping population [F2 (2005),
F2;3(2006) and F2;4(2007)] collected three and four weeks after inoculation ....... 72

Table 5.3: Information about all polymorphic simple sequence repeat (SSR) markers
ﬁ'om F2 population of Titan and PI 567598B ......................................... 85

viii

LIST OF FIGURES

Figure 1.1: Distribution of damage rating scores in F2 populations: a) 040129-1, b)
040129-2, c) 040130-1, d) 040130-2, e) 030104-3, and t) 030104-8 ............. 25

Figure 4.1: The damage rating distributions of: a) the F2 population of the cross between
Titan and PI 5675983 b-g) the 188 selected mapping population individuals for
2005 and 2006 three and four weeks after inoculation ............................ 53

Figure 4.2 A: Putative QTLs associated with Aphid resistance on linkage group J from
2006 week3 and week4 data. The LCD threshold was set at 3.0 ................ 61

Figure 4.23: Putative QTLs associated with Aphid resistance on linkage group J from
2007 week3 and week4 data The LCD threshold was set at 3.0.. .......62

Figure 4.2C: Putative QTLs associated with Aphid resistance on linkage group C1 from
2006 week3 and week4 data. The LCD threshold was set at 3.0. ................63

Figure 5.1: Linkage map of 188 F2 lines from cross Titan and PI 5675983 constructed
using JoinMap 3.0 with a 0D grouping threshold 3.0. The linkage groups were
named according to Song et a1. (2004) and the map distances between the
markers are given in cM (centiMorgans) ............................................. 77

ix

CHAPTER 1

INTRODUCTION

Soybean

Soybean, Glycine max (L.) Merr., (2n=2x=40) is a legume in the Fabaceae
family. The genus Glycine is divided into two questionably distinct subgenera: Glycine
and Soja. The ﬁrst consists of six or seven perennial species primarily ﬁom Australia.
The second consists of three annual species from Asia: Glycine max, Glycine soja, and
Glycine gracilis (Palmer et al., 1996). Soybean combines in one crop both the dominant
world supply of edible vegetable oil, and the dominant supply of high-protein feed
supplements for livestock (Rao, 2002). Other ﬁactions and derivatives of the seed have
substantial economic importance in a wide range of industrial, food, pharmaceutical, and
agricultural products (Johnson, 1987). As a source of protein, soybean is often less
expensive compared to animal protein on a cost per kilogram basis (Hymowitz and
Newell 1981).

The worlds leading producer of soybeans is the US. followed by Brazil and
Argentina (FAO, 2007). In 2006, these three countries produced 82% of the 236 million
tonnes of soybean produced worldwide. In Michigan, soybean is the number two crop, in

terms of acreage, 1.75 million acres was planted with in 2007 (Soy Stats 2007).

The Soybean Aphid
The soybean aphid (Aphis glycines Matsumura) belongs to the order Hemiptera

and family Aphididae. It is a small, light yellow or yellowish green aphid with two

distinct black comicles and a pale colored tail projection. Adult soybean aphids are about
1/ 16th of an inch (2 mm) long and may be winged or Wingless. Immature aphids look like
a miniature version of the Wingless adults and winged ones have a black head and thorax.
Soybean aphids have quickly established themselves as one of the most damaging

pests of soybean (Sun et al., 2000). Originally from Asia, aphids were ﬁrst detected in
2000 in the upper Midwest of the US; they have since spread to several states and some
Canadian provinces (Chen et a1. 2000). The soybean aphid is the only aphid in North
America that develops large colonies on soybeans.

Soybean aphids display a complex life cycle with alternation of sexual and
asexual generations and host plants (Ragsdale et a1. 2004). In North America, various
buckthom (Rhamnus carthartica L.,thmnus alnifolia L’ Hertier ) species are used as
primary hosts (V oegtlin et al., 2004). Soybeans are the secondary hosts of soybean
aphids. The observed life history of the aphid in North America is similar to that
observed in China and Japan, with the exception of the primary hosts, Rhamnus davurica
Pallus and Rhamnusjaponica Maxim which it uses as an over-wintering host (T akahashi
et al., 1993). In spring the Wingless mothers hatch from an egg and begin to produce
colonies of Wingless females, these then produce a third generation of aphids that are
winged emigrants which ﬂy in search of soybean, the summer host. All asexual
generations are entirely female and are clones of the mother. Winged females occur in the
_ fall as the temperature decreases and plant conditions deteriorate. They then migrate to
the buckthom where they produce Wingless females. At this time, winged males occur in
the soybean ﬁeld and migrate to the buckthom where they mate with the Wingless

females, which lay over-wintering eggs. The life cycle repeats the next spring.

Numbers of aphid generations range ﬁom 10 to 22 per year (Li et a1. 2000). In
China,Wu et a1. (2004) recorded a total of 18 generations per year with 15 of those
generations occurring on soybean. Wingless and winged female aphids produce an
average of 58 and 38 nymphs, respectively, at 26°C (Li et a1. 2000). Winged aphids play
a vital role in expanding the range of dispersal within and among ﬁelds and migration
between alternative host plants. Crowding of Wingless adults and poor host quality induce

winged aphid production (Lu and Chen 1993).

Symptoms and Damage on Soybeans caused by the Soybean Aphid

Plant damage occurs when large numbers of aphids remove signiﬁcant amounts
of water and nutrients as they feed on leaves and stems, causing leaves to wilt, curl,
yellow, and even drop ( Wu et al., 2004). Other symptoms include plant stunting, poor
pod ﬁll, reduced pod and sad counts, smaller seed sizes, and nutrient deﬁciencies,
resulting in overall yield and quality reduction (DiFonzo and Hines, 2002). Signiﬁcant
yield loss (8-25%) occurs when aphid densities peak at ﬂower initiation. Honeydew, a
sticky substance excreted by soybean aphids onto the leaves leads to the development of
sooty mold, which affects photosynthesis and results in yield and seed quality loss (Chen
and Yu, 1988). '

During the feeding process, soybean aphids are capable of transmitting viruses
including soybean mosaic, alfalfa mosaic, mungbean mosaic, peanut mosaic, and bean
yellow mosaic virus (CAB International, 2001). These viruses commonly occur together
and form a disease complex which leads to plant stunting, leaf distortion and mottling,

reduced pod numbers and seed discoloration (Glogoza, 2002). Soybean Mosaic Virus

(SMV) is transmitted in a nonpersistent manner and causes high yield loss. It is spread
mainly by infected aphids feeding on healthy plants. Epidemics of SMV are dependent
not only on the initial virus source but also on the abundance and development of aphid
vectors, especially winged aphids. Occurrence of winged aphids in soybean ﬁelds has
been found to be closely associated with the incidence of SMV (Quimio and Calilung,

1993).

Host Plant Resistance Modalities

Resistant varieties of crop plants have played an important part in controlling
many insect, mite and nematode pests. Fewer applications of insecticides are usually
needed to control insect pests when resistant varieties are used (Hoffmann et al., 1993).
The importance of developing crop plants that are resistant to major insect pest has
created the need for examination of the mechanism involved in resistance. The widely
recognized classiﬁcation proposed by Painter (1951), provides an acceptable illustration
of the possible basis of resistance. The three mechanisms that inﬂuence the ability of a
plant to grow productively in the presence of an insect are nonpreference, antibiosis, and
tolerance (Painter, 1951). One or more of these mechanisms can be in operation in a plant
considered as insect resistant.

Nonpreference refers to behavioral responses of insects to a plant. Kogan and
Ortrnan (1978) suggested the term antixenosis to describe the plant properties responsible
for this response. The plant characters that inﬂuence nonpreference include color, light
reﬂection, type of pubescence, leaf angle, odor and taste. For example; the yellow-green

cultivars of peas are less desirable to the pea aphid than are the blue-green ones. The

cabbage aphid is attracted most to plants withleaves that reﬂect low intensities of light
(F ehr, 1999). Antibiosis refers to plant characteristics and is considered to be the only
true form of resistance. It is the type of resistance in which a host plant has a detrimental
effect on the physiology and life history of an insect pest. These adverse effects may
include inhibited growth, death, and prolonged time to matrnity (Painter, 1951).
According to Smith (1989), if a plant deters feeding by an insect, the mechanism
of resistance may be either antixenosis or antibiosis. The critical question that separates
these two types of resistance is whether the insect is completely prevented from feeding,
thus starving to death (antibiosis) or would eventually feed on that plant when given no
choice (antixenosis). Often there is an overlap between the antibiosis and antixenosis
types of resistance. Complex types of resistance and different combinations of resistance
are expected to give more complete and durable control than one simple type. For
example, a resistant variety that expresses antibiosis and tolerance to an insect will give
excellent and durable control. On the other hand, such a combination might be difﬁcult to

select for and manage in a breeding program.

Soybean Aphid Resistance in North America

At the time the aphid arrived in 2000, no known sources of resistance were
available in soybean. In 2004, the ﬁrst aphid resistance sources were reported. After
screening 1,542 soybean accessions, Dowling and Jackson, two late maturity ancestral
cultivars were found to have antibiosis resistance to the aphids (Hill et. al., 2004). The .

next report of resistance was from our breeding program here at MSU in 2005. After

evaluating 2,147 soybean germplasm in choice tests we identiﬁed four accessions, PI
5675983, PI 5675413, PI 567543C, and PI 567597C, with resistance to the soybean
aphid (Mensah, et al., 2005). A no-choice test revealed that PI 5675983 and PI 5675413
possessed antibiosis resistance (Mensah et al., 2005). In 2006, Diaz-Montana et al.
compared the reproduction of soybean aphids on 240 soybean entries and found eleven
entries with fewer nymphs than the susceptible checks. In a follow-up experiment, they
identiﬁed K1639 and Pioneer 95397 as showing a strong antibiosis effect on soybean
aphids. Two more new sources of aphid resistance are PI 230977 and G93-9223 (PI
595099) with antibiosis and antixenosis resistance, respectively (Hesler et al., 2007).The
latest report of aphid resistance is of, three PIs (243540, 5673013 and_567324) identiﬁed
by Mian et al., (2008) after screening nearly 200 soybean genotypes in a greenhouse no-
choice test. PI 243540 was found to possess antibiosis resistance and PI 5673013 and PI

567324 possessed mainly antixenosis resistance.

Inheritance of Aphid Resistance

Resistance to insects is governed by genetic mechanisms like other plant traits
(Auclair, 1989). Understanding the inheritance of resistance is necessary for breeders to
develop an effective strategy for utilization of resistant germplasm in their breeding
programs (Momhinweg et al., 2002). Knowledge of the inheritance of insect resistance,
as with other economic plant traits, helps to design appropriate breeding procedures to
develop resistant cultivars. It is also useﬁil for the identiﬁcation of biotypes of insects that
may already exist or develop over time (Smith, 1989). Qualitative, or simply inherited,

traits require different breeding methods than quantitative traits controlled by many genes.

In many crops, the next logical step after the discovery of resistance is to
determine the mode of inheritance. There are many examples where this has been
conducted using a classical genetics approach. In spring barley (Hordeum vulgare L.)
line STARS-9577B, segregation data of F2 and BC1F1 populations suggested that
Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), resistance is controlled by
two dominant alleles (Momhinweg et al., 2002). Inheritance of resistance to aphid (Aphis
cra'ccivora Koch) in cowpea (Vigna unguiculata (L.) was found to be controlled by a
single dominant gene after analyzing data from parental, F1, F2, F3, and backcross
populations (Bata et al., 1987). Inheritance of resistance to a wheat midge, Sitodiplosis
mosellana (Géhin), was investigated in spring wheat derived ﬁ'om nine resistant winter
wheat cultivars and was found to be conferred by a single partly dominant gene
(Mckenzie et al., 2002). Resistance to the green peach aphid in the red leaf peach
rootstock cultivar ‘Rubira' was found to be controlled by a single dominant gene (Pascal
et al., 2002). Resistance to the soybean aphid in the cultivars Dowling and Jackson has

been found to be controlled by single dominant genes (Hill et al., 2006a, 2006b).

Biotypes of Insects

The resistance in many cultivars has been effective for only a short period of time
due to the emergence of new genotypes of the pest (F ehr, 1999). The protective
properties of insect resistant cultivars may be overcome by the development of resistance
in insect populations that possess an inherent genetic capability to over come plant

resistance (Smith, 1989). Typically, insect biotypes occur in nature as products of a

survival mechanism for the persistence of an insect species and develop as a result of
selection of parental populations in response to exposure to resistant cultivars (Smith,
1994). The failure to recognize the existence of biotypes may lead to severe infestations
of formerly resistant plants. The study of insect biotypes is a signiﬁcant part of insect
resistance programs as it provides tools for the analysis of insect plant relationships that
serve as the basis of breeding resistant plants (Saxena and Barrion, 1987). Identifying
insect biotypes can be a long and difﬁcult process. In many insects, biotypes may be
determined by the response of a group of differential host varieties to an insect population

(Smith, 1994).

Insect Resistance QTL

Linkage drag caused by co-integration of undesirable agronomic traits linked to
alleles associated with resistance QTL is a major obstacle to soybean breeders,
developing agronomically competitive cultivars with effective insect resistance (Boerma
and Walker, 2005). Genetic studies using classical techniques have identiﬁed >250
soybean loci since the discovery of the T locus for pubescence color by Piper and
Morse’s in 1910. In comparison, over 300 QTLs associated with various traits have been
identiﬁed in soybean using molecular markers since 1990 (Orf et al., 2004). Yencho et a1.
(2000) listed 233 insect resistance QTLs that have been mapped in six different crop
species. Although DNA marker technology is powerful, it nevertheless has limitations in
detecting QTLs with relatively small effects (i.e., ‘modiﬁer genes’). Of the soybean

QTLs reported in the literature, at least 162 appear to condition >1 0% of the variation in

phenotype, and only a small fraction of the total have actually been conﬁrmed. DNA
markers linked to important genes or QTLs can be used for MAS, thereby reducing the
need for phenotype-based selection. Tagging these genes with markers also makes it
possible to study them in different genetic backgrounds.

Resistance to aphids may be quantitative rather than qualitative in expression. For
instance, expression of resistance to the cabbage aphid, Brevicoryne brassicae (L.), in the
wild species Brassicafruticulosa Cirillo is quantitative (Pink et al., 2003). A quantitative
trait locus involved in adult plant cereal aphid resistance has also been detected and
mapped in barley (Moharramipour et al., 1997). Resistance to other insects in soybeans
is quantitative in expression and inheritance (Kilen and Lambert, 1998), including
resistance to the Mexican bean beetle, Epilachna varivestis (Multsant) (Rufener et al.,

1989), and resistance to the corn earworm, Helicoverpa zea Boddie, (Rector et al., 2000).

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11

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Agronomy, Madison, WI, USA

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

Palmer, R. G., T. Hymowitz, and R. L. Nelson. 1996. Germplasm diversity within
soybean. p. 1—35. In D. P.S. Verma and R. C. Shoemaker (ed.) Soybean:

Genetics, molecular biology and biotechnology. CAB. International, Wallingford,
Oxon, UK.

Pascal, T., F. Pfeiffer, J. Kervella, J. P. Lacroze, M. H. Sauge. 2002. Inheritance of green
peach aphid resistance in the peach cultivar ‘Rubira’. Plt. Breed.,.121: 459—461

Pink, D.A.C., N. B. Kift, P. R. Ellis, S. J. McClement, J. Lynn, and G. M. Tatchell. 2003.
Genetic control of resistance to the aphid Brevicoryne brassicae in the wild
species Brassicaﬁuticulosa. Plant Breed. 122:24—29.

Quirnio, G. M., and V. J. Calilung. 1993. Survey of ﬂying viruliferous aphid species and
population build-up of Aphis glycines Matsurnura in soybean ﬁelds: Philipp.
Entomol. 1993, 9 52—100.

Ragsdale, D.W., D. J. Voegtlin, and R. J. O’Neil. 2004. Soybean Aphid Biology in North
America. Ann. Entomol. Soc. Am. 97 : 204—208.

Rector, B.G., J. N. All, W. A. Parrott, and H. R. Boerma. 2000. Quantitative trait loci for
antibiosis resistance to corn earworm in soybean. Crop Sci. 40:233-23 8.

Rufener, G.K., S. K. St Martin, R. L. Cooper, and R. 3. Hammond. 1989. Genetics of
antibiosis resistance to Mexican bean beetle in soybean. Crop Sci. 29:618—622.

Saxena, R. C. and M. V. Velasco and A. A. Barrion.(l999) Morphological variations

between brown planthopper biotypes on Leersia hexandra and rice in the
Philippines, Int. Rice Res. Newsl., 8(3):3

12

Smith, C. M. 1989. Plant resistance to insects: A fundamental approach. John Wiley &
Sons, New York.

Smith, C. M. ,.Z R. Khan, and M. D. Pathak. 1994. Techniques for evaluating insect
resistance in crop plants. CRC Press, Inc.

Soy Stats. 2007. [Online] Available at: http://www.soystats.com/2007/page_30.htm

Sun, 3., S. B. Liang, and W. X. Zhao. 2000. Outbreak of the soybean aphid in Suihua
prefecture in 1998 and its control strategies. [Online] English version available at
http://www.ksu.edu/issa/aphids/reporthtml/trans40.htm; veriﬁed 21 June 2005.
Soybean Bull. 1:5.

Takahashi, S., M. Inaizumi, and K. Kawakami. Life cycle of the soybean aphid Aphis
glycines Matsumura, in Japan: Jpn. J. Appl. Entomol. 2001. 1993, 37 207-212.

Voegtlin, D. J ., S. E. Halbert, and G. Qiao. A guide to separating Aphis glycines
Matsumura and morphologically similar species that share its hosts: Ann.
Entomol. Soc. Am. 2004, 97 pp 227—232.

Wu, Z., D. Schenk-Hamlin, W. Zhan, D. W. Ragsdale, and G. E. Heimpel. The Soybean
Aphid in China: A Historical Review. Ann. Entomol. Soc. Am. 2004, 97 pp 209—
218.

Yencho 6., Cohen M., and Byme P. Applications of tagging and mapping insect
resistance loci in plants. Ann. Rev. Entomol. 2000. 45:393—422.

13

CHAPTER 2

INHERITANCE OF SOYBEAN APHID RESISTANCE IN PI 5675413 AND
P15675983

ABSTRACT

In a previous study, two soybean [Glycine max (L.) Merr.] plant introductions (PIS), PI
5675413 and PI 5675983, were found to possess antibiosis-type resistance to the
soybean aphid (Aphis glycines Matsumura). Plants with antibiosis resistance negatively
interfere with the reproduction of the aphid and thus control the insect. Field studies were
conducted to determine the inheritance of antibiosis resistance in P1 5675413 and PI
5675983. The two resistant PIS were crossed with susceptible soybean lines and the F1
and F2 plants and F23 families were evaluated for aphid resistance. All F1 plants were
found to be susceptible to soybean aphids. The plants in seven F2 populations segregated
in a 15 susceptible to 1 resistant ratio, which is the expected ratio for a trait controlled by
two recessive genes. The F23 families also segregated in a 15 susceptible to 1 resistant
ratio. Therefore, the segregation data suggest that two major recessive genes are involved
in the resistance in P1 5675413 and PI 5675983. This information can be used by
breeders to design efﬁcient breeding schemes for developing soybean cultivars with

antibiosis resistance to aphids.

14

INTRODUCTION

The soybean aphid (SBA), Aphis glycines Matsumura, was ﬁrst discovered in
eight Midwestern US. states in 2000. Since then it has spread throughout the north
central United States and parts of Canada (NCSRP, 2004) and has become one of the
major pests affecting soybean production in North America. SBA populations can
double very quickly (McComack et al., 2004), and aphid numbers can reach thousands
per plant. Aphid feeding reduces photosynthesis (Macedo et al., 2003) and reduces yield
components including plant height, number of nodes and pods per plant, seed Size, and
bean quality (DiFonzo and Hines, 2002; Ostlie, 2003). In efﬁcacy trials conducted in
Michigan during SBA outbreak years, yield in untreated plots was 18% to 40% less than
yield in treated plots (DiFonzo, 2006; Difonzo and Hines, 2002).

Insecticides are still the primary means of controlling SBA, which increase
production costs and human exposure to pesticides. In 2005, an outbreak year for SBA
across the Midwest, millions of acres were treated (NASS, 2006). Insecticide
applications also kill natural enemies of soybean aphids (Smith and Krischik, 1999) and
may increase populations of other soybean pests such as spider mites. Host-plant
resistance is the most effective means to control insect pests. Soybeans resistant to SBA
colonization would eliminate or minimize the need for insecticides, reducing cost,
environmental impacts, and exposure.

Since the discovery of SBA in the US, signiﬁcant effort has been made to identify
sources of resistance in soybean. Hill et a1. (2004) screened 1,542 soybean accessions and
identiﬁed seven, including Dowling and Jackson, with resistance to SBA. We evaluated

2,147 soybean germplasm accessions in choice tests and identiﬁed four PIS, PI 5675983,

15

PI 5675413, PI 567543C, and PI 567597C, with resistance to SBA (Mensah et al., 2005).
In a no-choice test, PI 5675983 and PI 5675413 were found to possess antibiosis
resistance (Mensah et al., 2005). In 2006, Diaz-Montana et al. compared the reproduction
of SBA on 240 soybean entries and identiﬁed eleven entries with fewer nymphs than the
susceptible checks. In a follow-up experiment they identiﬁed K1639 and Pioneer 95397
as showing a strong antibiosis effect on SBA. Recently, Hesler et a1. (2007) have also
found two aphid resistance sources, PI 230977 with antibiosis resistance and G93-9223
(PI 595099) with antixenosis resistance. Currently only the resistance in Dowling and
Jackson has been characterized; it was shown to be controlled by a single dominant gene
(Hill et al., 2006a, 2006b). The inheritance of the other 30ma of aphid resistance has
not yet been characterized.

Development of SBA-resistant cultivars is an objective in many public and
private soybean breeding programs in North America. For resistance sources to be useful
in developing resistant plants, the genes conferring resistance must be characterized. The
number of genes controlling resistance as wellias the nature of the resistance determines
the breeding method required to transfer this resistance into elite cultivars. The objective
of this current study is to determine the inheritance of SBA resistance in the two soybean

accessions PI 5675983 and PI 5675413 that exhibit antibiosis resistance.

16

MATERIALS AND METHODS

PI 5675413 was crossed with E00075 and PI 567598B was crossed with Titan
and E00075. Both Titan and E00075 were susceptible to soybean aphids. Each F1 plant
was harvested separately to develop F2 populations. The parental lines and F1 plants of
the cross Titan x PI 567598B were evaluated for SBA resistance in 2004 and the F2
populations from the same cross were evaluated in the ﬁeld during 2005. Parental lines,
F. plants, and F2 populations ﬁom the crosses 300075 x PI 5675413 and 300075 x PI
5675983 were evaluated for aphid resistance in the ﬁeld in 2005. The number of plants in
each F2 population is shown in Table 2.1. Evaluation of SBA resistance was carried out
in a 12.2- x 18.3-m aphid-proof cage in the ﬁeld on the Michigan State University
campus in East Lansing, MI. Two weeks after planting, when the plants were at the V2
stage (F ehr and Caviness, 1977), each plant was inoculated with two aphids according to
the method described by Mensah et a1. (2005). All aphids used in these tests were
obtained ﬁ'om nearby naturally infested soybean ﬁelds. The F1 plants were planted 30.5
cm apart with no replication and the parents were planted 5.1 cm apart with two
replications. Each F1, F2, and parental plant was rated for aphid damage two, three, and
four weeks after inoculation using a rating scale of 0 to 4 described by Mensah et a1.
(2005).

Seeds ﬁ'om 376 individual F2 plants in population 030104-8 (Table 2.1), which
was developed ﬁom a single F1 plant of the Titan x PI 5675983 cross, were harvested
individually during fall of 2005. The 376 F23 lines and the parents were evaluated for

aphid resistance in the ﬁeld during summer 2006. Depending on seed availability, up to

17

ﬁfteen F3 progenies from each F2 plant were planted. Resistance evaluations were
conducted in a ﬁeld cage as described previously, but using a modiﬁed version of the
rating scale described by Mensah et a1. (2005). The rating scale used for F1 and F2 plants
did not clearly distinguish between plants with low (one or two) versus moderate (tens of
aphids) infestation. In 2006 half steps were added to the original 0 to 4 scale (Table 5.1,
appendix). Over 3000 F3 plant were rated weekly for three consecutive weeks starting
three weeks after inoculation.

When the susceptible parents ﬁrst rated a score of 4.0, the data from that sample
date were used to classify the F2 or F3 plants as resistant or susceptible. A plant with a
rating of 1.5 or less was classiﬁed as resistant, while a plant with a rating larger than 1.5
was considered susceptible. The threshold of 1.5 was comparable to the threshold used to
identify susceptible plants in our previous study (Mensah at al., 2005). Chi-square tests
were performed to test the goodness of ﬁt of observed segregation ratios among F2 plants

and F23 families with different genetic ratios, with rejection at 0.05 levelof probability.

RESULTS AND DISCUSSION

All F 1 plants from two of the three crosses were found to be susceptible with a
rating greater than 1.5 (Table 2.2), suggesting that resistance to SBA is controlled by
recessive genes. Data for the F1 plants in the third cross (Titan x PI 5675983 ) were not
obtained due to poor infection in 2004 as a result of heavy rain and ﬂooding damage after
aphid inoculation. The overall frequency distribution of aphid colonization ratings in all

F2 populations was not normal, and was skewed toward the susceptible parents (Figure

18

2.1), suggesting that susceptibility was dominant over resistance. The distributions were
continuous, indicating that more than one gene was involved in aphid resistance in the
two PIS and the dominance of susceptibility over resistance was not complete. All the F2
populations segregated in a 15:1 susceptible/resistant ratio (Table 2.1), which is the
expected ratio for a trait controlled by two recessive genes with duplicate dominant
epistasis. In both cases when E00075 was crossed with P1 5675983 and PI 5675413 the
resulting F2 populations also ﬁtted the 15:1 susceptible/resistant ratio conﬁrming the
recessive nature of the resistance genes in a different population.

For the Titan x PI 567598B F23 families, on average, eight seeds per family
germinated. Out of the 376 F23 families 25 were found to be resistant, ﬁtting the 15:1
ratio with a P value of 0.258. Forty ﬁve out of 351 F23 families derived from susceptible
F2 plants segregated for resistance. The recessive nature of the resistance in P1 5675983
and PI 5675413 was conﬁrmed in the F23 families as all 25 resistant F2 individuals
produced resistant F23 families. Due to the recessive nature of resistance in PI 567598B,
it was expected that susceptible heterozygotes would segregate when the F 3 families were
tested for aphid resistance.

However, segregation was observed only in 45 out of F23 families. This low
number of F23 segregating families might be due to low seed yield from susceptible F2
plants and poor germination. Based on Fehr (1987), at least 11 plants are needed to have
a 95% chance of identifying one resistant plant with a 0.25 expected ﬁequency. On
average, we had only eight plants per family; therefore many families did not have the

minimum number of plants required to ﬁnd a resistant plant in a segregating F23 family.

19

The segregation data in the F2 populations and F23 families shows that two major
recessive genes are involved in aphid resistance in both PI 5675983 and PI 5675413.
However, from the results there is a possibility that other minor genes are also involved
in the resistance.

Insect resistance like all other traits can be controlled by either one or more
dominant or recessive genes. SBA resistance in the soybean cultivars Dowling and
Jackson is controlled by a single dominant gene (Hill et al., 2006a, 2006b). Our study
demonstrated that aphid resistance in the soybean PI 5675983 and PI 5675413 is
controlled by two recessive genes, suggesting different resistant genes from those in
Dowling and Jackson underlie the resistance in these two PIS. Little is known about the
mechanism of pest resistance in soybean, the involvement of a recessive allele in the
antibiosis might be a clue (Komatsu et a1. 2005).

Different genes and inheritance patterns for aphid resistance have also been
reported in other crops. In wheat, nine characterized genes (Dnl , Dn2, dn3, and Dn 4 to
Dn9) ,are involved in resistance to the Russian wheat aphid, Diuraphis noxia (Du Toit,
1989; Harvey and Martin, 1990; Liu et al., 2001; Marais and Du Toit, 1993; Marais et al.,
1998; Nkongolo et al., 1991a, 1991b; Schroeder-Teeter et al., 1994). Eight of the genes
are independent dominant genes each conferring resistance in a different resistance
source, while dn3 is a recessive gene conferring the aphid resistance in Triticum tauschii.
In barley, a single dominant gene controls the Russian wheat aphid resistance in the line
S13 (Robinson et al., 1992) and two dominant genes control resistance in the line

STARS-9577B (Momhinweg et al., 2002).

20

As with all host plant resistance to insects or pathogens, there is the concern that
the resistance will be overcome. In wheat, the resistance gene Dn4, found in many
varieties, was overcome by a new biotype of Russian wheat aphid found in Colorado in
2003 (Haley et al., 2004). In a follow-up experiment, Haley et a1. (2004) found that only
one of the nine resistance genes, Dn 7, conferred resistance to the new biotype. In 2006,
three new aphid biotypes were identiﬁed based on the foliar damage they caused; one
biotype was virulent to eight of the nine sources of Russian wheat aphid resistance in
wheat (Burd et al., 2006). Each of the eight sources carried different genes conferring
resistance to Russian wheat aphid. The adaptive ability of aphids in general to overcome
plant resistance through biotype differentiation highlights the need to explore the genetic
diversity of SBA resistance. Variation of SBA biotypes has been observed in the US
(Kim et al., 2007; Mensah et al., 2007). Some biotypes have overcome the resistance
from Dowling and Jackson but not the resistance ﬁ'om PI 5675983 and PI 5675413 (Kim
et al., 2007; Mensah et al., 2007). Therefore, different sources of resistance must be used
to develop SBA resistant cultivars.

In general, resistance controlled by multiple genes is more durable than the
resistance controlled by a single dominant gene (Duvick, 1999). Thus the resistance from
PI 5675413 and PI 567598B may be more durable than the single gene controlled
resistance from Dowling and Jackson. However, more effort will be required to
incorporate the resistance from these two PIS into elite germplasm because larger progeny
populations are required to recover at least one resistant progeny with the resistance.

The information on the recessive inheritance of the SBA resistance [detected in

this study is useﬁrl to breeders in developing Special schemes in breeding programs in

21

order to incorporate this resistance in elite breeding lines. In breeding for insect
resistance, backcrossing is the major approach for introducing resistance into an
otherwise superior cultivar. Selﬁng after each backcross can be used to select lines with
the recessive resistance gene. If markers associated with the genes are identiﬁed, marker-
assisted selection can be used to identify resistant lines faster, and therefore incorporation
of the recessive genes into new cultivars will be easier and faster (Chen and Line, 1999).
Genetic populations have been developed to test for allelism of genes controlling aphid
resistance in these two PIS. Research is ongoing to identify molecular markers associated

with the resistance genes in this study.

22

Table 2.1: Segregation of aphid resistance in F2 populations derived from susceptible x
resistant crosses

Population Susceptible Resistant Total Observed Expected by a P value

 

IDT . parent parent no. of 15:1 (R: S) of X2
plants ratio test§
- RI Si. R S
040129-1 E00075 PI 5675413 155 5 150 10 145 0.120
040129-2 E00075 PI 5675413 98 5 93 6 92 0.639

040130-1 E00075 PI 5675983 100 7 93 6 94 0.757
040130-2 E00075 PI 5675983 126 8 l 18 8 1 18 0.963

030104-3 Titan PI 5675983 415 26 389 26 389 0.990
030104—8 Titan PI 5675983 388 32 356 25. 363 0.148
0301 04-10 Titan PI 5675983 416 26 390 26 390 1.000

 

1“Each F2 population was developed from a single F1 plant. F2 populations developed
ﬁ'om different F1 plants of the same cross were considered different populations.
IR= Resistant, S= Susceptible

§If the P value 13 larger than 0. 05, the null hypothesis that the observed R: S ratio ﬁts the
expected 1: 15 ratio is not rejected statistically.

23

Table 2.2: F1 and parental lines classiﬁed as resistant to soybean aphid.

 

 

Total no. No. of
of plants resistant
Genotype tested plants Mean rating

PI 5675413 9 9 1.0
PI 5675983 12 12 1.0
300075 8 0 4.0
Titan 13 0 4.0
(300075 x P15675413) F1 6 0 3.3
(300075 x P15675983) Fl 12 0 3.0
(Titan x P15675983) F1 10 - -

 

24

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a)population040129-1
so
to 70‘
590'
Ego.
“am-
8 .
9:.
z
10‘
0.
c)popuation040130-1
E
‘6
B
E
3
2
11.5 2 2.5 3 3.5 4
DarnageRating
e)popdation0301o4-3
200
130
m
15150
— 140
e m. ..
3100 S
B a)“ t~ :
g m. 8 g
z 40- h." ¢
20- I
0'
11.522.533.54
DamageRating

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

b)population040129-2
so
, 45'
1n
‘5”
-35-
.
J .-
si‘s. : ..
§15« 5
z ro- i
5. ,
o-
1 1.5 2 25 3 3.5 4
DarnageRatlng
d)popu|ation040130-2
45
4o
3 l
535‘
E30-
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a ‘ 3
201 It
€1.2- a
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5.
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npopulation030104—8
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1zo~
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‘— ”d a
2 m, 3.: .
§ 5 5
:1 ‘0' E *
z
20- I
o-

 

 

d

1.5 2 25 3 3.5 4
‘ Damage Rating

 

Figure 2.1: Distribution of damage rating scores in F2 populations: a) 040129-1, b)
040129-2, c) M01304, (1) 040130-2, e) 030104-3, and f) 030104-8.

25

 

 

 

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Chen, X. M., and R. F. Line. 1999. Recessive genes for resistance to races of Puccinia
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Diaz-Montano J., Reese J. C., Schapaugh W. T., Campbell L. R. 2006. Characterization
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28

CHAPTER 3

VARIATION IN SOYBEAN APHIDS IN 2006: A CASE FOR THE PRESENCE
OF SOYBEAN APHID BIOTYPE DIVERSITY IN MICHIGAN

ABSTRACT

The soybean aphid, Aphis glycines (Matsumura), has over the past ﬁve years become one
of the most important pests of soybean Glycine max L. in Michigan. When this research
was initiated in 2006, there was no documentation of the existence of biotypes of soybean
aphids in North America. However in other aphid species, like the green bug and Russian
wheat aphid, biotypes have arisen after the release of aphid resistant crop genotypes.
With the testing of several soybean aphid resistant genotypes it is only a matter of time
that a new biotype would evolve. In a ﬁeld study in 2006, Dowling, a resistant check was
found to be susceptible to the soybean aphid. The objective of this study was to determine
if a new aphid biotype is present in Michigan. A greenhouse study was conducted to
compare the effect of the aphids which overcame the resistance in Dowling with random
aphids collected in the ﬁeld in 2006. Dowling was found to be susceptible to both aphid
colonies collected in 2006. These results were compared to data ﬁ'om a greenhouse study
conducted using aphid colonies that were collected in 2002 and maintained in a growth
chamber and greenhouse since 2002. Dowling was found to be resistant to the aphid
colony collected in 2002. These results indicate that, there is a difference in the virulence
reaction on Dowling by aphids collected in 2002 and 2006. This suggests that a new

soybean biotype may have evolved in Michigan.

29

INTRODUCTION

The soybean aphid has become one of the major economic pests affecting
soybean production in North America (Schmidt et al., 2007). In 2005, soybean aphid
outbreaks were reported in several states, with millions of acres treated with pesticides in
Minnesota, Indiana, and Michigan (O’Neal, 2006). The soybean aphid is the only aphid
in North America that develops large colonies on soybean (Plant Health Initiative, 2004).
Plant damage occurs when large numbers of aphids remove signiﬁcant amounts of water
and nutrients as they feed on leaves and stems, causing leaves to wilt, curl, yellow, and
even drop. Other symptoms of direct feeding damage include plant stunting, poor pod ﬁll,
reduced pod and seed counts, smaller seed Size, and nutrient deﬁciencies resulting in
overall yield and quality reduction (DiFonzo and Hines, 2002). Signiﬁcant yield loss (8-
25%) occurs when the soybean plants are heavily infested by the aphid during the early
reproductive stage (DiFonzo and Hines, 2002; Hunt and J arvi, 2005).

After a Six year observation period, the soybean aphid appears to be on a 2-yr
cycle, alternating years with signiﬁcant economic problems with years where populations
are very low or almost non existent (Ragsdale, 2006). The ﬁrst response to aphid control
was the use of insecticides, but these pesticides also killed natural enemies of soybean
aphids (Smith and Krischik, 1999). Millions of dollars were spent annually spraying
chemicals to control the aphids in infested soybean ﬁelds (Li et al., 2007).

Host-plant resistance however, is the most effective means of controlling insects as it
helps eliminate or minimize the need for insecticides.

Many soybean breeding programs in North America are working to identify

soybean genotypes with resistance to the soybean aphid. In 2004, Hill et al., reported

30

seven accessions, including Dowling (Maturity Group (MG) VIII) and Jackson (MG
VII), with antibiosis resistance to the aphids after screening 1,542 soybean accessions. In
2005, the breeding program here at Michigan State University (MSU), identiﬁed four
(MG III) plant introductions (PIS) PI 5675983, PI 5675413, PI 567543C, and PI
567597C, with resistance to the soybean aphid, after evaluating 2,147 soybean
germplasm in choice tests (Mensah, et al., 2005). Diaz-Montana et a1. (2006) compared
the reproduction of soybean aphids on 240 soybean entries and found eleven entries with
fewer nymphs than the susceptible checks. Hesler et a1. (2007) have also found two aphid
resistance sources, PI 230977 with antibiosis resistance and G93-9223 (PI 595099) with
antixenosis resistance. Recently, Mian et a1, (2008) found three MG IV PIS (243 540,
5673013 and 567324) to be resistant to soybean aphids and identiﬁed six others which
were moderately resistant after screening nearly 200 soybean genotypes (cultivars,
breeding lines and PIS) in a greenhouse choice test.

The aphid resistance in each of the two soybean cultivars Dowling and Jackson is
controlled by single dominant genes (Hill et al., 2006a, 2006b) Rag I and Rag
respectively (Hill et al., 2006a, Li et al., 2007). The aphid resistance in the germplasm
identiﬁed by Mensah et al., (2005) is controlled by two recessive genes. With deployment
of resistance sources there is always the concern that biotypes of the insects would arise
and overcome resistance. When resistance is controlled by a Single dominant gene it is
not uncommon for resistance to breakdown in a relatively short time. In other aphid
species, the use of cultivars with a Single aphid resistance gene has favored the selection
and rapid spread of aphid biotypes adapted to this resistance. For example, biotypes of

both Russian wheat aphid [Diuraphis noxia (Mordvilko)] and greenbug [Schizaphis

31

graminum (Rondani)] were found capable of overcoming resistance genes in new
cultivars (Burd and Porter, 2006; Haley et al., 2004). In breeding red raspberry for
resistance to the large raspberry aphid (Amphorophora idaei) using single major genes or
polygenic minor genes proved successful in controlling this virus vector aphid for more
than 30 years. Current surveys found that more than 75% of the A. idaei populations in
the United Kingdom consisted of biotypes with the ability to break the most widely
deployed resistance gene, A10 (Birch et al., 2006).

In other aphid species similar methods have been used to determine the presence
of biotypes. In a Russian wheat aphid study, the performance of two D. noxia sources was
compared on three wheat cultivars, Trego, Halt, and Stanton, and after characterizing
substantial differences in plant responses, it was determined that biotypes were present
(Jyoti and Michaud, 2005). In another study, Qureshi et al., (2005) evaluated colonization
of commercial wheat cultivars by the two biotypes and reported some differential
responses and conﬁrmed the presence of biotypes in Russian wheat aphid.

In the summer of 2006, Dowling (Hill et al., 2004) an aphid resistant cultivar
which has been used as a resistant check since 2002 in our breeding program was found
to be susceptible to aphids collected in natural occurring colonies in the ﬁeld. Since the
arrival of the soybean aphid there has been no documentation of the presence of biotypes,
but this observation led to our current hypothesis that biotypes of soybean aphids may
have arisen. The discovery of soybean aphid biotype diversity in Illinois and Ohio has
been reported recently (Kim et al., 2008). The objective of this current study is to

determine if soybean aphid biotypes have evolved in Michigan. The reaction of aphids

32

collected in 2006 and 2002 would be compared on selected resistant and susceptible

soybean genotypes.

MATERIALS AND METHODS

To determine the possible presence of soybean aphid biotypes in Michigan two
studies were conducted. The ﬁrst study compared the reaction of different resistance
sources to aphids collected from a susceptible Dowling plant with aphids collected in the
ﬁeld in 2006. In the second study, selected resistant and susceptible genotypes were
infected with aphids that have been kept in a growth chamber and greenhouse since 2002
to verify if the cultivar Dowling was still resistant. This information would help
determine genetic differences exist between aphids collected in 2002 from those collected
in 2006.

All studies were carried out in the Plant Science Greenhouse, MSU with
temperatures between 22 and 25°C. All soybean seeds were planted in a plastic pct 22
cm in diameter and 23 cm deep, the soil used in all cases was Baccto High Porosity
Professional Planting mix (Michigan Peat Company, Houston, TX). All plants were
inoculated at the V2 stage (Fehr and, Caviness, 1977) with two Wingless aphids each on

the partially expanded trifoliate with a camel-hair brush.

Aphid Culture

In study 1, two sources of aphids were used, Colony A consisted of aphids

collected from susceptible Dowling plant in the ﬁeld in 2006 and raised on Dowling

33

plants in the greenhouse. Colony 3 was made up of aphids collected randomly from a
seed cage in the ﬁeld in 2006. The aphids Were raised on Williams 82 (susceptible check)
in the greenhouse. In study 2 we used aphids which have been maintained in isolation in
a growth chamber and greenhouse since 2002. The colony was obtained from the Field

Crops Entomology Laboratory, MSU.

Plant materials

In the ﬁrst study to determine the difference between ﬁeld collected aphids and
aphids ﬁ'om the susceptible Dowling plant, the soybean genotypes used were: PI
5675983, PI 5675413, PI 567543C, and PI 567597C (resistant accessions from Mensah
et al., 2005), Dowling, Jackson, PI 71506 (resistant cultivars from Hill et al., 2004), and
two susceptible checks, Titan and Williams 82.

In the second study using aphids which had been in colony since 2002 the
following soybean genotypes were used: PI 5675413, Dowling, 030108-515 (Resistant),

Titan, Roundup-Ready (RR) Titan, 300075, Williams 82 (susceptible).

Location of Experiment and Screening Procedure

The ﬁrst study was set up as a no-choice (Davis, 1985) test in a factorial
experiment arranged in a randomized complete block design with three replications. The
two factors in the experiment were the nine soybean genotypes and two aphid colonies (A

and 3). A total of eight seeds per genotype were planted. In the no-choice test, each

34

genotype was isolated for the next by the use of a no-see-um mesh cage (Venture
Textiles, Inc., Braintree, MA) to prevent the two different sources of aphids ﬁom mixing.
The second was set up as a Choice test (Davis, 1985) in a randomized complete block
design with three replicates. Five plants per genotype were planted. In the choice test the

aphids were ﬂee to move from plant to plant among genotypes.

Data Collection

Plants were rated visually using a modiﬁed version of the method of rating as
described by Mensah et al., 2008 (Table 5.1, Appendix) which ranges ﬂour 0 for no aphid
present to 4 for totally covered with aphids. Data was collected starting from the third
week through the ﬁfth. When the susceptible parents ﬁrst rated a score of 4.0, the data
from that sample date were used to classify soybean genotypes as resistant or susceptible.
A damage index (DI) for each accession was calculated by the following formula
(Zhuang, 1999): D1 = 2(8cale value x No. of plants in the category)/ (4 x Total no. of
plants evaluated) x 100. The DI ranges between 0% for no infestation and 100% for the
most severe damage. A D1 of 38% or less was classiﬁed as resistant, whereas a D1 of 38%
or more was classiﬁed as susceptible. The 38% break point was chosen on the basis of the
observation that a soybean genotype with a D1 value less than 38% never Showed

symptoms of damage under high aphid pressure until the end of the season.
Statistical Analysis:

Analysis of variance (AN OVA) for choice and no-choice tests was conducted

using the PROC GLM procedure in the SAS V9.1 (SAS Institute, 2000). Means were

35

separated by least signiﬁcant difference (LSD) at the 5% probability level if their effects

were found to be signiﬁcant in the AN OVA.

RESULTS
Studyl

The effects of soybean genotype and the interaction between soybean genotype
and aphid colony was found to be signiﬁcant at P <0.0001 and P=0.0362, respectively.
The effect of aphid colony was not signiﬁcant (P=0.306). All the PIS found to be resistant
by Mensah et al., 2005, Jackson and PI 71506, showed antibiosis to both aphid colonies
(Table 3.1). However, PI 567543C, and PI 567597C which were formerly reported as
having antixenosis resistance was found to exhibit antibiosis resistance in the no choice
test. Dowling was found to be susceptible to both aphids from colony A and 3.
Interestingly, Williams 82 the susceptible check was less susceptible to colony A aphids
than Dowling. Titan, the other susceptible cultivar was not signiﬁcantly different from
Dowling (Table 3.1). These results indicate that all the aphids collected randomly in the
ﬁeld in 2006 can overcome the resistant gene Rag] in Dowling but not the Rag gene in

Jackson.

Study 2

In this study the damage index of each entry was calculated at three and then four
weeks after inoculation with aphids ﬁ'om the 2002 colony. The results Showed that,
Dowling was not signiﬁcantly different from P1 5675413 in both rating and both

genotypes were resistant. In week three, there was no signiﬁcant difference between the

36

four susceptible genotypes Titan, RR Titan, 300075, and Williams 82. However in week
four, 30007 5, the advanced breeding line was signiﬁcantly less susceptible than Titan,
RR Titan, and Williams 82. Over-all the plant damage in week four was not typically

different from that in week three (Table 3.2).

DISCUSSION

This research conﬁrms the suspected variation that exists between the soybean
aphid populations in Michigan in 2002 and 2006 based on their virulence reaction to
Dowling. The results obtained here and that from similar studies carried out in other
states Show that new soybean aphid biotypes are emerging. This supports the presence of
a new soybean aphid biotype in Michigan.

In their recent paper, Kim et a1. (2008), report that there are soybean aphid
biotypes that can overcome the aphid resistance in both Dowling and Jackson. Another
study in Ohio conﬁrms Dowling and Jackson are susceptible to the Ohio isolate of aphids
under greenhouse conditions (Mian et al., 2008). In our study Jackson is still resistant to
the soybean aphid variant found in Michigan in 2006 (Table 3.1). The breakdown of the
resistance in Dowling may have occurred prior to 2006 as a similar trend was observed
when some material from Illinois was tested in Michigan in 2005 (personal observation).
The report of biotypes occurring in soybean aphids is not unique to this aphid alone.
Multiple biotypes have been seen to occur in other aphid species such as Russian wheat
aphid and green bug (Burd and Porter, 2006). In the Russian wheat aphid biotype

variation was known to exist worldwide, but it was not observed in the U. S. until 2003,

37

when a biotype was identiﬁed that could overcome Dn4, the major resistance gene used
to protect wheat (Triticum aestivum L.) from this aphid (Haley et al., 2004).

There is a need to systematically collect and test soybean aphid isolates in North
America and other parts of the world to track potential changes in biotype variation in
soybean aphid. This year a project funded and coordinated by Monsanto Company, St.
Louis, Mo. will help answer the extent of biotypic variation in soybean aphids in the US.
In this project all aphid resistant sources would be tested with aphids in states where
aphid resistance research is being carried out no-choice tests (Dechun Wang, pers.
comm). DNA-based techniques are increasingly being applied to explore the genetic
differences between insect biotypes. These techniques are proving particularly valuable
for the study of aphids which are characterized by low levels of intraspeciﬁc genetic
variation as revealed by allozyrnes (Hales et al., 1997). For example, restriction analyses
of mitochondrial DNA have revealed consistent differences between green bug
(Schizaphis graminum) biotypes found on different sorghum cultivars (Powers et al.,
1989) and between alfalfa aphid ( Therioaphis trifolii ) biotypes using different legume
crops (Sunnucks et al., 1997). Additionally, differences in microsatellite proﬁles have
been identiﬁed in the English grain aphid (Sitobion avenae) collected ﬁom wheat (De
Barro et al., 1995). Similar molecular work needs to be conducted to determine if indeed
soybean biotype diversity exists in North America Based on results of current ﬁeld
studies to detect biotype variation.

In 2005, we reported PI 567543C and PI 567597C as having antixenosis
resistance, but our current results (Table 3.1) shows that they possess antibiosis

resistance. For a genotype exhibiting antixenosis type resistance the expectation is that

38

their DI values would be signiﬁcantly higher than rating obtained for P15675983 and PI
5675413. This difference in resistance mechanism was also observed by Kim et al.,
(2008) where they classiﬁed all the resistance sources ﬁom Michigan (Mensah et al.,
2005) as showing strong antibiosis. This change in resistance mechanism in P1 567543C
and PI 567597 C, from antixenosis to antibiosis can be attributed the fact that these aphid
biotypes have a different reaction to these two genotypes. It is also possible that the plant
defenses have developed antibiotic cues which can now adversely affect the aphid’s
ability to develop on them.

The identiﬁcation of new biotypes is of critical importance in accurately
identifying genetic sources of resistance crop plants (Smith, 1994). With these reports of
soybean aphid biotype diversity emerging, the identiﬁcation of more effective sources of
resistance to aphids is encouraging for resistance breeding efforts. New sources with
multiple genetic resistance such as that present in P1 5675983 and PI 5675413 controlled
by two recessive genes (Mensah et al., 2008) are needed to help maintain durability of

resistance to soybean aphids.

39

Table 3.1: The average Damage Index (DI) at 4 weeks after inoculation based on three
Eplications in a no-choice test carried out in Study 1, Fall 2006 the greenhouse.

 

 

 

Entry Damage Index (%)
Colony A Colony 3
PI 567543C 2 a1" 0 a
P1567597C ‘ 1 a 2 a
PI 5675413 6 a 0 a
PI 5675983 0 a 0 a
PI 71506 4 a 0 a
Titan 76 bed 77 bed
Jackson 0 a 7 a
Dowling 65 be 67 be
‘Williams 82’ 44 b 74 bed
Mean 22.0 25.2

 

1' Means followed by the same letters in the DI columns are not signiﬁcantly different by,
the least signiﬁcant difference test (P=0.05); Colony A: Dowling Aphids; Colony 3:
Random 2006 Aphids

40

Table 3.2: Damage Index (DI) based on results obtained in Study 2-resistant sources

tested in the greenhouse, winter 2006 at 3 and 4 weeks after inoculation using aphids
originally collected in 2002.

 

 

 

Entry Damage Index (%)
Week 3 Week 4
300075 3 75 b1‘ 80 b
030108-515 20 a 20 a
P15675413 13 a 13 a
Titan 81 b 88 c
R Titan 81 b 88 c
Dowling 13 a 13 a
Williams 82 75 b 88 c ,
Mean 55 51

 

'1 Means followed by the same letters in a column are not Signiﬁcantly different by the
least signiﬁcant difference test (P=0.05)

41

REFERENCES

Davis, F. M. 1985. Entomological techniques and methodologies used in research
programmes on plant resistance to insects. Insect Sci. Appl. 6:391—400.

DiFonzo, C., and R. Hines. 2002. Soybean aphid in Michigan: Update from 2001 season,
Michigan State University Extension Bulletin 3-2746.

Birch, A.N.E., A.T. Jones, 3. Fenton, G. Malloch, I. Geoghegan, S.C. Gordon, J. Hillier,
and G. Begg. 2006. ISHS Acta Horticulturae 585: VIII International Rubus and
Ribes Symposium. Resistance-Breaking Raspberry Aphid Biotypes: Constraints
to Sustainable Control through Plant breeding.

de Barro, P. J ., Sherratt, T. N., Brookes, C. P., David, O. and Maclean, N. 1995. Spatial
and temporal genetic variation in British ﬁeld populations of the grain aphid
Sitobion avenae (F .) (Hemiptera: aphididae) studied by RAPD-PCR Proc R Soc
3, 262: 321-327.

Burd, J .D., and DR. Porter. 2006. Biotypic diversity in greenbug (Hemiptera:
Aphididae): Characterizing new virulence and host associations. J. Econ.
Entomol. 99:959—965.

Hales, D., F. J. Tomiuk, K. Wiihrmann, and P. Sunnucks. 1997. Evolutionary and genetic
aspects of aphid biology: a review. Eur J Entomol, 94: 1—55.

Haley, S. D., F. 3. Peairs, C. 3. Walker, J. 3. Rudolph, and T. L. Randolph. 2004.
Occurrence of a new Russian wheat aphid biotype in Colorado. Crop Sci.: 44,
1 589-1 592.

Hill, C. 3., 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. 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.

Hunt, T., and K. Jarvi. 2005. Focus on Soybeans 11 [Online]. Available at
http://cropwatch.unl.edu/archives/ZOOS/cropOS-7.htm#aphids. Accessed 6 July,
2007.

Jyoti, J. L., and J. P. Michaud. 2005. Comparative biology of a novel strain of Russian

wheat aphid (Homoptera: Aphididae) on three wheat cultivars. J. Econ. Entomol.
98:1032—1039.

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Kim, K. S., C. 3. Hill, G. L. Hartman, M.A. R. Mian, and B. W. Diers. 2008. Discovery
of Soybean Aphid 3iotypes.Crop Sci. 48: 923-928.

Li, Y., C.B. Hill, S.R. Carlson, 3.W. Diers, and G.L. Hartman. 2007. Soybean aphid

resistance in the soybean cultivars Dowling and Jackson map to linkage group M.
Mol. Breed. 19:25-34.

Liu X., C. M. Smith, 3. S. Gill, V. Tolmay. 2001. Microsatellite markers linked to six
Russian wheat aphid resistance genes in wheat. Theor. Appl. Genet. 102:504—510.

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

Mensah C, C. Difonzo, D. Wang. 2008. Inheritance of soybean aphid resistance in P1
5675413 and PI 5675983. Crop Sci. (in press)

Mian R. M. A., R. 3. Hammond, and S. K. St. Martin, 2008. New Plant Introductions
with Resistance to the Soybean Aphid. Crop Sci 48: 1055-1061.

O'Neal, M. 2006. 2005 Wrap-Up. [Online]. Available at

http://www;ipm.iastate.edu/ipm/icm/2006/1-23/wrapup.html. (Veriﬁed 6 June,
2008).

Plant Health Initiative. 2004. Soybean aphid (Aphis glycines) [Online]. Available at
http://www.planthealth.info/aphids_basics.htm (Veriﬁed June 2008)

Powers, T. 0., S. G. Jensen, S. D. Kindler, C. J. Stryker, and L. J. Sandall. 1989.
Mitochondrial DNA divergence among greenbug (Homoptera: Aphididae)
biotypes. Ann Entomol Soc Am, 82: 298—302.

Qureshi, J .A., and J .P. Michaud. 2005. Comparative biology of three cereal aphids on
TAM 107 wheat. Environ. Entomol. 34:27—36.

Ragsdale, D.W. 2006. North Central Soybean Research Program .Plant Health Initiative.

June e-newsletter. www.planthealth.info/e_news/e_news_jun06.htrn (veriﬁed 31
Mar. 2008).

SAS Institute. 2002. SAS/STAT release 9.1. SAS Inst., Cary, NC.

Schmidt, N.S., M. E. O'Neal, J .W. Singer. 2007. Alfalfa living mulch advances biological
control of soybean aphid. Environ. Entomo.36 (2): 146-424.

Smith S. F. and V. A. Krischik. 1999. Effects of systemic Imidacloprid on Coleomegilla
maculata (Coleoptera: Coccinellidae). Environ. Ento. 28: 1189—1 195

43

\

Sunnucks, P. and D. Hales. 1996. Numerous transposed sequences of mitochondrial
cytochrome oxidase I-11 in aphids of the genus Sitobion (Hemiptera: Aphididae).
Mol Biol Evol, 13: 510—524.

Zhuang, 3. 1999. Biological studies of Chinese wild soybean. lst ed. Science Publisher,
Beijing, China (In Chinese).

44

CHAPTER 4

IDENTIFICATION OF QTLS ASSOCIATED WITH SOYBEAN APHID
RESISTANCE IN P1 5675983

ABSTRACT

The soybean aphid (Aphis glycines Matsumura) has become a very important pest of
soybeans in the US. Since it was ﬁrst reported in 2000. Soybean accession PI 5675983,
is a source of aphid resistance identiﬁed in 2005. The aphid resistance in P1 5675983 is
controlled by two recessive genes. The objectives of this study were to identify and map
quantitative trait loci (QTL) associated with aphid resistance in P1 5675983. One
hundred and eighty-eight F2 individuals randomly selected from a cross between Titan
and PI 5675983 were genotyped with 109 polymorphic simple sequence repeats (SSR)
markers. The F2 mapping population was screened for aphid resistance in the ﬁeld in
2005. In 2006 and 2007, the F23 and F 24 lines were evaluated for aphid resistance in the
ﬁeld. Single marker analysis (SMA) revealed 24 markers on linkage groups (LGS) A2,
32, Dla, le, D2, 3, G, J, K, M, and O that were signiﬁcantly (PS 0.05) associated with
aphid resistance. QTL mapping by composite interval mapping (CIM) identiﬁed a QTL
on LG J that explained from 22 to 32.5 % of the phenotypic variation in the ﬁeld. The
SSR markers ﬂanking these resistance genes can be used in marker-assisted selection for

aphid resistance in soybean breeding programs.

45

INTRODUCTION

Soybean aphids were frrst reported in the USA in July 2000. Since that time the
insect has rapidly spread to the major soybean production areas in the USA and Canada
(Plant Health, 2004). Plant damage occurs when large numbers of aphids remove
signiﬁcant amounts of water and nutrients as they feed on leaves and stems, causing
leaves to wilt, curl, yellow, and even leaf drop (DiFonzo and Hines, 2002). Yield losses
caused by soybean aphid were over 50% in Minnesota in severely infested ﬁelds in 2001
(Ostlie, 2002) and up to 52% in China (Wang et al., 1994).

Many soybean breeding programs in the US. are currently conducting research
on aphid resistance. To date only three programs have successfully found resistance to
the soybean aphid, determined the inheritance of resistance and mapped the location of
the resistance gene(s). Single dominant genes were found to control resistance to the
soybean aphid in the cultivars ‘Dowling’ and ‘Jackson’ (Hill et al., 2004, 2006a, 2006b).
The gene in Dowling was named as Rag! (Hill et al., 2006a). Rag] and the resistance
gene (Rag) in Jackson were mapped to a similar genomic region of linkage group (LG) M
using SSR markers (Li et a1. 2007). Mensah et al. (2005) identiﬁed four soybean
accessions among 2,147 with aphid resistance. Accessions PI 5675413 and PI 5675983
have been shown to be controlled by two recessive genes. Two QTLS controlling the
aphid resistance in P1 5675413 have been mapped to LGs F and M, respectively (Zhang
et al., 2008). Three new soybean aphid resistant accessions have been published recently,
PI 243540, PI 5673013 and PI 567324 (Mian et al., 2008). In a report to the USDA, Mian

and Redinbaugh (2007) reported using SSR markers to map a gene for aphid resistance

46

that is different from that in the cultivars Dowling and Jackson. With the current reports
of soybean aphid biotype diversity in North America (Kim et al., 2008, Mensah et al.,
2007, Main et al., 2008), there is the need to map the location of more soybean aphid
resistant genes so that markers ﬂanking them can be used in marker assisted selection and
gene pyramiding. The use of conventional breeding alone would delay the release of
aphid resistant soybeans. During the last few years, molecular marker aided-selection has
been used successfully for the breeding of crops with improved quantitative traits
(Dubcovsky, 2004). Identiﬁcation of molecular markers that are closely linked to the
aphid resistance genes in P1 5675983 would enable the use of marker-assisted selection
for aphid resistance in segregating populations and would facilitate the incorporation of
the resistance genes into adapted northern soybean breeding lines. Over 1,000 SSR
markers have been mapped to an integrated genetic linkage map of the soybean (Song

et al., 2004) and these markers have been successfully employed in marker-assisted
selection by many breeding programs. The objective of this study is to map the aphid
resistance genes in P1 5675983 and to identify ﬂanking markers that could be used in

marker-assisted selection.

MATERIALS AND METHODS

Plant Materials

A population of 388 F2 individuals developed from a single F1 plant from a cross
between Titan, an aphid susceptible cultivar, and PI 5675983 were used for QTL

detection. The initial cross was made in the summer of 2003. A total of one hundred and

47

eighty-eight resistant and susceptible F2 individuals were randomly selected for use as the
mapping population. The 188 individuals were advanced to F23 and F23 in 2006 and 2007

respectively.

Field Planting, Inoculation and Data collection

Three hundred and eighty-eight F2 seeds and the two parents of the cross between
Titan and PI 5675983 were planted in a 12.2- x 18.3-m aphid-proof polypropylene cage
with the 0.49-mm size mesh (Redwood Empire Awning Co., Santa Rosa, CA), in the
ﬁeld at the Agronomy Farm, MSU, East Lansing, MI in June 2005. Two weeks after
planting, when the plants were at the V2 stage (F ehr and Caviness, 1977), each plant was
inoculated by gently placing two Wingless aphids with a camel hair brush on the newly
emerged trifoliate (Mensah et al., 2005). Aphids used in this study were collected from
natural colonies in nearby naturally infested soybean ﬁelds. The F2 plants were planted
approximately 2 cm apart and the parents were planted 5.1 cm apart with two
replications. The F2 and parental plants were rated for aphid damage two, three, and four
weeks after inoculation using a modiﬁed version of the rating scale of 0 to 4 described by
Mensah et al., (2005) (Table 5.1, appendix). Data collected at weeks 3 and 4 were used to
identify DNA markers associated with aphid resistance.

In 2006 and 2007, depending on seed availability, up to twelve F23, F23 seeds per
family and parents were planted in the summer of each year. The aphids used for
inoculation were collected from naturally occurring colonies in the ﬁeld in the respective
years. Inoculation and data collection were conducted as described above for the F2
population. The aphid resistance score was determined as the mean of the scored plants in

each line.

48

DNA Extraction and Marker Analysis

The unopened trifoliate from each individual was harvested from each F2 plant in
the ﬁeld and kept on ice. The leaf samples were kept at -80°C for 24 hours and then
lyophilized for approximately 72 hours. The DNA was extracted with the CTAB
(hexadecyltrimethyl ammonium bromide) method described by Kisha et a1. (1997).

The population and parents were genotyped using SSR marker pairs. The SSR primer
sequences were obtained according to Song et a1. (2004). A total of 1059 SSR markers
were screened for polymorphism between the two parents. The DNA ampliﬁcation of
SSR markers was performed using 15111 polymerase chain reaction (PCR) consisting of
1.0 x PCR buffer (10 mM Tris-HCl, 50 mM KC1,'0.01% Gelatin, pH=8.3), 3.0 mm
MgCl2, 0.2 mM each of dATP, dCT P, dGTP, and dTTP (Sigma-Aldrich, St. Louis, M0),
0.3 uM each of forward and reverse primers, 100 ng of genomic DNA and 1 unit of
Thermus acquaticus (Taq) DNA polymerase. The PCR ampliﬁcation conditions consisted
of an initial denaturing step of 94°C for 4 min, followed by 43 cycles of 25 sec. of
denaturing at 94°C, 25 sec. of annealing at 47°C, and 25 sec. of extension at 68°C, with a
ﬁnal seven minute extension at 72°C before cooling down to 4°C in :1 MJ TetradTM
thermal cycler (MJ Research, Waltham, MA). Gel electrophoresis was performed using
non-denaturing polyacrylanride gels as described by Wang et al. (2003). After
electrophoresis, gels were photographed under UV light and scored. The SSR markers
were scored co-dominantly as ‘a’ = homozygous for the marker allele from the resistant
parent, ‘h’ = heterozygous for the marker, or ‘b’ = homozygous for the marker allele

from the susceptible parent. Situations where it was difﬁcult to distinguish ‘a’ and ‘h’

49

were scored as‘d’ and those where ‘b’ and ‘h’ could not be distinguished were scored as

6 9

C .

Linkage Map Construction

Analysis of linkage between the aphid resistance genes and associated SSR
marker loci, and calculation of their relative map positions, was performed with J oinMap
3.0 (Van Ooijen and Voorrips 2001) using the Kosambi mapping function. A logarithm
(base 10) of the odds (LOD) score of 3 or higher was used to identify those loci linked to
aphid resistance. The best position of each marker was searched by comparing the
goodness-of-ﬁt for each tested position to determine the order and distance among

markers within each linkage group.

QTL Analysis

Analysis of variance (ANOVA) was performed for phenotypic data from the ﬁeld
data using the GLM procedure of SAS (1999). QTL analysis was performed in WinQTL
Cartographer V2.5 (Wang et al., 2005). The trait data used in the analysis was the aphid
rating scores at three and four weeks after inoculation. Single marker analysis was
performed and markers showing signiﬁcance (p_<_ 0.05) were identiﬁed. Composite
interval mapping (CIM) was performed to detect QTLS for aphid resistance using QTL
Cartographer V2.5 with the standard model Zmapqtl 6. The CIM analysis uses markers
other than the interval being tested as cofactors to control the genetic background (Zeng,

1994). The forward and backward regression method was used to select markers as

50

cofactors. The walk speed chosen for CIM was 2 cM and a window size of 10 cM. The
empirical LOD threshold at 5% probability level was determined by a 1,000-permutation
test (Churchill and Doerge, 1994). QTLS were graphically displayed with line maps using

MapChart (V oorrips, 2002).

RESULTS

Phenotypic Data

Titan and PI 5675983 consistently showed signiﬁcant difference in aphid
resistance in each trial for both three-week and four-week ratings. The resistant parent PI
5675983 always had a signiﬁcantly (P < 0.05) lower score than Titan. In general, the
four-week rating score was higher than the three-week rating score for the same line in all
three years of evaluation (Table 5.2, Appendix). The damage rating for the F2 population
(Titan x PI 5675983) showed continuous variation and approximately normal
distribution, suggesting that aphid resistance is a quantitative trait controlled by multiple
genes and ranged from 0.5 to 4 (Fig 4.1 a). From within this population all the resistant
lines and random selection of susceptible lines were chosen to generate the mapping
population of 188 individuals. The phenotypic data for the mapping population was
approximately normally and continuously distributed in all three years for data collected
four weeks after inoculation (Fig. 4.1 c, e and g). This indicates that more than one
recessive gene may control aphid resistance in P1 5675983. The data collected three
weeks after inoculation in 2005 and 2006 was skewed towards the resistant parent and

ranged frOm 0 to 3 ( Fig.4.] b and d). In 2007, three weeks post-inoculation, the lines

51

were quite evenly distributed over the lower part of the rating scale and ranged from 0.5

to 3.5 (Fig.4.le).

52

Figure 4.1

a) Distribution of damage rating for F2 population
(Titan X PI 5675983) in 2005 four weeks after inoculation

Number of Plants
00
o
‘ PI 5675983

   

5.1 1.5 2 2.5 3 3.5 4
Damage Rating

b) Distribution of damage rating for F2 mapping population
(188 individuals) in 2005 three weeks after inoculation

1 20 / PI 5675983

 

8 1
g 1001‘
g 80- E
33 i-
E 40 1 I
= 20
z 0 L .5 7 ,- _ L. ,
512 3 3. 5 4
Damagze Stating

53

Fig 4.1 (cont’d)

c) Distribution of damage rating for F2 mapping population
(188 individuals) in 2005 four weeks after inoculation

501

A
C?
‘— PI 5675983

N
‘?
‘ Titan

.8
‘3

Number of Plants
co
9

 

0
IA
_s

1 .5 2 2.5 3 3.5 4
Damage Rating

11) Distribution of damage rating for F23 mapping population
(188 individuals) in 2006, three weeks after inoculation

60 1 PI 5675983

or
c?

40i
30“
20‘

10‘ i

Number of Plants
Titan

 

 

s1 1 .5 2 2.5 3 3.5 4
Damage Rating

Figure 4.1 (cont’d)

e) Distribution of damage rating for F2,; mapping population
(188 individuals) In 2006, four weeks after inoculation

 

   

60 - m
8 50 g
540 a
a a 5
§ 30: a: 1::
3 20 ‘ {
g1o- l
2 0 4
51 1.5 2 2.5 3 3.5 4
Damage Rating
f) Distribution of damage rating for F23 mapping population
(188 individuals) in 2007, three weeks after inoculation
501
40 _ PI 5675983

30‘
201
10"

‘ Titan

1

Number of Plants

 

51 1 .5 2 2.5 3 3.5 4
Damage Rating

55

Figure 4.1 (cont’d)

9) Distribution of damage rating for F2,, mapping population
(188 individuals) in 2007, four weeks after inoculation

g 401 g Titan
Vi \

g 30“ {g
o _ E:
.9 2°- l
S 1o~
Z

0..

 

   

51 1.5 2 2.5 3 3.5 4
Damage Rating

Figure 4.1: The damage rating distributions of: a) the F2 population of the cross between

Titan and PI 5675983; b-g) 188 individuals of mapping population for 2005, 2006 and
2007 three and four weeks after inoculation.

56

Identification of QTL for Soybean Aphid Resistance

Of the 1050 SSR markers tested for polymorphism between the two parents, only
109 that were polymorphic, easy to score and had good ampliﬁcation were used to
genotype the mapping population. Out of 109 polymorphic markers 58 were placed into
23 linkage groups that were segments of the 20 linkage groups on the consensus map by
Song et a1. (2004). The remaining markers were unanchored but had some markers
signiﬁcantly associated with aphid resistance ﬁom SMA. The total map distance of the
23 linkage groups was 760 cM, with an average interval length of 10.8 cM and covering

38% of the soybean genome.

In the SMA, 24 markers were found to be signiﬁcantly associated with aphid
resistance in both years (p50.05). In 2005, at three and four weeks after inoculation ten
and nine markers respectively were Signiﬁcantly associated with aphid resistance with
four signiﬁcant in both weeks. Six markers were signiﬁcantly associated with resistance
in 2006. Satt529 and Sattl7l were signiﬁcant in both weeks three and four weeks after
inoculation. In 2007, seven and six markers were associated with aphid resistance three
weeks and four weeks after inoculation respectively. Five markers were signiﬁcantly
associated with aphid resistance in both weeks. Only SattZ80 and Satt529 on LG J were
consistently associated with aphid resistance in 2005, 2006 and 2007 (Table 4.1, 4.2). In
the CIM analysis, empirical signiﬁcance threshold was computed as LOD score of 5.05
using 1000 permutations in the mapping population in 2005. However, none of markers
that were signiﬁcantly associated aphid resistance in 2005 had a LOD score equal or

greater than 5 .05. The putative QTL on LG J had the highest LOD score of 4. l 7 (Fig.

57

4.2A) and accounted for 23.0% of aphid resistance variation three weeks after inoculation
in 2006. This putative QTL on LG J was also observed in 2007 week 4 with a LOD of
3.08 and an R2 of 32.5 % (Fig. 4.2 A). Another putative QTL (LOD = 4.19 ) and 112 of
46.6% was detected on LG C1 in 2005, three weeks after inoculation close to the SSR
marker Sat_l 78 (Fig.4.2C). In 2007, CIM analysis the empirical signiﬁcance threshold
computed was 4.65 after 1000 permutations. The only QTL identiﬁed in 2007 was in
week 4 with a LOD of3.29 and R2 of 22.0% (Fig. 4.213) and signiﬁcant in SMA at

p<0.0001 (Table 4.1, 4.2). This putative QTL on LG J was closer to Satt529.

58

Table 4.1: Markers signiﬁcantly associated with soybean aphid resistance in
P1 5675983 in single marker analysis in 2005, 2006 and 2007 at three weeks after

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

inoculation.
Marker Linkage Position 2005 week3 2006 week3 2007 week3
Group (cM)
Satt34l A2 77.69 0.007** NS NS
Satt304 32 65.55 0.024* NS NS
Sat_355 32 66.23 0.036* NS NS
SattO70 32 72.81 NS NS 0.01 1*
Satt321 D1 a 50.16 NS NS NS
SattO95 le 25.60 0.015* NS NS
SattOOS le 75.29 NS NS NS
SattZ7l le 137.05 NS NS NS
Satt208 D2 67.91 NS NS NS
Satt699 E 41.24 NS NS NS
Satt685 E 56.69 NS NS 0010*
Sattl 63 G 0.00 NS NS 0025*
Satt280 I 38.70 0.011* 0.286 0.032*
Satt686 1 40.50 0.019* NS NS
Satt529 J 41.29 0.004** 0.011* 0.014*
Satt285 J 25.51 NS NS NS
Satt380 J 43.11 NS NS 0016*
Satt215 J 44.81 NS NS NS
Satt628 K 49.59 NS NS NS
Satt273 K 56.62 NS NS 0039*
Satt435 M 38.94 NS 0044* NS
Sat_038 0 112.17 0.05* NS NS
SattZl6 - NS NS 0.352
Satt171 - NS 0.009** NS

 

 

 

 

 

 

NS= not signiﬁcant 0.05 probability level. Markers signiﬁcant at 5%, 1%, 0.1% and 0.01% levels are
indicated by *, **, ***, and **** respectively. Linkage group names and relative position for the markers

were assigned according to the Soybean Composite Map (Song et al., 2004).

59

 

Table 4.2: Markers signiﬁcantly associated with soybean aphid resistance in

P1 5675983 in single marker analysis in 2005, 2006 and 2007 at four weeks after
inoculation.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Linkage Position
Marker Group (CM) 2005 week4 2006 week4 2007 week4
Satt34l A2 77.69 NS NS NS
Satt304 32 65.55 0012* NS NS
Sat_ 355 32 66.23 NS NS 0.684
SattO70 32 72.81 NS NS 0.112
Satt321 D1 a 50.16 NS 0027* NS
Satt095 le 25.60 0005** NS NS
SattOOS le 75.29 NS NS NS
Satt27l le 137.05 0024* NS NS
SattZOS D2 67.91 0008" NS NS
Satt699 E 41.24 NS 0012* NS
Satt685 E 56.69 0.053 0024* 0034*
Satt163 G 0.00 0024* NS 0026*
Satt280 J 38.70 0008** NS 0006"
Satt686 J 40.50 NS NS NS
Satt529 J 41.29 0.001 ** 0.004** 0000****
SattZSS J 25.51 0.001*** NS 0.114
Satt3 80 I 43.11 NS NS 0020*
Satt215 J 44.81 0030* NS NS
Satt628 K 49.59 0012* NS NS
Satt273 K 56.62 NS NS 0.492
Satt435 M 38.94 NS NS NS
Sat_038 0 112.17 NS NS 0.083
Satt21 6 - - 0021* NS 0011*
Satt17l - - NS 0003" NS

 

 

 

 

 

 

 

NS= not signiﬁcant 0.05 probability level. Markers signiﬁcant at 5%, 1%, 0.1% and 0.01% levels are
indicated by *, **, ***, and **** respectively. Linkage group names and relative position for the markers
were assigned according to the Soybean Composite Map (Song et al., 2004).

60

LGJ

0.0 $311280 0

 

 

 

—e— week3

 

- -I — week4

 

 

 

ﬂee/n

QXGGM

 

 

 

 

 

 

41.0 Satt529 __

 

 

Figure 4.2A: Putative QTLS associated with Aphid resistance on linkage group J based
on phenotypic data from 2005 week3 and week4 data. The map distances between the
markers are given in cM (centimorgans). The linkage groups are named according to
Song et a1. (2004). The LOD threshold was set at 3.0.

61

LGJ

$811280 0,—‘le_00,# 01

 

 

 

. .. + week3
E 3. - -I -
29. _ week4

 

 

 

 

 

 

 

 

Satt529

 

Figure 4.2 B: Putative QTL associated with Aphid resistance on linkage group J based
on phenotypic data from 2007 week 3 and week4 data. The map distances between the

, markers are given in cM (centimorgans). The linkage groups are named according to
Song et a1. (2004). The LOD threshold was set at 3.0.

62

 

LGC1

 

 

 

 

 

 

 

 

 

 

 

 

0.0 Satt136 °_ "‘ N‘ “i “1 "‘
24.3 Satt713
+ week3
—_|— week4
. 60.2 Sat_172

 

Figure 4.2 C: Putative QTL associated with Aphid resistance on linkage group C1 based
on phenotypic data ﬁ'om 2005 week3 data. The map distances between the markers are
given in cM (centimorgans). The linkage groups are named according to Song et a1.
(2004). The LOD threshold was set at 3.0.

63

DISCUSSION

Markers associated with soybean aphid resistance were detected in all three years
of this study. The markers found to be signiﬁcantly associated with aphid resistance in P1
5675983 have not been previously reported. The markers SattZ80 and SattSZ9 on LG J
were consistently associated with aphid resistance in SMA in all three years and at two
screening dates. In all but one year (2006) and one data collection time (2005 week3), a
putative QTL was identiﬁed on LG J between Satt280 and SattSZ9. The putative QTL on
LG J was also tested in 44 resistant lines derived from a cross with P1 5675983 as the
resistant parent but in different genetic backgrounds. Both markers ﬂanking the QTL
were found to be associated with resistance in all 44 lines (Menghan Liu, pers. Comm).

The ﬁrst report of markers associated with aphid resistance ( Li et al., 2007),
using SSR markers and data for aphid resistance from F23 populations developed from
crosses between Dowling and the two susceptible soybean cultivars ‘Loda’ and ‘Williams
82’, and between Jackson and Loda. The resistance genes Rag] (Hill et al., 2006a) and
Rag ﬁ'om cultivar Jackson segregated 1:2:1 in the F23 populations and mapped to the
same location on LG M between the markers Satt435 and Satt463. This suggests that the
two resistance genes maybe allelic or tightly linked. The markers associated with aphid
resistance in P1 5675413 (Mensah et al., 2005), have been recently mapped using a
population of 228 recombinant inbred lines (RILS). Using CIM two, QTLS controlling the
aphid resistance in P1 5675413 were found on LGs F and M, respectively (Zhang et al.,
2008). Mian and Redinbaugh, (2007) reported using SSR markers to map a gene for

aphid resistance that is different from the aphid resistance genes from cultivars Dowling

64

and Jackson (Hill et al, 2004). This resistance gene is from a new aphid resistance source
described in Mian et al., (2008).

The QTLS found in this study will assist breeders in marker-assisted selection
(MAS) when breeding for aphid resistance using P15675983. Currently, MAS is being
used in breeding for soybean aphid resistance, using the markers ﬂanking Rag] and Rag
genes to accelerate the breeding process and reduce the cost associated with aphid
resistance bioassays. After a year and a half of MAS, aphid resistant 3C3F2 lines
containing Rag] have been released to public and private soybean breeders (Li et al.,
2007). I

The initial expectation was to identify two QTLS associated with aphid resistance
in P1567 5983 corresponding to the two recessive genes controlling aphid resistance in
the germplasm accession. During the QTL mapping of aphid resistance in this population
one big challenge encountered was the surprisingly low number of polymorphic markers
between Titan and PI 5675483. Being a wide cross the expectation was to have more
polymorphic markers but this was not observed due to technical difﬁculties encountered
while screening for polymorphism. Only one locus was mapped and may be attributed to
the mapping population being used was on an individual plant basis. To correct this
situation an F 43 populations of the same cross (Titan x P15675983) was phenotyped in
the greenhouse and genotyped with markers found in this study to be associated with
aphid resistance. Only the QTL on LG I was detected. In the study by Zhang et al., 2008,
they found that the two resistant genes in P1 5675413 were expressed differently between
ﬁeld and greenhouse trials. Only one gene was expressed in the greenhouse while both

genes are expressed in the ﬁeld.

65

The results from this study will guide our ﬁrture investigation of QTLS underlying aphid
resistance in P1 5675983. Less than 10% of the available SSR markers were mapped and
many of them were unlinked, therefore more marker data needs to be obtained. In the
future careﬁrl consideration must be given to the type of mapping population chosen for
different patterns of inheritance. The knowledge of markers associated with aphid
resistance in different sources is very essential as it will be useful in gene pyramiding
(Mittal et al., 2008). Combining different sources of aphid resistance is important to
develop durable management programs, especially with the rapid development of new
aphid biotypes in response to resistance gene deployment (Kim et al., 2008, Mensah et al.
2007). Although these reports are the ﬁrst few evidence of biotypes of soybean aphids in
North America, in the Russian wheat aphid many biotypes have arisen in response to the
deployment of aphid resistance genes (Harvey et al., 1997, Haley et al., 2004). Gene
pyrarniding would be useful to introduce genes for resistance to multiple biotypes of the
soybean aphid, as and when new sources of resistance are identiﬁed in different
environments. Combining aphid resistance genes may decrease the problem of aphids
overcoming resistance since the pest would need to deal with each resistance gene. With
the resistance genes in P1 5675413, PI 5675483, Dowling and Jackson being different

this is a very good opportunity to stack these genes as new biotypes evolve.

66

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69

APPENDIX

70

Table 5.1: Visual rating scale used to establish the Damage Index (DI) of a plant.
Damage Number of

 

Rating Aphids Aphid Colony Plant Charactenstrcs
0.0 0 - Normal and healthy
0.5 S 10 No colony Normal and healthy
1.0 S 100 Young leaves Normal and healthy
1.5 101-150 Young leaves Normal and healthy
2.0 151-300 Young “2:11am tender Normal and healthy
Both young and old
2.5 301-500 leaves, undersides and Normal and healthy
tender stem
Both young and old '
3.0 501-800 leaves, undersides of Leaves shiny, slightly curled,
leaves and hard stems
Leaves curled and slightly
On all leaves and stems,
3.5 2800 few cast skins yellow, plants stunted, no sooty
mold,
plants sttmted, leaves severely
4.0 2800 On all leaves and stems curled, yellow, covered with

many cast skrns sooty mold

71

Table 5.2: Phenotypic data for 188 individuals of mapping population [F2 (2005),
F2,3(2006) and FE(2007)] collected three and four weeks after inoculation.

 

 

2005 Fz'l’ 2006 F 2:33: 2007 F 2341
Individual Week 3 Week 4 Week 3 Week 4 Week 3 Week 4

1 1.5 2 0.7 1.5 0.7 1.1
2 2 3.5 0.7 1.8 - -
3 2.5 3.5 1.0 2.1 1.1 1.5
4 3 3 1.0 2.4 - -
5 2.5 3 0.7 2.7 1.6 1.8
6 2.5 3 - - - -
9 2 3.5 2.5 3.0 2.6 2.0
10 2 4 1.7 2.3 1.1 1.6
11 3 3.5 0.8 2.0 1.6 3.0
12 2.5 3 2.0 3.0 2.7 3.0
13 2.5 3 0.7 1.0 0.5 2.5
14 2 3 1.2 1.5 - -
17 l 2 0.5 0.7 0.5 1.2
18 3 3 5 1.5 2.0 1.7 2.8
19 2 3 2.0 2.0 2.0 2.0
20 l 2.5 1.5 1.5 1.9 2.8
22 2.5 3 5 0.9 1.2 1.7 2.9
24 2 3 1.0 1.5 0.8 1.1
27 2.5 3.5 1.3 1.8 1.1 3.0
28 1 2 0.9 1.0 1.4 2.5 '
32 l 2 1.3 1.8 1.5 2.4
33 1.5 2 1.0 2.0 0.8 2.3
34 l 2.5 2.0 3.0 3.0 3.6
37 2 3 - - 3.0 3.8
38 1 2 2.5 3.0 2.5 2.0
40 l 2 1.0 1.2 0.7 1.9
43 1 2 0.8 1.3 1.2 1.0
44 1 2 0.8 1.0 1.5 2.9
45 1.5 2.5 1.0 1.3 2.7 2.0
46 l 1.5 1.0 0.9 1.8 2.3
47 l 2 1.5 2.3 1.9 2.0
49 2 3.5 2.5 3.5 2.5 4.0
51 1 2 1.0 2.2 - -
52 l 2.5 2.3 3.0 3.5 3.0
53 l 2.5 2.0 2.5 2.5 2.5
55 l 3 - - - -
56 2 2.5 2.6 3.5 - -
57 l 1.5 - - - -
58 l 1.5 - - - -
59 2 3.5 2.0 2.8 2.7 3.5
61 l 1.5 , 2.5 2.8 3.0 3.5
62 1 1 2.0 2.5 - -
66 0.5 2.5 2.0 2.9 2.7 3.3

 

T: Damage rating of individual F2 plant, I: Mean Damage rating for up to 15 plants

72

Table 5.2 cont’d

 

 

2005 F11 2006 F23 2007 F 2:4
Individual Week 3 . Week 4 Week 3 Week 4 Week 3 Week 4

67 0 3 - - - -

68 1 2.5 2.5 3.0 3.5 4.0
70 0 1 1.3 1.9 3.0 4.0
73 l 2 2.5 2.9 3.3 2.7
74 2 2.5 2.0 2.5 - -

75 2 2.5 1.0 1.2 1.4 2.1
77 l 2.5 1.2 2.0 2.0 3.0
80 1.5 2 1.5 2.3 0.5 0.9
81 1.5 2.5 1.7 2.7 1.0 1.2
83 2 3 1.0 2.2 - 1.4 2.0
84 1 2 1.5 2.9 2.5 3.0
85 l 1.5 2.5 3.2 2.8 4.0
86 l 1.5 2.0 2.4 2.1 3.7
87 3 3.5 2.1 2.6 2.7 4.0
88 3 4 2.5 3.0 3.5 4.0
89 l 1.5 - - - -

92 0.5 1 - - - -

93 1 2.5 2.0 2.6 2.0 2.3
94 0.5 1 2.5 3.3 2.0 2.0
95 0.5 l 0.5 1.0 0.5 1.0
96 l 2 1.0 1.9 1.0 2.5
98 2 2.5 2.0 3.2 2.0 3.4
99 l 1.5 2.0 2.4 1.0 2.0
100 l 2.5 1.5 2.7 2.8 4.0
101 1 3.5 2.0 3.1 - -

102 1 2 1.5 1.2 0.5 4.0
103 1 2 2.0 2.5 3.0 4.0
105 3 4 2.5 3.2 3.1 4.0
106 1 3.5 1.5 1.9 2.0 3.0
107 1.5 2 1.3 1.3 2.6 2.7
108 l 2 2.0 2.6 2.1 4.0
109 l 2.5 1.0 1.7 2.4 3.0
110 l 2.5 1.0 2.2 1.0 1.8
111 1.5 2 2.0 2.8 2.7 3.3
112 2 3 2.0 2.6 - -

113 2 3 1.0 1.0 2.2 2.8
114 1.5 2.5 1.0 1.8 1.0 1.4
115 2 2.5 1.5 2.2 - -

116 1.5 2 1.0 1.8 - -

117 l 2 1.0 2.5 0.5 3.0
118 1 2.5 1.0 2.2 - -

119 l 1.5 1.0 1.0 - -

 

’r: Damage rating of individual F2 plant, I: Mean Damage rating for up to 15 plants

73

Table 5.2 cont’d

 

 

2005 F21 2006 F223: 2007 F 2241
Individual Week 3 Week 4 ‘ Week 3 Week 4 Week 3 Week 4
121 1.5 2 1.0 2.2 2.1 - 2.5
122 l 2 0.5 1.3 1.5 2.1
123 1.5 2 1.0 2.5 - -
124 l 2 1.5 2.5 - -
126 l 1.5 - - - -
128 3 4 2.5 3.0 3.5 4.0
129 3 4 2.5 3.0 3.0 4.0
131 l 2 0.5 1.5 - -
132 3 3.5 2.3 3.1 - -
133 3 3.5 1.3 1.5 2.2 2.1
135 1 1.5 2.0 3.0 2.4 2.8
137 l 2.5 1.0 2.0 2.7 2.5
138 1 2.5 1.0 2.2 2.5 4.0
139 2 3 1.0 2.3 - -
140 3 4 2.0 3.0 - -
142 2 2.5 2.0 3.3 3.5 4.0
144 2 3 2.0 2.9 3.5 3.8
145 2 3.5 2.0 2.6 2.4 3.0
147 l 2 2.5 3.5 3.3 2.3
149 1 2 2.0 2.8 3.4 4.0
150 0.5 1.5 1.5 1.5 2.7 3.3
151 2 3 1.0 2.4 3.4 1.8
154 1 2 - - - -
157 l 1.5 1.2 1.3 1.0 1.5
158 l 1.5 1.0 1.0 1.5 1.2
159 0.5 1.5 - - - -
160 3 4 2.5 3.5 3.5 4.0
163 2 3 1.5 2.2 2.7 3.0
164 l 1.5 1.0 1.3 2.2 2.9
165 l 2.5 2.0 3.2 3.0 3.0
169 1 3 1.0 1.0 - -
170 0.5 2.5 - - - -
175 1 2 2.0 3.0 - -
177 0.5 1.5 2.0 2.7 3.0 3.5
181 0.5 3 1.9 2.4 2.8 3.0
182 l 3 1.5 2.1 3.0 3.0
183 2 3 1.5 2.5 - -
184 2 3 1.0 2.7 - -
187 l 2 2.0 2.8 3.5 3.0
188 1 3 1.5 2.1 2.9 4.0
189 2.5 4 2.0 3.2 3.0 3.0
190 3 4 - - - -
191 ' 2 3 2.0 2.5 - -

 

T: Damage rating of individual F2 plant, I: Mean Damage rating for up to 15 plants

74

Table 5.2 cont’d

 

 

2005 F21 2006 F231: 2007 F2241
Individual Week 3 Week 4 Week 3 Week 4 Week 3 Week 4

192 2 3 1.5 2.0 2.5 3.5
193 2 3 1.2 2.3 1.5 3.0
195 1.5 2.5 1.0 2.1 2.4 2.5
198 l 2 1.0 1.9 1.0 2.0
199 l 2 2.2 2.7 3.0 1.6
200 l 1.5 - - - -

201 2 3.5 1.8 2.7 2.7 4.0
203 l 2 2.0 2.6 1.7 2.5
204 l 2 . - - - -

207 1 1.5 1.5 1.3 3.0 1.5
213 l 1 - - - -

214 1 1 1.5 2.3 2.0 1.5
216 1 1.5 - - - -

217 1 2 1.8 2.7 1.5 2.0
218 1 2 2.0 3.0 2.8 2.0
231 l 2 1.3 1.8 2.9 2.5
235 1 3 2.0 2.7 2.5 3.5
236 1.5 3 2.0 2.7 2.7 3.0
237 1.5 3 1.2 2.2 3.0 4.0
239 1 2 2.0 2.7 3.2 3.5
240 l 2 1.0 2.0 1.0 2.0
242 1 2 0.8 1.9 3.0 3.0
247 1 2 1.3 1.9 2.7 3.0
249 l 2.5 1.0 2.5 2.0 3.0
250 l 2 2.0 2.8 3.0 2.5
251 1 1.5 2.0 3.0 3.5 3.5
253 l 2 2.0 3.1 - -

255 2 3.5 2.0 3.0 3.0 3.0
258 2 3 1.5 1.9 2.6 3.0
265 2.5 3.5 1.5 2.0 3.0 3.0
266 2 3.5 2.0 3.0 - -

267 2 3 1.0 2.5 2.8 2.5
268 2 3 1.0 1.9 3.0 4.0
270 1.5 3 1.0 2.2 - -

274 3 4 1.0 1.9 - -

293 2 3 2.0 3.0 1.5 1.5
302 2 3 2.0 2.8 3.0 3.0
306 1.5 3 2.5 3.2 3.5 4.0
327 l 1.5 1.0 1.8 1.2 2.4
329 l 2 2.3 2.8 - -

349 1 3 1.0 2.5 1.5 2.0
355 1.5 3 1.5 1.9 - -

386 1.5 2.5 1.0 2.4 - -

 

T: Damage rating of individual F2 plant, I: Mean Damage rating for up to 15 plants

75

Table 5.2 cont’d

 

 

2005 F21 2006 F231 2007 F2241:
Individual Week 3 Week 4 Week 3 Week 4 Week 3 Week 4

387 I 2 1.5 1.8 2.1 1.5
402 2 3.5 1.0 1.8 - -
403 1.5 2 1.0 1.8 3.5 4.0
404 2 2.5 . 1.5 2.3 1.0 2.5
407 1.5 2 2.0 2.5 1.5 4.0
503 I 2 1.5 2.6 - -
505 l 2 1.5 2.0 - -
508 I 2.5 1.5 2.0 2.9 -
512 I 2 1.5 2.0 2.8 -
519 1.5 2 2.5 4.0 3.0 -
280 2 2.5 2.0 2.8 3 3.0
340 3 4 - - - -
400 2 3.5 1.5 1.5 2 3.0
487 2 3 2.5 3.3 3 3.5
492 2 3 - - - -
521 l 1.5 - - - -

T 2.5 3.0 4.0

N 0.7 1.0 1.5

 

1: Damage rating of individual F2 plant, I: Mean Damage rating for up to 15 plants

76

LGE LGE LGC2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0 Satt452 0.0 Satt045 0.0 SattZ89
12.5 Sattl 85
15.5 Sattl l7 .
18.9 Satt656
29.8 Satt49l
35.3 Sat1297
46.1 Satt706
64.7 Satt685
106.5 Satt606

 

 

 

Figure 5.1: Linkage map of 188 F2 lines from cross Titan and PI 567598B constructed
using JoinMap 3.0 with a 0D grouping threshold 3.0. The linkage groups were named
according to Song et al. (2004) and the map distances between the markers are given in
cM (centiMorgans)

77

LGK

 

 

 

 

 

 

0.0 Satt020
18.3 Satt601
41.3 Sattl68
66.0 Satt304
Fig 5.1 (cont’d)

0.0

18.4

LGF

 

 

 

 

 

 

LGK
Satt215 0.0
Sat_366 20.7
49.1

 

 

 

78

Sattl78

SattSSZ

Satt273

LGAl LGJ ' LGle

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0 Satt21] 0.0 Satt280 0.0 StagaOOZ
20.3 Satt27l
24.0 Satt529
27.3 Satt236
43.9 Satg002
51.6 SattS45
Fig 5.1 (cont’d)

79

LGC1

 

0.0

 

24.3

 

 

 

60.2

Sattl36 0.0
Satt7l3

28.1
Sat_l72 53-4

Fig 5.1 (cont’d)

 

 

 

 

 

LGle.

Sat_l63 0.0
Sat_3 08 3 0.2
Sattl 63

80

 

 

 

 

SattO95

SattZl6

LGL

 

 

 

 

 

0.0 Satt418 0.0
23.3 Satt306
27.1
48.4 Sat_l9l
Fig 5.1 (cont’d)

 

 

 

 

LGE

 

Satt720 0.0

Satt65 l

 

33.5

81

 

 

Satt699

Satt685

LGF LGM

 

 

 

 

 

 

 

 

 

 

 

0.0 Satt297 0.0 Satt418
16.7 Satt656
34.7 Satt289 333 83‘8“;
60.8 Satl9l
67.9 Satt316
Fig 5.1 (cont’d)

82

LGle LGH LGle

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0 SattOOS 0.0 Satt279 0.0 Satt701
33.2 Satt703 _
36.0 Sat_l38 33 1 Sattooz
67.2 Satt669
Fig 5.1 (cont’d)

83

Table 5.3: Information about all polymorphic simple sequence repeat (SSR) markers
from Fmpulation of Titan and PI 567598B.

 

 

 

 

 

 

 

 

Marker Integrated map a , h' b X2 Signiﬁcance
name (cM) ' ' levelT
Linkage Group A1
Sattle 71.39 39:74:39 0.6 -
SattZ36 93 .23 25:42:27 1.1 -
SattS45 95.96 39:74:39 0.1 -
Linkage Group A2
SattS89 33.96 58:92:48 4.8 *
Sattl77 36.77 34:32:23 9.7 ***
Sattl87 54.92 -
Satt341 77.7 16:29:36 16.4 ****
Sat_138 123.257 25:14:51 21.6 ****
Linkage Group 31
Satt665 96.36 36:70:48 3.1 -
Linkage Group 32
Sattl68 55.2 50:88:46 0.5 -
Satt304 65.55 47:67:72 18.5 *****
Sat_355 66.235 11:17:16 3.4 -
SattOZO 72.13 41:95:50 1 -
SattO66 78.844 25:14:51 57.7 ****
SattO70 72.808 40:18:16 35.1 ****
SattO63 93.488 22:22:47 38.0 ****
Satt474 75.346 25:51:18 1.7 -
Satt601 67.23 18:51 :24 1.6 -
Linkage Group C1
Satt66l 74.36 34:77:46 1.9 -
Satt136 75.11 48:96:44 0.3 -
Satt36l 75.52 22:66:60 21.2 *****
Satt7l3 88.95 49:98:35 1.9 -
Linkage Group C2
Sat_246 91.81 16:26:14 95.2 *****
SattB76 97.83 50:39:36 20.8 *****
SattZ89 112.35 34:59:68 14.4 ****
Satt3l6 127.69 64:56:38 21.9 *****
Linkage Group Dla -
Satt321 50.16 35:79:41 0.1 -
Sat_346 53.671 44:00:00 132 *****
SattZ95 55.221 19:39:34 7 **
SattS80 62.367 29:40:25 2.4 -
SattO77 77.49 23:33:38 13.1 ****
Satt408 106.69 35:89:46 2 -

 

1 “-“ means not signiﬁcant at 0.05 probability level

0.001 probability levels, respectively.

-***
9 9 9

and *** means signiﬁcant at 0.05, 0.01, and

Table 5.3 (Cont’d)

 

 

 

 

 

 

Marker Integrated map . . 2 Signiﬁcance
a.h.b X
name ( cM) levelT
Linkage Group le
SattO95 25.6 54:72:48 5.6 *
Satt701 40.04 26:63:24 1.6 -
SattOOS 75.29 36:78:51 3.2 -
SattOOS 75.29 36:78:51 3.2 -
Satt350 76.6 35:19:29 25.3 *****
Satt703 98.74 26:49:37 3.9 -
StagaOOZ 126.45 13:38:22 2.3 -
SattZ7l 137.06 38:72:58 8.2 **
Linkage Group D2
SattOOZ 47.7 32:63 :39 1.2 -
Satt669 67.7 26:47:49 15.1 *****
Satt397 69.296 35:33:16 12.4 ****
Satt311 84.62 -
Linkage Group E
Satt720 20.80 63:83:29 13.7 ****
Satt651 32.10 55:94:29 8.2 **
Satt606 39.77 17:36:40 16.1 ****
Satt699 41.24 44:91 :46 0.1 -
Satt602 41.68 37 :0:48 87.8 ****
Sat_172 42.74 19:66:6 22.2 ****
Sat_380 43.29 39:28:21 19.0 ****
Satt706 43.36 19:58:39 4.3 -
Satt49l 43.64 22:46:25 0.2 -
Sattl85 44.76 61 :1 :29 10.9 ***
Satt452 45.10 65:3:25 11.6 ***
Sattl 17 45.78 22:46:26 0.4 -
SattO45 46.65 20:46:28 1.4 -
Satt685 56.69 51:69:52 6.7 **
SattSS3 67.92 50:0:39 91.2 ****
Linkage Group F
Satt656 22.67 43:83:49 0.9 -
Linkage Group G
Sattl63 0.00 31:68:74 29.3 *****
Satt275 2.20 51 :91 :45 0.5 -
Sat_l 68 3.90 62:8:23 9.7 ***
Sat_l63 10.06 16:49:29 3.8 -
Satt356 12.18 -

 

“-“ means not signiﬁcant at 0.05 probability level; *,**, and *** means signiﬁcant at 0.05, 0.01, and 0.001
probability levels, respectively

85

Table 5.3. (Cont’d)

 

 

 

 

 

 

 

 

Marker Integrated a'h°b X2 Signiﬁcance
name map( CML ' ' levelT
Linkage Group G
Sat_315 27.48 15:16:63 89.9 ****
Sat_308 43.09 14:56:24 -
Sat_358 45.49 68:0:26 13.2 ****
Sat_ 223 61.64 66:2:26 12.0 ****
Linkage Group H
SattZS3 67.17 22:21 :50 44.8 ***"
SattZ79 68.5 137:00100 41.1 ***"
Satt353 84.8 44:79:55 3.6 -
Linkage Group 1
Satt650 63.33 50:106z3l 7.2 **
Satt623 95.519 -
Linkage Group J -
SattZ85 25.51 31:44:18 3.9 -
SattZSO 38.23 33:87:50 3.5 -
Satt686 40.67 48:80:46 1.2 -
SattSZ9 41.19 37:98:53 3.1 -
Satt622 42.35 22:46:25 0.2 -
Satt380 43.11 39:28:21 19.0 ****
SattZlS 44.81 19:43:22 0.3 -
Sat_366 52.10 25:36:30 4.5 -
Linkage Group K
Sattl78 40.80 39:97:52 2 -
SattSSZ 46.44 43:76:50 2.3 -
Satt628 49.59 70:74:41 16.5 *****
Satt673 50.80 40:104:43 2.5 -
Satg002 51.45 28:103155 10 ***
SattZ73 56.62 44:96:43 0.5 -
Linkage Group L
Sat_191 23.1 30:62:55 12.1 ****
Satt418 30.93 35:94:42 2.3 -
Satt313 34.54 46:80:51 1.9 -
Linkage Group M
Satt636 5 48:90:45 0.1 -
Satt435 38.94 55:93:40 2.4 -
Sat_244 48.85 24:29:69 66.8 *****
Satt323 60.05 25:21 :23 10.7 ***
Satt306 80.01 34:85:36 1.5 -
SattZ50 107.7 45:87:42 0.1 -

 

“-“ means not signiﬁcant at 0.05 probability level; *,**, and *** means signiﬁcant at 0.05, 0.01, and 0.001

probability levels, respectively

86

Table 5.3. (Cont’d)

 

 

 

Marker Integrated a'h'b X2 Signiﬁcance

name map( cM) ' ' levelf
Linkage Group N

Satt675 34.67 24:46:44 11.3 ****

Satt660 72.6 54:62:42 9.1 **

SattZSS 76.49 48:74:37 2.3 -

Linkage Group
Unknown

Sat_178 37:36:21 10.6 ***

SattOZ4 69:41 :49 42.3 *****

SattOS9 127:19z39 200.5 ***"

SattO98 50:94:43 0.5 -

Satth9 42:84:62 6.4 **

Sattl71 50:92:43 0.5 -

SattZl6 34:40:20 6.3 *

SattZ97 45:95 :47 21.9 *****

 

“-“means not signiﬁcant at 0.05 probability level

probability levels, respectively

-***
9 9 9

87

and *** means signiﬁcant at 0.05, 0.01, and 0.001

   

l11111111111111);“5111111111l