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This is to certify that the
thesis entitled
Cytogenetic evaluation of a wheat—rye
derivative for resistance to leaf rust
presented by

Rafael Antonio Bravo

has been accepted towards fulfillment
of the requirements for

Pho Do degree in Farm Crops

6m

Major professor

 

Date July 17 , 1962

0-169

 

LIBRARY

Michigan State
University

 

 

 

CYTOGENETIC EVALUATION OF A WHEAT—RYE DERIVATIVE

FOR RESISTANCE TO LEAF RUST

BY

Rafael Antonio Bravo

AN ABSTRACT OF A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Farm Crops

1962

(_:7/7(C‘3
Approved Q 7 Y (k:~3\ -. \

 

 

CYTOGENETIC EVALUATION OF A WHEAT—RYE DERIVATIVE

FOR RESISTANCE TO LEAF RUST

By Rafael Antonio Bravo

The cytology of selection 82 a l—2—6—2, a wheat—rye
derivative which has excellent mature plant resistance to
leaf rust at East Lansing, Michigan, was investigated. This
wheat—like selection, as shown by Sebesta (1958), has 44
chromosomes with the two extra chromosomes being telo—
centric and carrying a factor(s) for leaf rust resistance.

To obtain some information on the homology of the extra
chromosomes, the wheat and the rye complements, crosses
were made between 82 a l—2—6—2 and two of its ancestors,
Rosen rye and Cornell 595 wheat. The formation of hetero—
morphic bivalents in the first type of cross may be taken
as evidence for a degree of homology between the extra
telocentric and the rye chromosomes. Tetravalent forma-
tion was observed in 13 % of the pollen mother—cells of the
F1 between 82 a 1-2—6—2 x Cornell 595. This was attributed
to possible rearrangements within the wheat genome, as
three different varieties (Honor, Yorkwin, and Cornell 595)

were used in the evolutionary process of this selection.

 

Rafael Antonio Bravo

Selection 82 a 1—2—6—2 has shown some variation in
leaf rust resistance at East Lansing due to the instability
of the two telocentric chromosomes. Methods of trans—
ferring a small segment of the telocentric chromosome
containing the leaf rust resistance to a wheat chromosome
were studied. Dry seed of several plant selections of 82
a 1—2—6—2 were irradiated with the source of fast neutrons
at Oak Ridge National Laboratory. The dosage delivered was
72 minutes at a total flux of 7.7 x 108 N/ cm.2/ sec. This
treatment proved very effective in inducing translocations,
as judged from the number of multivalent associations
found in both N and N . In comparison, no multivalent

l 2

configurations were found in control (non—irradiated)

plants. The N2 was inoculated with an oil suspension of
leaf rust spores. One resistant plant (4—35) with 42 chro—
mosomes was found in this second generation. Its resis—

tance was attributed to a possible transfer of the factor(s)

for leaf rust resistance from the telocentric rye chro-
mosomes to the wheat complement. The possibility of the
telocentric chromosomes being different in some cases is
suggested. Leaf rust would depend not only on the presence

of one or two extra telocentric chromosomes, but also on

the kind present.

l

 

Sggt 5.1;! m mbnld -

CYTOGENETIC EVALUATION OF A WHEAT—RYE DERIVATIVE

FOR RESISTANCE TO LEAF RUST

By

Rafael Antonio Bravo

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Farm Crops

1962

C/i/éi
ii
ACKNOWLEDGMENT

The author wishes to express his gratitude to Drs.
Everett Everson and Fred C. Elliott for their guidance and
assistance throughout the investigation and to Dr. Carter M.
Harrison for his assistance during the preparation of the
manuscript.

Dr. Albert Ellingboe helped with advice and assistance,
and Ray Hart and Joe Clayton gave help during the inocula—
tion procedure.

Thanks are extended to the National University of
Colombia, the National Academy of Sciences of the U. S. A.,
and to Michigan State University, for their generous finan—
cial assistance during the course of this study.

The author is very thankful to his wife and children

for their encouragement and inspiration.

 

 

LIST OF TABLES . . .

LIST OF FIGURES .

INTRODUCTION .

REVIEW OF LITERATURE

MATERIALS AND METHODS

RESULTS .

DISCUSSION . . . . .

TABLE OF

SUMMARY AND CONCLUSIONS . .

LITERATURE CITED

CONTENTS

Page

iv

18
23
42
58

62

 

Table

LIST OF TABLES

Distribution of bivalent and
multivalent associations in
82 a 1—2—6-2 x Rosen rye . .

Distribution of multivalent
associations in N1 plants . .

Distribution of multivalent
associations in N2 plants

iv

Page

26

28

29

 

Figure

l.

10.

LIST OF FIGURES

Page

Pedigree of leaf rust resistant selection

82 a l—2—6—2 used in this study. (R)

resistant and (S) susceptible to leaf

rust reaction . . . . . . . . . . . . . . . . l6

Spikes from selection 82 a 1—2—6—2 and
from a 82 a l—2—6—2 X rye hybrid plant . . . 22

Somatic chromosomes in a root tip cell
from the 82 a l—2—6—2 x rye plant No. 9 . . . 33

Prophase I showing 21 I, 2 II and 1 III
in a microsporocyte from hybrid plant
No. 3 . . . . . . . . . . . . . . . . . . . . 33

Prophase I showing 24 I and 2 II in a
microsporocyte from hybrid plant
No. 3 . . . . . . . . . . . . . . . . . . . . 33

Prophase I showing 23 I, 1 II and 1 III
in a microsporocyte from hybrid plant
No. 3 . . . . . . . . . . . . . . . . . . . . 33

Organized metaphase plate showing 28
chromosomes in a microsporocyte from
hybrid plant No. 10 . . . . . . . . . . . . . 34

Prophase I showing 27 I and 1 II in a
microsporocyte from hybrid plant No. 2 . . . 34

Prophase I showing 25 I and 1 chain of
4 chromosomes in a microsporocyte from
hybrid plant No. 2 . . . . . . . . . . . . . 34

Prophase I showing 20 I, 2 II and l
association of 5 chromosomes in a micro—
sporocyte from hybrid plant No. 5 . . . . . . 34

 

 

Figure

ll.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Metaphase I showing 1 I, 19 II and 1
IV in a microsporocyte from 82 a
1—2—6—2 x Cornell 595 . . . . . . . . . .

Metaphase I showing 20 II and 1 IV in a
microsporocyte from N1 plant 2—15—1 . . . .

Metaphase I showing 20 II and 1 ring of
4 in N1 plant l—6-3 . . . . . . . . . . . .

Metaphase I showing 1 fragment, 1 I, 18
II, 1 chain of 6. N1 plant 2—15—1 . . . . .

Metaphase I showing 1 fragment, 14 II,
1 III and 1 chain of 12. N1 plant
l—l7—3 . . . . . . . . . . . . . . . . . .

Metaphase I showing 1 I, 10 II, 1 III
and 2 chains of 10 chromosomes in a
microsporocyte from N1 plant 2—24—7 . . .

Metaphase I showing 2 I, 17 II, 1 ring
of 4 and 1 chain of 4 in a microsporo—
cyte from N2 plant 3—123 . . . . . . .

Metaphase I showing 2 I, 18 II and 1
chain of 6. N2 plant 5—114 . . . . . . . .

Metaphase I showing 18 II and 1 chain
of 8 chromosomes. N2 plant 3—101 . . .

Metaphase I showing 14 II and 1 chain
of 16 chromosomes. N2 plant 3-205

Metaphase I showing 2 I, 15 II, 1 IV
and 1 chain of 6 in N2 resistant plant
4-35 . . . . . . . . . . . . . . . . . .

Page

35

36

36

36

36

37

38

38

38

38

39

vi

Figure Page

22. 42 chromosomes (1 divided) at Anaphase
I in N2 resistant plant 4—35 . . . . . . . 39

23. 42 somatic chromosomes in a root tip
cell from a germinating seed from plant
4—35 . . . . . . . . . . . . . . . . . . . 39

24. 42 chromosomes in a root tip cell from a
germinating seed from N2 susceptible
plant 4—70 . . . . . . . . . . . . . . . . 4O

25. 43 chromosomes in a root tip cell from a
germinating seed from N2 susceptible
plant 5—125 . . . . . . . . . . . . . . . 4O

26. 44 chromosomes in a root tip cell from a
germinating seed from N2 susceptible
plant 5—126 . . . . . . . . . . . . . . . 4O

27. 44 chromosomes in a root tip cell from a
germinating seed from N2 resistant
plant 2—259 . . . . . . . . . . . . . . . 41

28. Double ”satellited“ iso—chromosome in a
root tip cell from a germinating seed
from N2 resistant plant 5—180 . . . . . . 41

 

 

INTRODUCTION

The transfer of desirable characteristics such as disease
resistance to wheat through intergeneric and interspecific
crosses, has been of particular interest to cytogeneticists,
especially since genome homologies were demonstrated.

In 1930 McFadden suggested the possibility of transfer—
ring desirable characters from emmer, durum, einkorn, rye,
Aegilops and other related grasses to common wheat. Such
transfers are very difficult to execute by conventional
hybridization methods, as emphasized by Riley (1960) and
Unrau (1960) and different approaches are required.

New possibilities in the intergeneric and interspecific
transfer of characters to wheat species have become avail—
able through the use of alien addition and substitution
races. The process, by which a nonhomologous chromosome is
substituted into the genome of another species is known as
alien—chromosome substitution, whereas the addition of an
alien chromosome to the normal complement of the host spe—
cies is the alternative. Several investigators including
Florell (1931), O‘Mara (1940, 1947, 1951, 1953), Sears (1953),
Hyde (1953) Chapman and Riley (1955) and Jenkins (1958) have

contributed to the development of this approach.

 

Whole chromosome substitutions or additions have usually
been found to be unsatisfactory from a practical point of
view, apparently because so much genetic material is intro—
duced from the donor species that undesirable characters are
included with desirable factors. Genetic recombination of
this desired character with the desirable genes cannot be
effected due to the lack of chromosome pairing. In the trans-
fer of leaf rust resistance from Aegilops umbellulata to com-

mon wheat and stem rust resistance from Aqropvron elongatum

 

to common wheat, Sears (1956) and Elliott (1957) have demon—
strated a method of introducing small segments of chromo—
somes from alien species, by irradiation techniques.

For several years a wheat—rye selection 82 a 1—2—6-2,
originating at Cornell University, has demonstrated a high
degree of resistance to leaf rust, Puccinia rubigo—ygra tri:
gig; (Eriks) Carl.,at East Lansing, Michigan. Sebesta (1958)
found that this selection, a wheat-rye derivative, has two
extra telocentric chromosomes, presumably derived from the
rye parent. He further demonstrated that the telocentric
chromosomes carry a factor(s) for leaf rust resistance.
Research at East Lansing has demonstrated that this selec—

tion loses its leaf rust reSistance due to the instability

 

of these two extra telocentric chromosomes. It would be
highly desirable to transfer the segment(s) containing the
factor(s) for leaf rust resistance to the wheat complement to
stabilize the character. Furthermore the quality of this
selection may be enhanced by the loss of the extra rye chro-
matin linked with the resistance factor(s).

One of the objectives in the present work was to study
the relationship between the extra chromosomes and the
wheat complement, through the analysis of the F1 of the
cross between the 44—chromosome material and Cornell 595, a
wheat variety with 42 chromosomes. Similarly the study of
the homology between the extra telocentric and the rye com—
plement was an objective of this study, by an analysis of
the F1 of the cross between selection 82 a 1—2—6—2 and
Rosen rye, one of the original parents of this selection.

Another objective was to study methods of transferring
the factor(s) for leaf rust resistance from the extra telo—
centric chromosomes in selection 82 a 1-2—6—2 to the wheat
complement, through possible induction of translocations by

means of neutron irradiation.

 

REVIEW OF LITERATURE

The cytology of wheat and related species included in
the Sub—Tribe Triticinae was reviewed by Sears (1948).
Genome homologies are given for the genera Triticum,
Aegilops, Agropvron, Secale and Haynaldia.

Sebesta (1958) reviewed the cytology of wheat, rye,
and the F1 between them, as well as the constant interme-
diate hybrids, and hybrids of later generations. Certain
aspects of wheat and rye cytology pertinent to this thesis,
including meiotic behavior in the F1 hybrids and haploids

in both wheat and rye will be reviewed.

Haploids in wheat and rye.

Since Gaines and Aase (1926) described a haploid plant
in Triticum compactum, a number of such plants have been
found, not only in Triticum compactum but also in T. mono:
coccum and I, vulgare. Sebesta (1958) reviewed the early
work with haploid wheat plants. McGinnis and Unrau (1960)
found that in a haploid plant of Triticum vulgare, 57.6 %
of the metaphase cells showed one to three bivalents.
Homologous pairing was assumed if a chiasma was actually
observed or if the appearance of a disjoining pair indica—

ted the previous formation of a chiasma. Person (1955)

 

 

made an analytical study of chromosome behavior in a hap—
loid wheat plant. He describes two types of chromosome
associations: 1. Side—by—side (s—s) in twos (”secondary
association,” "secondary pairing“); 2. End-to-end associa—
tion (e—e) in groups of two or more. He showed that the
proportion of total univalents forming e-e associations

was relatively unaffected by changes in the number of biva—
lents and concluded that homology was not a factor in the
formation of e—e— associations. Instead, homology was con—
sidered a determining factor in s—s associations, which
divide at the metaphase plate.

Riley and Chapman (1957) also dealt with the problem of
homologies in pairing in haploids and polyhaploids of
Aegilops and Triticum. They concluded that the presence of
chiasmata associations in this type of material may be
accepted as evidence of some degree of segmental corres—
pondence, and accept Person's view that the end—to—end
associations do not depend upon homology. That this type
of association may be due to the fusion of heterochromatin,
was suggested by Kostoff (1938). Four kinds of haploids

are mentioned by Riley (1958) in Triticum vulgare varo

 

Holdfast, as derived from lines used in obtaining additions
and substitutions of single pairs of rye chromosomes.
Haploid plants have also been found in rye. Muntzing
(1937), in temperature induced doubling studies, found a
haploid rye plant among the progeny from five spikes exposed
to —30C for 30 minutes after cross pollination. Nordens—
kiold (1939) also found and described the cytology of a
haploid rye plant. Generally no associations were found
between the seven univalents, but in some cases, there was
some bivalent formation. The bivalents were rod—shaped and
the two chromosomes were joined by a chiasma. Levan (1942)
described the meiotic process in a haploid rye plant, find—
ing mostly bivalents and trivalents. Quadrivalents were
found in 4 out of 2628 cells. The results were interpreted

as a consequence of non—homologous pairing.

Wheat x rye hybrids.

 

Since Wilson (1876) described the first wheat x rye
hybrids, a number of workers have been interested in this
type of cross. A review of the early work is given by
Vakar and Krot (1934), Aase (1935 and 1946) and Sebesta

(1958).

 

 

Thompson (1927), studying the cytology of hybrids
between wheat and rye, found that although occasionally 1 or
2, and more rarely 3, bivalents were formed, the usual fea-
ture was the complete absence of chromosome pairing.

Longley and Sando (1930) found no bivalent formation in
a hybrid between Triticum vulgare and Secale cereale.
Kagawa and Chizaki (1934) made a cytological analysis of
hybrids between Triticum compactum and I. spelta in crosses
with Secale cereale. In most cases they observed 28 uni—
valent chromosomes at metaphase, but often found from 1—3
bivalents, formed by two chromosomes of similar size,
paired end to end.

Ledingham and Thompson (1938) studied the cytology of
non—amphidiploid derivatives of wheat—rye hybrids. In the
F1 they found occasional bivalents, of the I'loose” type,
which separated very slowly. In the F2 they found 3 groups
of material, with 28, 42 and 49 chromosomes respectively.
In the 28—chromosome group a few bivalents of the end—to-
end type were formed. Secondary pairing of bivalents was
also observed, and it was explained as due in some cases to
duplication of wheat chromosomes. They found neither evi—

dence that the individual rye chromosomes could be ”added

 

 

permanently to the wheat complement, or substituted for wheat
chromosomes, nor that by crossing over or translocation com-
posite wheat—rye chromosomes can be produced or at least
cause a visible effect.”

Chin (1946) crossed a variety of Chinese wheat and rye,
finding at meiosis from 0—3 bivalents, although most of the
28 univalents remained unpaired. He suggests that the biva—
lent formation may be due to pairing of intrageneric genomes.

Nakajima (1952) makes an analysis of intergeneric F hybrids

1
between Triticum and Secale. He used Triticum durum, T.
spelta and T, vulgare, in crosses with Secale cereale. One
plant of the cross Triticum spelta X Secale cereale was
studied and he found at first metaphase from 0-4 bivalents
and 20—28 univalents. Some trivalents but no tetravalents
were found. In the cross Triticum vulgare x Secale cereale
bivalents were observed at M I, in a range of 0—4. In rare
occasions ring bivalents were observed, and formation of
interstitial chiasma was infrequent. He thought that the
bivalents were due to the autosyndesis between the genomes
of Triticum. Of the 11 bivalents found in this last hybrid,
7 are explained as the result of autosyndesis between A and

B genomes of Triticum, 3 are formed from the conjugation of

   

 

R genome of Secale and l bivalent by the conjugation of chro—
mosomes in the D genome of Triticum. This means that there
is no syndesis between genomes of Triticum and Secale.
Nakajima (1953) in crosses between Triticum pyramidale (28
chromosomes) and Secale cereale found from 0—4 bivalents at
M I., and from 21-13 univalents. The occurrence of bivalents
is explained as the product of autosyndesis between the AB

genomes of Triticum pyramidale.

Behavior of telocentrics and iso—chromosomes.

 

The origin and behavior of telocentrics and iso—
chromosomes has been reviewed by Darlington (1939, 1940)
and by Sears (1944, 1952a, b). Only a few pertinent cita—
tions will be made here.

According to Darlington (1939) telocentrics originate
by misdivision of the centromere in univalent chromosomes.
He points out several types of misdivision leading to the
formation of telocentrics, that is, of chromosomes with a
terminal centromere. These telocentrics are functional, as
they can divide normally at both first and second anaphase.
but they are unstable. The telocentric may give origin to
iso—chromosomes with identical arms, by a process described

by Darlington (1940).

 

10

Sears (1944) found both telocentric and iso—chromosomes
in the offspring of trisomic and monosomic plants of Triti—
gum vulgare. He points out that the frequency of telocen—
trics and iso—chromosomes among the offspring of certain
monosomics is rather high, and might result from pollen com—
petition rather than from a high frequency of misdivision.
In another paper (1952a) he describes the manner of origin
of telocentrics and iso—chromosomes in wheat, and states
that they are due to a number of types of misdivision of
univalent chromosomes at the first division of meiosis. At
the second division some iso-chromosomes might give rise to
telocentrics by a new misdivision, and new telocentrics may
be found by misdivision of chromosomes that divided nor-
mally during the first division. The behavior of these
chromosomes was also analyzed by Sears (1952b) using iso—
chromosomes and telocentrics for the long arm of chromosome
IX of Chinese Spring wheat. He found that telocentric chro—
mosomes behave normally when they are present as a pair, or
when a normal homologue is present. There might be post—
meiotic conversion of a telocentric into an iso-chromosome
and also somatic loss of both telocentrics and iso—

chromosomes.

 

11

Transfer of characters from related
species and genera to hexaploid wheat.

As the chromosome homologies between different species
of Triticum and related species were studied and clarified,
the possibility of the transfer of desirable characteristics
from those species to hexaploid wheats has been of special
interest in wheat breeding.

Hayes (1920) found that by growing large populations it
was possible to obtain stem rust resistance in crosses be—
tween Triticum vulgare and both Triticum durum and Triticum
dicoccum. Harrington and Smith (1929) also emphasized the
need for large segregating populations in order to obtain
42—chromosome hybrids resistant to stem rust, in the early
segregating generations of a I, dicoccum x T. vulgare cross.
McFadden (1930) using hybridization was successful in trans—
ferring desirable characteristics from Yaroslav emmer to
common wheat.

In the search for new sources of desirable characteris-
tics, crosses between species of Triticum and related gen-
era have been made. As the common methods of hybridization
are effective only when there is pairing of the parental

chromosomes, the synthesis of alien addition races has been

 

12

suggested as a method of securing the addition of foreign
germplasm to the wheat genome in a stable manner. Florell
(1931a, b) found cytological evidence for the possibility
of adding a pair of rye chromosomes to the wheat complement
in a constant combination, through hybridization between
wheat and rye and backcrossing to the wheat parent. O'Mara
(1940) discusses the possibility of combining characters of
two species or genera by the addition of one or more chromo—
somes from one species to the diploid complement of the
other. The proposed method involves the production of an
amphidiploid between two species or genera, A and B, and
subsequent backcrossing to one of the parents. By this
method he secured plants which had the normal wheat comple—
ment plus a disomic rye chromosome.

As Haynaldia shows a marked resistance to stem and leaf
rust of wheat, Hyde (1953) was interested in obtaining the
addition of Haynaldia chromosomes to common wheat. He
accomplishedfhis by the use of an amphidiploid (AABBVV)

between Triticum dicoccoides and Haynaldia villosa as a

 

 

bridge for the cross with Triticum aestivum (= Triticum
vulgare). A series of backcrosses to Triticum aestivum

and selfing of different types of progeny. gave origin to

 

13

"addition monosomics” and ”alien addition races.” Sears
(1953) used the same bridge technique for adding the chro—

mosomes of Haynaldia villosa (L) Schur. to common wheat, a

 

technique that he also used to add individual chromosomes
to wheat from Aegilops umbellulata.

Chapman and Riley (1955) found in the progeny of an
amphidiploid between Triticum vulgare var. Holdfast and
Secale cereale var. King II, a 28—chromosome plant that
arose parthenogenetically; this plant was crossed with T.
vulgare and a single Fl plant was obtained, with 49 chromo—
somes (21 II from wheat and 7 I from rye). Selfing of this
gave origin to a 44—chromosome plant, with two identical
rye chromosomes. Evans and Jenkins (1960) described the
addition of individual ”Dakold” fall rye chromosomes to
”Kharkov" winter wheat and their subsequent identification.
Bhattacharyya et al. (1961) made a detailed karyotypic
analysis of the Dakold rye chromosome additions to Kharkov
winter wheat and identified these addition lines in rela—
tion to the wheat karyotype.

As obtaining addition and substitution lines does not
assure that genetic recombination will occur between the

alien material and the wheat chromosomes, new methods have

 

 

14

been used to secure the transfer of desirable characteris—
tics, mainly disease resistance, from the foreign material
to the wheat complement. Sears (1956), after using the
amphiploid of Triticum dicoccoides x Aegilops umbellulata
as a bridge for combining the genomes of this last species
and Triticum aestivum, obtained plants with the whole wheat
complement and one Aegilops iso-chromosome. X-rays were
used to irradiate these plants and both reciprocal and
intercalary translocations were obtained. Plants homo—
zygous for the small intercalary translocation were secured
and they carried the factor for leaf rust resistance.
Elliott (1957) reported the transfer of resistance to stem
rust from Agropyron elongatum to hexaploid wheat, after the
irradiation of a 49—chromosome plant obtained by crossing a
resistant octoploid plant (derived from the original cross

between Agropyron elongatum and common wheat) and the sus—

 

ceptible variety Idaed.

Another method for obtaining genetic recombination is
proposed by Riley and Chapman (1958) who suggest that ”inter—
generic pairing may well take place in the absence of the HH
chromosome and alien genes could then be introduced into the

wheat chromosomes by normal recombination.” The H

 

 

15

chromosome (in the variety Holdfast) is supposed to control

the normal diploid behavior in hexaploid wheats.

Previous work with selection 82 a 1-2—6-2

and related lines.

 

Since the original cross was made at Cornell University,
different lines of wheat—rye hybrids have been obtained pos-
sessing a high degree of resistance to leaf rust.

Originally the wheat variety Honor, susceptible to leaf
rust, was crossed with the resistant rye variety Rosen, as
seen in the pedigree in Fig. l. The F1 was backcrossed to
Honor and selection 369 a 2—2—44 was obtained in the F4
generation. This selection was crossed with Yorkwin in 1935
and a single disease resistant plant, selection 779 b
10-8—6 was obtained. The progeny of a cross made in 1942
between Selection 779 b 10—8—6 and Cornell 595, gave origin
to selections 82 a 1—2—4—7 and 82 a l—2—6—2 (Sebesta, 1958).
Both of these lines have been selfed more than 15

generations.

 

16

Honor (S) Rosen rye (R)

F4 369 a 2-2-44 (3) X Yorkwin (S)
779 b 10-8—6 (R) X Cornell 595 (S)
82 a 1-2—6-2 (R)

Figure l. Pedigree of leaf rust resistant selection
82 a 1-2—6—2 used in this study. (R) =
resistant and (S)= susceptible leaf rust
reactions.

Jensen and Kent (1952), trying to explain the disease
resistance (leaf rust and mildew) of selection 82 a 1—2—4—7
proposed ”the transfer, in the original hybrid, of one or
more segments of rye chromatin by crossing over or recipro—
cal translocation, followed by a novel recombination of
genes in the final hybrid.” Jones and Jensen (1954) made
crosses between Alaskan and each one of three wheat-rye
selections derived from the head selections of 779 b 10—8—6.
They concluded that the hairy neck character was inherited

as a simple Mendelian character, and suggested the

 

17

possibility of a pair of wheat-rye chromosomes resulting
from translocation or crossing over as responsible for
the results obtained. Sebesta (1958) made a cytological
study of the selections 82 a 1—2—4—7 and 82 a 1—2—6—2, dis—
covering that completely leaf rust resistant, moderately'
resistant and completely susceptible plants of these selec—
tions had 44, 43 and 42 chromosomes, respectively. In the
case of the 44 and 43—chromosome plants, the extra chromo—
somes were telocentric, with the exception of their being
iso—chromosomes.in the case of some completely resistant
plants with 43 chromosomes. In the progeny from moderately
resistant plants with 43 chromosomes, he found resistant,
moderately resistant and susceptible plants. The latter
ones had lost the extra telocentric chromosomes. Sebesta
concluded that the extra chromosomes were apparently the
same in both selections 82 a 1—2-6-2 and 82 a 1—2-4—7, and
that they were the carriers for the factor or factors, for

leaf rust resistance.

 

 

l8
MATE RI ALS AND METHODS

Several plant selections of 82 a 1—2-6—2 were used in
the course of this work. Approximately 200 seeds from each
one of them were irradiated with fast neutrons at the Oak
Ridge, Tennessee, Laboratory of the Atomic Energy Commission,
through the courtesy of Dr. T. S. Osborne. The dosage deliv—
ered was 72 minutes of fast neutrons at a total flux of 7.7
x 108 N/cm.2/sec. A tabulation of technical details con—
cerning the source of irradiation is given by Heinz (1961).
Seed of the irradiated material and the non—irradiated
(control) was space planted in the field in September, 1960,
at a distance of one foot between rows and plants. Samples
for meiotic studies were taken in the summer of 1961 and
fixed in Newcomer's solution.

Whole spikes were collected at the early boot stage,
three to four hours after sunrise and stored in a refriger-
ator until further use.

Spikes of the N’ and control populations were harvested

1
in late summer of 1961. Four or five seeds of each spike

were planted in small peat pots in early September of 1961

and vernalized for 7—8 weeks in a cold room at 38°F., and

 

 

19

transplanted to 4-inch pots in the greenhouse. Meiotic
samples were taken in December 1961, and fixed as whole
spikes in Newcomer's solution. Samples were taken after
three to four hours of illumination each day, and stored
in the refrigerator until needed. After heading, the N2
population was inoculated with leaf rust spores from a
field collection that had been increased on the susceptible
wheat variety Little Club. The method of inoculation was
described by Rowell and Olien (1957).

Plants of susceptible varieties (Little Club, Genesee)
were included among N2 plants to serve as a control for
the inoculation procedure.

In the summer of 1961 flowers of non-irradiated mate—
rial of selection 82 a 1—2—6—2 were emasculated and polli—
nated with pollen from Rosen rye. Out of 693 flowers pol—
linated, 15 hybrid seeds were obtained. These seeds were
planted in small peat pots, vernalized for two months in a
cold room at 380 F and then transplanted to 4 inch pots and
grown under greenhouse or growth chamber conditions. Mei—
otic samples were taken after three to four hours of illu—
mination each day, in the growth chamber, and fixed in

Newcomer‘s until examined.

 

 

20

Crosses between control plants with 44 chromosomes and
Cornell 595, a wheat variety with 42 chromosomes were also
made and handled in the same way as the wheat—rye hybrids.

From anthers of all the material fixed in Newcomer's

N 82 a l—2—6—2 x rye F and 82 a

(control population, N l

1’ 2’
1-2—6—2 X Cornell 595 F1)’ temporary slides were prepared
with propionic carmine and meiotic associations analyzed.
Photographs were taken of suitable material. Root tips
were secured from some of the hybrid 82 a 1—2—6—2 x rye
plants and pretreated with hydroxyquinoline for 24 hours
(Markarian, 1957). They were then fixed in 3:1 (absolute
alcohol: glacial acetic acid) and stained in Feulgen. The
stain was prepared following the modification suggested by
Wilson (personal communication):

1 gram of basic fuchsin was dissolved by pouring over
it 200 c.c. of boiling distilled water; this was shaken and
cooled to 500 C. and filtered; to the filtrate 3O c.c. of
l/N HCl were added and then 3 grams of potassium metabisul—
phite. The solution was allowed to bleach for 24 hours in

the dark, in a stoppered bottle, and then 0.5 grs. of Norit-

A were added and after some time it was filtered again.

231513an :iflid'd I5 .398.

' .xsmoowsJ '1;

 

 

21

Seed from both resistant and susceptible N2 plants were
germinated in moist blotting paper in Petri dishes in a ger—
minator under controlled light and temperature conditions
(8 hours light, 16 darkness at 30 and 200 C of temperature
respectively). When primary root tips were about 4—6 mm.
long, they were excised and pretreated with actidione for
12 hours (Wilson, 1950; Bowen and Wilson, 1954) or with
hydroxyquinoline for 24 hours. Both pretreatments gave
satisfactory results. The root tips were then fixed in 3:1
absolute alcohol: glacial acetic acid for a minimum of 12
hours and were stained in Feulgen, after hydrolysis for 10-

12 minutes in l/N HCl at 600 c.

 

 

 

Figure 2. Spikes from selection 82 a 1—
the left and from a 82 a 1—2
hybrid plant on the right.

 

RESULTS

Observations on Rosen rye and Cornell 595.

No detailed analyses were made for meiotic associations
on Rosen rye or Cornell 595, but meiotic behavior was ob—
served in some plants of both species. Rosen rye has 14
somatic chromosomes. Meiosis is normal, seven bivalents
always being found at Metaphase I, and the chromosome sep—
aration at Anaphase I being regular. The 42 chromosomes of
Cornell 595 form 21 bivalents at Metaphase I, the chromo—

some distribution at Anaphase I also being normal.

Observations in plants of selection 82 a 1—2—6-2.

Plants of the control population of selection 82 a
1-2—6—2 were found to have 43 or 44 chromosomes, the extra
chromosomes being telocentric. Of 20 plants that were exam—
ined, 6 had 43 and 14 had 44 chromosomes. In 238 cells
analyzed to determine meiotic associations, only univalents
and bivalents were found. No multivalent configurations
were present in the cells studied. The wheat chromosomes
almost always formed 21 pairs at Metaphase I, with very few
exceptions where more than two univalents were found. The

telocentric chromosomes (1 or 2) were usually found off the

23

 

24

metaphase plate, unpaired in the case of two extra telocen—
tric being present, but a few plants showed a tendency for
them to form pairs, in which case 22 pairs were found at M
I. Sometimes one pair of telocentrics showing an end-to-end
association was found slightly off the equatorial plate. At
early anaphase I the distribution of chromosomes was normal,
almost always being 22—21 or 22—22, in 43 or 44—chromosomes
plants, respectively.

At advanced anaphase or telophase I the univalents, al—
ready divided or dividing, shOWed a tendency to lag. The
second division was not observed in detail, but the pres-
ence of micronuclei at the tetrad stage indicated that the
lagging chromosomes sometimes were not included in the

daughter nuclei.

82 a 1—2-6—2 x rye hybrids.

Of 693 flowers of control plants of 82 a 1—2—6-2 pol—
linated with Rosen rye (2n = 14), a total of 15 hybrid
seeds were obtained, all with characteristic shrunken endo-
sperms. Ten of them germinated, were wheat—like in appear-
ance and grew normally, showing the hairy neck character-

istic and being completely sterile. The spikes of these

25

hybrids were intermediate in length between wheat and rye,
with awns of intermediate length, in contrast with the
spikes of plants of selection 82 a 1—2—6—2, which were awn—
less. Figure 2 shows the differences in spike length,
presence of aWns and general morphology of spikes of 82 a
1—2—6—2 and of one of the hybrids. Some of these hybrids
had 28 and some 29 chromosomes. Figure 3 shows the 29
somatic chromosomes from plant No. 9. Meiotic associations
were analyzed for several of the hybrid plants. In most of
the cells the univalent chromosomes did not pair at prophase
and were scattered throughout the cell. No regular meta—
phase plates were observed, except in one case where the
chromosomes were well aligned at the equatorial plate, as
seen in Figure 7.

At “anaphase” of the first division the univalents went
to either pole, frequently in about the same numbers. When
associations of two chromosomes forming true chiasmata
were observed, they frequently remained at the equatorial
region after the rest of the univalents had moved to the
poles. When the members of these associations began sepa-

ration, they were considerably stretched, forming a long

 

 

26

chromatin thread until separated. Usually there was no
division of the univalents during the first division of
meiosis.

In rare cases chromosome bridges were observed at telo—
phase I. Chromosome behavior at the second division was
not observed in detail, but some irregularities were found.
Formation of micronuclei was particularly notable, their
number per tetrad ranging from 0—9. Although most tetrads
were normal in appearance ”tetrads" with more than four
cells were commonly observed.

Out of 941 cells, from 4 different plants observed,

210 (22.32 %) had bivalent or multivalent associations.
The total number of associations recorded was 286, distri—
buted as shown in Table I.

Table I. Distribution of bivalent and multivalent
associations in 82 a 1—2—6—2 X Rosen rye.

 

 

Type of association Total number % Mean NO“
per cell

II 235 82.17 .48

III 48 16.78 .098

IV 2 .70 .004

V 1 .35 .002

 

 

Fl

 

27

Of the 235 bivalents found, 54 (22.98 %) had chiasmata
formation and 181 (77.02 %) were of the end—to—end type.
Of the 54 ”true” bivalents formed (with chiasmata) 25 were

heteromorphic, having one arm shorter than the other.

Observations in 82 a 1—2—6—2 x Cornell 595.

Ten plants from this type of cross were studied cyto-
logically and meiotic associations determined. All of them
had 43 chromosomes, one univalent always being found at M I.
Anaphase distribution was normal and the telocentric chromo—
some was found lagging at advanced anaphase and in telophase
I. Of 138 cells examined for meiotic associations at dia—
kinesis or M I, 18 multivalent configurations (13 %) were
found, 16 being open tetravalents, one a ring of four chro—
mosomes and one a trivalent. The mean number of tetravalent
associations per cell was 0.1232, the mean frequency for

the trivalent being 0.007 per cell.

Observations in N1 plants.

 

Out of 493 cells observed in 30 plants of the N1’ it
was found that 277 (56.19 %) had trivalents or associations

of higher order. The total number of multivalent associa-

tions was 411, distributed as shown in Table II.

 

 

28

Table II. Distribution of multivalent associations
in N plants.

 

 

1

M N .
Type of association Total number % ean 0
per cell

III 14 3.41 .028

IV 350 85.16 .71

VI 23 5.60 .047

VIII 14 3.41 .028

X 6 1.46 .012

XII 4 .97 .008

 

Presence of fragments was observed in 23 cells from 11
plants. A total of 28 fragments was observed, the average
number of fragments per cell being .057.

The anaphase I was rather regular and normal, the
chromosomes being distributed in about the same numbers,
but in some cases there was some chromosome stickiness.
This was probably responsible for the tendency of some

associations to move to a given pole as a group.

Observations in the second generation (N2).

 

Of 941 cells from 70 plants of the second generation

which were analyzed for meiotic associations, 396 (42.08 %)

 

29

had associations of three or more chromosomes. The total
number of multivalents scored was 480 and their distribu-

tion is given in Table III.

Table III. Distribution of multivalent associations
in N2 plants.

 

 

Type of association Total number % g::nc:I1
III 27 5.63 .029
IV 407 84.78 .43
VI 14 2.92 .015
VIII 23 4'79 '024
x 6 1.25 .006
XII 2 0.42 .002
XVI l 0.21 .001

 

Meiosis in the N2 was normal and the chromosome distri—
bution at A I was regular, although univalent chromosomes
and fragments were found lagging at late anaphase and telo—
phase I. Fragments were found in 53 cells from 18 plants.
Up to 4 fragments per cell were found in one single cell.

A total of 67 fragments were found in the 941 cells exam—

ined, this giving an average number of .07 fragments per

 

 

30

cell. About 10 % sterility was found in plants of this

second generation at harvesttime.

Results of the inoculation of N2 plants with leaf rust.

Whereas all of the plants of susceptible varieties
(Little Club, Genesee ) used as controls for the inocula-
tion procedure developed leaf rust, only 39 plants, of a
population of approximately 2,200 plants of the second gen—
eration developed leaf rust pustules. Some plants were only
slightly affected, but several presented a heavy infection.
All of the plants with pustules on the leaves were classi-
fied as susceptible, some of them being classified as
”very susceptible.”

Due to sterility of some plants and to environmental
conditions (mostly cloudy days) when meiotic samples were
taken, only a few of the susceptible plants were useful in
the determination of chromosome number and meiotic behavior.
Germinating seeds from some were used to ascertain chromo—
some numbers in the N3. Meiosis was observed in pollen
mother—cells from plant 5-121, classified as very suscep—
tible. This plant was found to possess 43 chromosomes, one

being a univalent. Two multivalent associations (one chain

 

31

of 4 and one chain.ofJJD were observed among others.
Plants 2—391, 4-29, 4-40 and 5—177, the first classified
as susceptible and the last three as slightly susceptible,
also showed 43 chromosomes.

Of 69 resistant plants analyzed for meiotic behavior in

N one was found to have 42 chromosomes (plant 4-35), 22

2,
had 43 chromosomes and 46 had 44 chromosomes. Meiotic be—

havior in these plants was summarized before, as they were

the basis for the analysis of the multivalent associations

in the second generation. Chromosome number was determined
at early M I or at A I.

As stated before, a 42—chromosome resistant plant was
found in this N2 population. Chromosome number for this
plant was first determined from N3 germinating seeds, and
this number was confirmed upon cytological examination of
pollen mother-cells from one spike of the second genera—
tion. In most cells two univalents were found, but up to
seven were observed in one single cell, which indicates a
rather high degree of asynapsis between some chromosomes.
As usual, from one to three open bivalents were observed,
but in one cell five were found. Chains and rings of four

chromosomes were observed, this being an indication of

 

segmental interchanges in this plant. Figures 21 and 22
show some aspects of the meiotic process in this leaf rust

resistant, 42—chromosome plant, in the N2.

Observations in mitosis in N3 seeds.

 

Root tips of N3 germinating seeds were examined for
chromosome number in both resistant and susceptible plants.

In seeds from 15 susceptible plants 3 had 42, 7 had 43 and

5 had 44 chromosomes. Mitotic chromosomes of some of these

plants are shown in Figure 24 through Figure 26.

In seeds from 92 resistant plants, 2 had 42, 18 had 43
and 72 had 44 chromosomes. Mitotic chromosomes of one of
these resistant plants are shown in Figure 27. Of the 42—
chromosome plants found, this chromosome number could be
confirmed in the N2 meiotic sample of one plant (4—35).

Root tip cells from germinating seed of some of the
control plants, 82 a l-2—6—2,were also examined, which
were inoculated at the same time as N2 plants. One sus—
ceptible plant was found in this group and the chromosome
number was 44. Chromosome number from eleven of these

control resistant plants was also 44.

32

 

 

1 ‘ ‘ "'

f

I?!‘
V\
I

<

c ‘EN‘ I

P»
<~

 

’\
/
<5

\

(

Fig. 3. Somatic chromosomes in
a root tip cell from the 82

a-l—2—6—2 x rye plant No. 9.

750x

\ \)‘
(

. f
Fig. 5. Prophase I showing
24 I, and 2 II in a

microsporocyte from hy—
brid plant No. 3. 970x

3

 

Fig. 4.
21 I,

(
~4
\“L V,

g.
\‘r'
w 13'

33

Prophase I showing
2 II and 1 III in a

microsporocyte from hy-
brid plant No. 3.

23 I,

*r -‘

i

a

0

970x

.
w

0"!

o
.

Fig. 6. Prophase I showing
1 II and 1 III in a

microsporocyte from hy—
brid plant No. 3.

970X

 

 

34

i ,13» 4')

JBZE,;~
’ _ ”“3
9.. <
I ‘ I
1 ‘ ds

 

 

 

 

Prophase I showing
27 I and 1 II in a micro-
sporocyte from hybrid
plant No. 2. 970x

Fig. 7. Organized metaphase Fig. 8.
plate showing 28 chromo—
somes in hybrid plant No.
10. 970x

 

  

 

7«———— M

 

Fig. 9. Prophase I showing 25 Fig. 10. Prophase I showing
I and 1 chain of 4 chromo— 20 I, 2 II and l associa-
somes in a microsporocyte tion'of 5 chromosomes in
from hybrid plant No. 2. microsporocyte from hybrid
970X plant No. 5. 970x

 

 

 

 

 

Fig. 11. Metaphase I showing 1 I, 19 II and 1 IV
in a microsporocyte from 82 a 1-2—6-2 X Cornell
595. 1455X

 

 

A'.’ l

{5?
‘ g.
\ 0

.}.$
1‘

3..

O

 

Fig. 12. Metaphase I showing
20 II and 1 IV in a micro—
sporocyte from N plant
2—15—1. 970x

j a."
l .' .‘
9. .
oaqj’sa
u: 1.7-

Fig. 14. Metaphase I showing
1 fragment, 1 I, 18 II, 1
chain of 6. N plant
2—15—1. 970x

36

W“!
"M“
S.

‘6 ‘

Fig. 13. Metaphase I showing
20 II and 1 ring of 4
in N1 plant 1—6—3. 970x

Fig. 15. Metaphase 1 showing
1 fragment, 14 II, 1 III
and 1 chain of 12. Nl
plant 1-17-3. 970x

 

 

 

Fig. 16. Metaphase I showing 1 I, 10 II, 1 III and
2 chains of 10 chromosomes in a microsporocyte
from N1 plant 2—24~7. 1455X

37

 

 

38

. Wu, 4-»
5 w _\ 2:

Fig. 17. Metaphase I showing Fig. 18. Metaphase I showing
2 I, 17 II, 1 ring of 4 and 2 I, 18 II and 1 chain of

1 chain of 4 in a micro— 6. N2 plant 5—114. 970x
sporocyte from N2 plant '
3—123. 970x

‘0. I

‘
1 *‘
NJ, ‘

r
" c

I
‘ N
1 .
Fig. 19. Metaphase 1 showing Fig. 20. Metaphase I showing
18 II, and 1 chain of 8 14 II and 1 chain of 16
chromosomes. N2 plant chromosomes. N2 plant

3—101. 970x 3—205. 970X

 

 

39

 

Fig. 22. 42 chromosomes (1
2 I, 15 II, 1 IV and 1 divided) at Anaphase I in
chain of 6 in N resistant N2 resistant plant 4—35.
plant 4-35. 97 X 750x

Fig. 21. Metaphase I showing

"(21,;

qt.

Fig. 23. 42 somatic chromosomes
in a root tip cell from a ger—
minating seed from plant 4-35.
750X

 

 

 

(
f‘t‘ n

 

Fig. 24. 42 chromosomes in a
root tip cell from a ger—
minating seed from N2
susceptible plant 4—70.
750X’

Fig. 26.

40

b \
,‘f‘. “‘3’“!
5‘} ‘étgc;

1 ”’J’II‘QF’
”‘

Fig. 25. 43 chromosomes in
a root tip cell from a
germinating seed from N
susceptible plant 5—125.
750x

44 chromosomes in

 

a root tip cell from a
germinating seed from N
susceptible plant 5-126.
750x

 

 

 

Fig. 27. 44 chromosomes in a root tip cell from a germinating
seed from N2 resistant plant 2—259. 1455X

 

Fig. 28. Double ”satellited" iso—chromosome in a root tip
cell from a germinating seed from N2 resistant plant 5—180.

1455X

 

 

 

 

42
DISCUSSION

Cytological observations in Rosen rye and Cornell 595
wheat indicated that structural homozygosity is a common
feature in these two varieties. Only bivalents are found
at Metaphase I and the meiotic process is completely normal.
No multivalent associations, indicating any degree of struc-
tural heterozygosity were found in this material.

Observations in control plants of selection 82 a 1—2-6—2
indicate that through the process of crossing, backcross—
ing, selfing and selection, after the original cross Honor
wheat x Rosen rye was made, it has reached a state of struc—
tural homozygosity. Only univalents, usually 1 or 2, and.
21 bivalents were found at meiosis. These results agreed
with those found by Sebesta (1958) in several wheat—rye
selections derived from the same original cross. The extra
univalents are usually telocentric and presumably they are
derived from the original rye parent. In the course of
the evolution of this wheat-rye derivative some interchange
between the rye and the wheat chromatin may have taken
place. This was suggested by Jensen and Kent (1952) and

is evidenced by the presence of the hairy neck character

43

in plants with 42 chromosomes. Therefore the telocentric

chromosomes might have some structural heterozygosity and

will be referred to as rye chromosomes. Even if some
chromosome rearrangements have taken place in this mate—
rial since the cross was made, they now seem to be in a
state of structural homozygosity. No special configura—
tions indicating some degree of structural heterozygosity
are seen at meiosis in plants of the non—irradiated (con—
trol) population of this leaf—rust resistant material.

The wheat chromosomes always form 21 pairs at Metaphase I

with very rare instances when some asypnasis occurs and

several univalents (besides the extra telocentric rye
chromosomes) are present. Even in the case of the extra
chromosomes, their lack of pairing is not unexpected as
they are operating in a foreign genome and under this
condition may lack their characteristic pairing ability
(O'Mara, 1940).

Although the loss of these telocentrics should occur
frequently (Darlington, 1940; Sears, 1952a) they have
shown some stability in the selection 82 a 1—2—6—2 and

they are usually transmitted to the progeny of plants

possessing them. This persistence might be due to some

 

 

44

selective advantage, other than leaf rust resistance,

which is not critical for wheat survival. The extra telo—

centric rye chromosome may have received a small wheat
segment carrying some factor necessary to the normal func—
tioning of the wheat complement.

In an attempt to establish any homology between the
telocentric chromosomes and both the wheat and the rye
complements, crosses between 82 a 1—2—6—2 control plants
and Rosen rye, and also Cornell 595 were made. The results
of the cross to Rosen rye established a relatively high
number of cells (22.32 %) with bivalent and multivalent
associations. Bivalent formation was especially notable,

82.17 % of the total number of associations found. This

could be taken as an indication of a degree of homology

between the extra rye chromosome (in 29 chromosome hy—
brids) and one of the 7 normal rye chromosomes, if always
27 I and one heteromorphic bivalent were formed. However
this was not the case, as not only bivalents but other

types of associations were observed (see Table I). If

the extra rye chromosome was involved in bivalent asso—
ciation, some autosyndetic pairing between the wheat or

the rye chromosomes or some pairing between normal wheat

 

 

45

and rye chromosomes occurred. Allosyndetic pairing is not
likely in wheat—rye hybrids, as established by many inves—
tigators. Although hybrids between wheat and rye are made
with relative ease, no homology between wheat and rye chro—
mosomes have been demonstrated, and it seems that normal
pairing and recombination between them is almost impossible

(Unrau, 1960) and all attempts to transfer desirable char—

 

acteristics from Secale to Triticum by conventional methods
have failed.

Some of the pairing found in 82 a 1—2—6—2 X Rosen rye
may be attributed to autosyndesis. As only one bivalent
per cell would be expected to form between the telocentric
and one normal rye chromosome, and as many as three biva-
lents in the same cell were found, the two other associa—
tions have to be explained on the basis of autosyndetic
pairing. Bivalent and multivalent associations have often
been reported in haploid wheat and rye plants, as well as
in 28 chromosome wheat—rye hybrids. As pointed out by
Darlington (1937) in ”haploid,” ”triploid” and hybrid de— ;
rivatives from allopolyploid species, "chromosomes derived
from the same parental gamete pair with one another,“ even

if this pairing is not frequent in the polyploid parent.

 

 

46

So it is clear that autosyndesis may occur between homoeo—
logous chromosomes that normally do not pair with each
other in the normal polyploid. The absence of homologous
chromosomes in the case of the haploids and in interspeci—
fic hybrids may promote ”abnormal” pairing of homoeo—
logous chromosomes. This autosyndetic type of pairing has
been given as an explanation for the formation of bivalent
and multivalent associations in wheat and in rye haploids
and in wheat—rye hybrids by many investigators, including
Chin (1946), Kihara and Nishiyama (1928, 1937), Levan
(1942), Nakajima (1952, 1953), O'Mara (1953) and Thompson
(1931). However, Sears and Okamoto (1958) point out that
homoeologous chromosomes show little tendency to pair, as
the bivalent frequency in haploids normally averages about
one. The presence of heteromorphic bivalents would support
the assumption of pairing between a telocentric chromosome
and a normal rye chromosome. Out of 54 ”true” bivalents
(with chiasma formation) found, 25 were heteromorphic,
with one arm shorter than the other. This may be taken

as evidence for the homology between the telocentric and
one normal rye chromosome. However, the possibility of a

heteromorphic wheat bivalent, formed by intragenomic

 

 

 

 

47

pairing cannot be discarded as Love (1939) found a number
of chromosome rearrangements, including the formation of
heteromorphic bivalents, in advanced generations from
vulgare-like derivatives of pentaploid wheat crosses.

The results obtained in the cross 82 a 1—2—6-2 x
Cornell 595, where tetravalent associations, almost always
of the "chain” type were found in 13 % of the cells stud-
ied, lead to the conclusion that some structural rearrange—
ment has taken place in the wheat complement, at least in
relation to Cornell 595, the last of the wheat ancestors
in the pedigree of selection 82 a l-2—6—2. Even in the
case that no transfer of rye to wheat chromatin has become
involved in the evolution of this selection, chromosome
interchanges between Honor, Yorkwin, and Cornell 595,
successive wheat parents of this wheat—rye derivative,
might lead to multivalent configurations, as pointed out
by Elliott (1958) and demonstrated by Love (1951), who
found some multiple associations in plants of intervari—
etal wheat crosses. The three possible crosses among the
varieties Honor, Yorkwin and Cornell 595 could be made and
the respective Fls studied for evidence of structural dif—

ferences. These would be reflected in the formation of

 

 

48

multivalent associations at Metaphase I. But even in the
case of structural homology between these varieties, as
they exist today, chromosome rearrangements between them
might have taken place, in the evolutionary process of 82 a
1—2—6—2 and this arrangement would be reflected, again, in
multivalent configurations in the cross 82 a 1—2—6—2 x
Cornell 595.

That some transfer of rye chromatin to the wheat has
occurred, was first suggested by Jensen and Kent (1952),
who proposed that leaf rust as well as mildew resistance
of selection 779 b 10—8—6 (parent of 82 a 1—2—6—2) could
have developed through the transfer of ”one or more seg—
ments of rye chromatin by crossing over or reciprocal
translocation, followed by a novel recombination of genes
in the final hybrid.” The fact that 42-chromosome deriva—
tives of these wheat-rye selections show the hairy neck
characteristic, normally carried in one rye chromosome
(O'Mara, 1951; Riley and Chapman, 1958) supports this
hypothesis that a segment of rye chromatin has been trans—
ferred to the wheat chromatin. However Sebesta (1958)
demonstrated that leaf rust resistance was carried in the

extra telocentric chromosomes and that there was no

 

 

49

association between leaf rust and the hairy neck charac—
teristic. Although the possibility of the transfer of a
small segment of wheat chromatin to the telocentric rye
chromosome cannot be ruled out, the results obtained in the
present work do not give clear evidence for this hypothesis.
As pointed Out before, the tetravalent associations found
can be due to structural rearrangements within the wheat
genome, modified by possible chromosome rearrangements
between the three wheat parents of 82 a 1—2—6—2. Assuming
that a small segment was translocated from the wheat
chromatin to the ”rye” telocentric, it would have been so
small as to not have a competitive ability for pairing
with the normal wheat chromosomes. No pairing between the
extra univalent and the wheat chromosomes was observed in
the hybrid plants 82 a 1-2—6—2 X Cornell 595. If a defi—
nite homology existed between the extra chromosome and
those from wheat, trivalent formation would be expected,
which occurred in only one case.

The results obtained from the cytological analysis of
microsporocytes of the N1 and N2 generations, after neutron
irradiation of 82 a 1—2—6-2, proves the effectiveness of

this type of radiation to induce chromosome breaks and

 

50

rearrangements. The dosage delivered to dry seeds was
effective in inducing translocations as judged by the num—

ber of multivalent associations found in the N where,

1
out of 493 cells studied, 56.19 % had associations of more «4
than three chromosomes. Especially notable was the number

of tetravalents formed, as 85.16 % of the multivalent asso—

ciations were of this type, indicating that one interchange

 

had taken place in most of the cases. In comparison, no
multivalent configurations were found in 238 cells of con—
trol plants examined during meiosis. The presence of asso—
ciations of 6 and 8 chromosomes in chains or rings, demon—
strated that two or three interchanges had taken place,
as indicated by Osborne and Elliott (1955). Associations
of 10 and 12 chromosomes were also found although their
formation was not too frequent (1.46 and 0.97 %). This
high number of interchanges between chromosome segments in
the irradiated population of 82 a 1-2—6—2 increased the
probability of transferring the desirable portion of the
telocentric ”rye” chromosome carrying the gene(s) for
leaf rust resistance to any of the wheat chromosomes.

It was observed that the N1 had a decreased vigor in

relation to the control population. This was perhaps due

 

 

51

to an overall effect of the reshuffling of chromosome seg—
ments, when some favorable linkage groups were broken.
Some sterility was noted but the amount not calculated.

In the second generation 42.08 % of the cells analyzed
presented trivalents or associations of higher order. This
high a percentage of residual heterozygosity in the N2
reflects the fact that many different translocations were
induced by the neutron radiation. The percentage of tetra—
valents was highest, being 84.78 % of all associations
found.

The results of inoculating the N2 population with an
oil suspension of leaf rust spores confirmed the high de—
gree of resistance possessed by the wheat—rye selection
82 a 1-2-6-2. The resistance to leaf rust in this type of

material was shown to be due to the presence of extra telo—

II II

centric rye chromosomes by Sebesta (1958). Only five
meiotic samples from susceptible N2 plants were suitable
for study. All of these plants had 43 chromosomes; one
was classified as very susceptible, one as susceptible,
and the other three as slightly susceptible (or moderately

resistant). According to Sebesta the first two plants

should have been moderately resistant. In this study some

 

 

 

52

43—chromosomes N2 plants were found to be highly resistant
to leaf rust. This observation does not agree with Sebes—
ta's results for he found these plants were only moderately
resistant. These 43—chromosome plants occurred in the N2
population which had a high number of interchanges as seen
above. Sebesta worked with non—irradiated material such as
the control in this experiment. It appears that these
plants are a product of chromosomal rearrangements. The
results also indicate that the degree of expression of leaf
rust in 43—chromosome plants might be affected by the en-
vironment. Although the environmental conditions under
which the inoculation was done were kept as constant as
possible, the possibility of slight changes in humidity,
light, temperature, etc., cannot be ruled out. This might
have influenced the degree of expression of leaf rust.

The possibility also exists that the telocentric chro—
mosomes of plants with 43 and 44 chromosomes are not
always the same. Not only the number of chromosomes but
the kind of extra telocentric(s) carried by a plant should
determine its degree of resistance to leaf rust. The
factor(s) for resistance, originally carried in one arm

of a rye chromosome, may be present or absent in a

 

53

telocentric chromosome. Only when this factor is present
will the plant be resistant. This also would explain the
finding of several 43-chromosome plants among the N2 re-
sistant material. Although the resistance of 43—chromosome
plants might be due to the presence of one iso-chromosome
(Sebesta, 1958), the frequency of this type of chromosome
was not common. Resistance of 43—chromosome plants cannot
be always attributed to the presence of iso-chromosomes.
More likely the telocentric chromosomes might be differ—
ent and the factor(s) for leaf rust resistance may be
present or absent in a given telocentric, as explained
before.

In root tip cells from germinating seeds of the N3
generation, a variable number of chromosomes was found,
this being an indication of the lack of complete stabil—
ity of the 44—chromosome number in this type of material.
When plants with 43 or 44 chromosomes are selfed, they
may give origin to 42, 43 or 44—chromosome plants. It
must be recalled that leaf rust readings were made only
in the N2 generation. Chromosome numbers in germinating
seeds of the N generation were determined, but no leaf

3

rust readings were made in this third generation. In

 

 

54

seeds from 15 N2 susceptible plants, it was found that 3
had 42, 7 had 43 and 5 had 44 chromosomes. The first three
very likely came from 42-chromosome susceptible plants and
the last ones from 43—chromosome plants (slightly suscep—
tible).

Progeny from resistant plants also showed a variable

chromosome number. Out of 92 examined, only two showed 42

 

chromosomes, all the others having 43 or 44. This indi—
cates that most of these plants came from N2 plants having
at least 43 but more likely 44 chromosomes, unless they
came from 42-chromosome plants fertilized by pollen carry—
ing 22 chromosomes which is unlikely as outcrossing in
wheat is very infrequent under greenhouse conditions.

Of special importance to this study was the finding
of a leaf rust resistant plant having 42 chromosomes. This
number was found in cells from root tips of the N3 genera—
tion and also in microsporocytes of the N2. Since all 42
chromosome plants not carrying the extra chromosomes which
confer the leaf rust resistance of 82 a 1—2—6n2 are sus-

ceptible, it can be concluded that at least one inter—

change took place between the telocentric ”rye“ chromo—

some and one of the wheat chromosomes and that it is

 

 

55

possible that the gene(s) for such resistance were incor—
porated in the wheat complement. Although the results must
be interpreted with caution, when consideration is given to
the high number of translocations observed in both N1 and
N2 populations, and the meiotic irregularity of this plant
(4—35) in the N2, it is possible that the desired type of

interchange took place. An N3 population with seed from

this N2 resistant plant should be grown and inoculated with

 

leaf rust to see if both its disease resistance and chro—
mosome number remain constant. After a few generations the
desired translocation should become homozygous and a stable,
42—chromosome, leaf-rust resistant line should be avail—
able. It could be crossed with susceptible, 42—chromosome
wheat varieties to study leaf rust reaction in F1 and sub—
sequent generations.

The occurrence of a point mutation, resulting in the
resistance to leaf rust of plant 4-35 cannot be ruled out.
Although a point mutation could not be distinguished cyto-
logically from a very small translocation, the two could
be identified by the coverage of resistance to different
physiologic races of the pathogen. As pointed out by Jen—

sen and Kent (1952), selection 82 a 1-2-6-2 is highly

 

56

resistant (in the seedling stage) to races 9 and 19, and
susceptible to races 5, ll, 15, 58, 93, 105 and 126, and
segregating for susceptibility to races 21 and 45. If
resistance of plant 4—35 is due to a translocation, its
segregates should show the same pattern of resistance to
the mentioned races; if due to a point mutation, the reac—
tion to physiologic races of leaf rust should be different.

As stated before, the possibility exists that the telo—
centric chromosomes found in selection 82 a 1—2—6—2 are
not always the same. There might be at least two types,
corresponding to the original arms of the rye chromosome
responsible for leaf rust resistance. This may be sup—
ported by the finding of a pair of double satellited iso—
chromosomes (satellites at both ends, see Figure 28) in
cells from root tips of germinating seed from N2 plant
5—180. Unfortunately, the whole complement could not be
studied in a single field. A very careful study of the
karyotype of both 82 a 1—2—6—2 and of Rosen rye would be
necessary to determine the possible origin of the telo-
centric involved in leaf rust reSIStance in this

selection.

 

 

57

As suggested before, the problem could be clarified
in two ways:

1. By making crosses between 82 a 1—2-6-2 and suscep-
tible wheat varieties, inoculating the F2 or backcrosses
with leaf rust to study segregation and by cytological

study of the segregates carrying the extra telocentric

rye chromosome. A11 42—chromosome plants in this segre—
gate should be susceptible.

2. By obtaining a series of monosomic addition lines
from Rosen rye to a susceptible hexaploid wheat variety.
This would help in identifying the rye chromosome respon—
sible for leaf reelstance (if this variety is still resis—
tant as it was when chosen as parent in the original
wheat—rye cross), and would provide new germplasm for
further work in the problem of transfer of disease resis—

tance, especially by the radiation—induced translocation

technique demonstrated by Sears (1956) and Elliott (1957).

58

SUMMARY AND CONCLUSIONS

The cytology of selection 82 a 1—2-6-2, a wheat—rye
derivative which has excellent mature plant resistance to
leaf rust at East Lansing, Michigan, was investigated.

This wheat-like selection, as shown by Sebesta (1958), has
44 chromosomes, with the two extra chromosomes being telo-
centric and carrying a factor(s) for leaf rust resistance.

Crosses between plants of this selection and both Rosen
rye and Cornell 595 wheat were made, in an attempt to
determine the homologies between the extra chromosomes and
the wheat and rye complements. Associations of two, three,

four, and five chromosomesvere observed in the cross 82 a

1—2—6—2 x Rosen rye. The formation of bivalents was pre—
dominant (82.l7 %). Chiasmata were observed in 54 of the
bivalents and 25 of them were heteromorphic. The presence

of this type of bivalent may be taken as evidence for a
degree of homology between the extra telocentric chromo—
some and one of the rye chromosomes. However the possi—
bility of heteromorphic wheat-rye bivalents cannot be ruled
out, and it was very difficult to make a distinction between

rye and wheat chromosomes at melOSlS of the hybrid plants.

 

 

 

59

Tetravalent formation was observed in 13 % of cells anal—
yzed from the cross 82 a 1-2-6—2 x Cornell 595. It was
concluded that homologies between the extra telocentric
and the wheat genome were not apparent, since the extra
univalent was unpaired in each cell examined, except in
one with a trivalent. The tetravalent formation was prob—
ably due to structural heterozygosity in the wheat comple—

ment of 82 a 1—2—6—2, in relation to Cornell 595

 

chromosomes.

Selection 82 a 1—2—6-2 has shown some variation in
leaf rust resistance at East Lansing due to the instabil—
ity of the two telocentric chromosomes. Methods of trans-
ferring a small segment of the telocentric chromosomes
containing the leaf rust resistance to a wheat chromosome
and eliminating the remainder of the telocentric chromo—
somes were studied. Dry seed from several plant selec-
tions of 82 a 1—2—6—2 were irradiated in an attempt to
transfer leaf rust resistance to the wheat complement. As
determined from the analySIS of multivalent associations
in pollen mother-cells from the N1, 56.19 % of them had
trivalents or associations of high order, with tetravalents

being predominant (58.16 %‘. In the N2 the Situation was

 

 

60

similar, with 42.08 % of the cells studied showing multi-
valent associations and again tetravalents being predomi—
nant (84.78 %). This proved that at least one interchange
was obtained in a high percentage of the cells analyzed.
The results of the inoculation of N2 plants with an oil
suspension of leaf rust spores demonstrated the high degree
of resistance of this type of material, as about 98 % of
the population was resistant. When chromosome number in
root tip cells of germinating seeds from N2 resistant
plants was examined, seeds from plant 4—35 proved to have
42 somatic chromosomes. This chromosome number was con—
firmed in the meiotic sample from the N2 of this plant. A
number of irregularities, as multivalent associations,
unpaired univalents at M I and lagging chromosomes at A I
and T I were found. This suggests the possibility that
some chromosomal rearrangements have taken place as a
result of the neutron irradiation and that the desired

transfer of the leaf rust resistance factor(s) from the

rye telocentrics to the wheat complement may have taken
place. N3 and further generations of this plant should be

grown and inoculated with leaf rust to determine if it

keeps its resistance and if the 42 chromosome number is

 

 

   

stable. If this is the case, it would be a useful source

 

of germplasm for further work.

 

62
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