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SUPPLEMENTARY MATERIALS
Selection of the A-A translocation and markers
Construction of new B-A-A translocations and successful screening for them
requires that two conditions be met. First, it is necessary to select an A-A translocation
wherein one of the chromosome arms involved in the interchange is the same arm as that
borne on the simple B-A chromosome and that the breakpoint in the arm of shared
homology of the A-A translocation be distal to the breakpoint of the A chromosome arm
borne on the B-A. The greater the distance between these two breakpoints, the greater the
extent of shared homologous region of that arm and the greater the likelihood of synapsis
and crossing over within that region resulting in a recombinant B-A-A chromosome.
Second, the breakpoint in the A chromosome arm of the distal-most segment of the B-AA translocation has to be proximal to the location of the marker locus in the tester stock
used to screen for the new B-A-A translocation. This assures that the distal-most segment
can bear the dominant allele of the marker locus and that the endosperm marker
phenotype can be uncovered in the hypoploid endosperm.
Selection of suitable B-A/A-A plants for pollen to cross onto tester stocks
Consideration must be given to whether or not a potential pollen parent plant
carries the simple B-A chromosome, and also whether or not it carries the A-A
translocation chromosomes. Because the B-A stock plants used as female parents of the
initial cross are grown from kernels containing embryos that are hyperploid for the B-A
chromosome (two copies of the B-A chromosome) most of these plants will carry the BA chromosome. The A-A stocks used as pollen parents of the initial cross are grown
from kernels containing embryos that are either homozygous (T/T) or are heterozygous
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(N/T) for the A-A translocation. In the former case all progeny plants grown from
kernels produced by the initial cross will carry the A-A translocation and will be
heterozygous for it (N/T). But in the latter case, where the pollen parent used to make the
initial cross is N/T then half of the progeny plants will carry the A-A translocation and be
heterozygous for it (N/T) and half will not carry the translocation (N/N). In either case, it
is important to verify that the progeny plants which are to be used to cross onto testers
carry the A-A translocation. Verification of the presence of an A-A translocation or an
inversion in a maize stock can readily be accomplished by examination of the pollen of
candidate plants. This is easily done by using a pocket microscope to score pollen
sprinkled onto a black bottle cap from a freshly extruded anther whose tip has been
pinched off (DOYLE 1993). Because plants heterozygous for an A-A translocation
produce microspores that are duplicate-deficient for chromosome segments and
microspores with a complete balanced set of chromosomes in a 1:1 ratio, plants that are
heterozygous for an A-A translocation (N/T) can be readily identified by their
approximately 50% pollen abortion frequency. Whereas normal pollen grains are opaque
and appear like white pearls, aborted pollen grains appear empty, translucent, collapsed,
or otherwise grossly abnormal (DOYLE 1993).
The B-A/A-A pollen parent plants need to carry both the B-A and the A-B
chromosomes
The sources of pollen parents to be crossed onto appropriate kernel trait tester
stocks are plants grown from kernels produced by crossing plants containing A-A
translocations onto plants carrying simple B-A translocations. The latter plants are
hyperploid for the B-A and contain two B-A chromosomes and one corresponding A-B
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chromosome. The plants containing A-A translocations are identified by pollen
semisterility, and therefore are heterozygous for the translocated chromosomes.
Consequently, among the progeny of the cross of the B-A stock by the A-A stock most
will contain the simple B-A chromosome and approximately half of them will also carry
the A-B chromosome. Furthermore, among these same progeny, approximately half of
the plants will carry the A-A translocation, and these will be heterozygous for the
translocation and can be identified by their pollen semisterility. In most cases this
semisterility approaches 50% but in those instances where one or both of the
translocation breakpoints are located in the distal region of the chromosome arms the
percent of aborted pollen may approach 25% (See BURNHAM 1978 for a detailed
discussion). In most cases, the identification of plants carrying an A-A translocation is
not difficult, and approximately half the plants in a 20 kernel family planted as a sourse
of B-A/A-A pollen parents will be identified as carrying the A-A translocation by their
pollen semi-sterility.
In addition to carrying the A-A translocation, the pollen parents need to carry both
the simple B-A chromosome and the A-B chromosome in order to be suitable for creating
B-A-A stocks carrying the A-B chromosome. The compound B-A-A stocks need to carry
the A-B chromosome in order to be euploid and for the B-A-A to regularly undergo
nondisjunction during the second pollen mitosis which results in the production of the
hyperploid and hypoploid sperm.
Selection of testers for screening for new B-A-A translocations
The most useful tester stocks for crossing onto with pollen from B-A/A-A
heterozygotes are stocks carrying either in the homogyzous or heterozygous condition a
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recessive allele for one of the eight aleurone color factors, while all of the other of the
factors are present in the homozygous dominant condition. These color factors, their
recessive aleurone phenotypes, and their chromosome arm locations are bz2, bronze, 1L;
a1, colorless, 3L; c2, colorless, 4L; a2, colorless, 5S; pr1 red, 5L; c1, colorless, 9S; bz1,
bronze, 9S; and r1, colorless, 10L. These tester stocks are conveniently used in
combination with the presence of a dominant R1 allele such as R1-scm 2 that conditions
the scutellum region of the embryo to have a purple color when all of the color factors are
represented in the embryo by at least one dose of their dominant allele (ROBERTSON
1967). These are the kinds of stocks most widely used in creating the new B-A-A
translocations reported here. For example, a c2 tester used to screen for new B-A-A
recombination events where the distal A chromosome segment of the new B-A-A will be
a distal portion of chromosome arm 4L, will have a genotype of Bz2/Bz2, A1/A1, c2/c2,
A2/A2, Pr1/Pr1, C1/C1, Bz1/Bz1, and R1-scm2/R1-scm2. Pure breeding purple kernel
stocks produce ears, when self pollinated, whereon all of the kernels have a purple
aleurone comprising the outermost layer of the endosperm. If they are homozygous for
the R1-scm2 allele, all of their embryos will be purple colored.
Most of the colorless endosperm inbred stocks (with yellow or white kernels)
used for hybrid corn seed production, and also used to propagate cytogenetic stocks such
as the A-A translocation stock collection, are homozygous recessive for both the c1 locus
on 9S and the r1 locus on 10L. This is a complicating factor when aiming to create new
B-A-A translocations involving distal regions of chromosome arms 9S or 10L. This is
because commonly the A-A translocation stock that is crossed onto a simple B-A stock to
make a B-A/A-A heterozygote is c1/c1, r1/r1 and thus the B-A/A-A stock will carry both
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the c1 and r1 recessive alleles. Consequently when the B-A/A-A stock’s pollen is
crossed onto a 9S (c1/c1) or 10L (r1/r1) tester; one-half or more of the resulting kernels
may be colorless, i.e. c1/c1 or r1/r1. This may be avoided by converting A-A
translocations into pure breeding purple kernel stocks and employing simple B-A stocks
that are homozygous for the dominant C1 and R1 alleles.
The aleurone color stocks are limited in their usefulness because the aleurone
color loci are not located at the ends of the chromosome arms bearing them. They are
only useful when screening for new B-A-A stocks where the distal A segment of the new
B-A-A includes the gene locus for the aleurone color factor borne on that arm of interest.
Ordinarily, the dominant color condition allele is present for that locus in the A-A stock
used to make the B-A/A-A heterozygous (except for c1 and r1, as noted above). When
this is the case then the new B-A-A chromosome will carry in its distal A segment the
dominant allele of the tester stock locus. This means that the hyperploid sperm
containing two copies of the new B-A-A will provide the dominant color factor so that
the hyperploid tissue product of double fertilization will be purple colored while the
hypoploid product of fertilization will lack anthocyanin and be colorless (white or
yellow).
All kernel tester traits are limited in their use by the general rule that they are
useful for detecting new B-A-A translocations only when the breakpoint of the A
chromosome arm of interest (in the A-A chromosome translocation stock) is proximal to
the location of the tester gene locus on that arm. In the example described above of the
production of the new B-A-A TB-1Sb-4L 002-19 1S.87 4L.42, the new B-A-A event was
discovered as a colorless endosperm/colored embryo kernel. This was possible because
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the c2 locus on chromosome arm 4L is distal to the breakpoint at the cytological position
of 0.42 on 4L and therefore the dominant C2 allele is borne on the distal 4L segment of
the B-A-A chromosome. The egg cell of the tester stock contained a recessive c2 allele
but the hyperploid sperm containing two doses of the C2 bearing B-A-A chromosome
fertilized the egg to produce an embryo that was C2/C2/c2 and therefore colored. The
hypoploid recessive c2 polar nuclei were fertilized by the hypoploid sperm nucleus
lacking any allele at the c2 locus and therefore the resulting endosperm was colorless
because it lacked the dominant C2 allele that is required for anthocyanin synthesis.
There are two additional classes of kernel trait markers that are useful in
screening for new B-A-A translocations. First are the genes that in the mutant recessive
allelic state block or reduce carotenoid synthesis resulting in white or lemon-yellow
colored kernels instead of the full yellow kernel normally exhibited by kernels carrying
one or more doses of the dominant Y1 allele. These include some of the genes with
mutant viviparous phenotypes. Particularly useful have been vp5 on chromosome arm 1S
in our study, and w3 on chromosome arm 2L (RAKHA and ROBERTSON 1970).
Additional markers with reduced yellow pigmentation include al1 on 2S, cl1 on 3S, lw1
on 1L, lw2 on 5L, vp1 on 3L, y10 on 3L, vp9 on 7S and y9 on 10S (See BECKETT 1991
for further descriptions including seedling factors for confirming the presence of a B-A or
B-A-A chromosome). All of the above noted mutant loci result in an albino or light
colored seedlings except for y9. The latter shows a normal seedling phenotype in our
nurseries, but in some environments it produces pale green seedlings. Two additional loci
are useful as markers and both are embryo or seedling lethals. These are the anl1 locus
on chromosome arm 5S which results in a light colored or gray endosperm and an
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unviable embryo, and the w2 locus on chromosome arm 10L which results in a mutant
kernels in a purple kernel stock displaying a mottled purple aleurone phenotype, and
producing albino seedlings. Both anl1 and w2 are distal to the more commonly used a2
and r1 loci on chromosome arms 5S and 10L, respectively, and are therefore useful for
detecting new B-A-A translocations involving breakpoints distal to the a2 and r1 loci.
Second are the numerous genes represented by the defective kernel (dek)
mutations that have been found to be located thoughout all of the chromosome arms
except for 8S (NEUFFER et al. 1986; NEUFFER and SHERIDAN 1980; SCANLON et
al. 1994). These mutations affect to varying degrees both endosperm and embryo
development. For many of the mutations, embryo development is so seriously impaired
that the embryos or resulting seedlings suffer lethality (SHERIDAN and NEUFFER
1980, 1981). These lethal types of dek mutations are especially useful when they are
used as tester stocks because any contaminating self pollinated kernels or mutant
phenotype kernels that are produced by previously unknown allelism of testers will fail to
produce normal plants. Some of the dek mutations, especially those resulting in a
collapsed endosperm phenotype, result in such a severely reduced endosperm that kernels
with mutant (hypoploid) endosperm and normal (hyperploid) embryos may require
germination and initial culturing in the greenhouse.
Dosage and deficiency analyses using B-A-A stocks
Simple B-A chromosome stocks can be used as pollen parents to produce kernels
with endosperms that are hypoploid (2X) or hyperploid (4X) for the A chromosome
segment borne on the B-A chromosome [which is the segment distal to the breakpoint in
the A chromosome; i.e., 0.05 to 1.00 (the end) of 1S in TB-1Sb], and with embryos that
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are hypoploid (1x) or hyperploid (3x) for that A segment. The possibilities are more
complex when using B-A-A stocks as pollen parents in making crosses. Such crosses
produce kernels with endosperms that are hypoploid (2X) or hyperploid (4X) for the two
A chromosome segments borne on the B-A-A chromosome (TB-1Sb-4L 002-19) [with
the 1S segment from 0.05 to 0.87 and the 4L segment from 0.42 to 1.00 comprising the
A-A region of the B-A-A chromosome], and whose embryos are, respectively, hyperploid
(3X) or hypoploid (1X) for these chromosome segments. This results in the B-A-A
pollen parent contributing either a duplicate or deficient segment for a chromosome
region of two different A chromosomes. Furthermore, although a compound B-A-A
chromosome stock can produce progeny with hyperploid and hypoploid tissues for both
A chromosome segments, the first segment (attached to the B chromosome) is always an
interstitial region of an A chromosome while the second segment (the distal-most of the
A segments of the B-A-A) is always a distal region extending to the end of the
chromosome arm of the A chromosome. Therefore tissues (endosperm or embryo) that
are hyperploid or hypoploid for the B-A-A chromosome will be either duplicate (with an
extra copy) or deficient (lacking a copy) for an interstitial region of one A chromosome
arm and for a distal region of another A chromosome arm. These features of hyperploid
and hypoploid doses and their consequences for duplication and deficiency of
chromosome regions are shown in Supplementary Materials Figure 2.
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REFERENCES FOR SUPPLEMENTARY MATERIALS
BECKETT, J. B., 1993 Locating recessive genes to chromosome arm with B-A
translocations, pp. 315-327 in The Maize Handbook, edited by M. FREELING and V.
WALBOT, Springer-Verlag, New York.
BURNHAM, C.R. 1978 Cytogenetics of interchanges, pp. 673-692 in Maize Breeding
and Genetics, edited by D.B. Walden. John Wiley and Sons, New York.
DOYLE, G.G., 1993 Inversions and list of inversions available, pp 346-349 in The Maize
Handbook, edited by M. FREELING and V. WALBOT. Springer-Verlag, New York.
NEUFFER, M. G. and W .F. SHERIDAN, 1980 Defective kernel mutants of maize I.
Genetic and lethality studies. Genetics 95: 929-944.
NEUFFER, M. G., M. T. CHANG, J. K. CLARK, W. F. SHERIDAN, 1986 The genetic
control of maize kernel development, pp. 35-50 in Regulation of Carbon and Nitrogen
Reduction and Utilization in Maize, edited by J. C. SHANNON, D. P. KNIEVEL, and
C. D. BOYERS. Am Soc Plant Physiol, Rockville, MD.
RAKHA, F. A. and D. S. ROBERTSON, 1970 A new technique for the production of AB translocations and their use in genetic analysis. Genetics 65: 223-240.
ROBERTSON, D. S., 1967 The use of R2scm to facilitate the transfer of maize
chromosomal segments. J. Heredity 58:152-156.
SCANLON, M. .J., P. S. STINARD, M. G. JAMES, A. M. MYERS, D. S.
ROBERTSON, 1994 Genetic analysis of 63 mutations affecting maize kernel
development isolated from Mutator stocks. Genetics 136: 281-294.
SHERIDAN, W. F.and M. G. NEUFFER, 1980 Defective kernel mutants of maize. II.
Morphological and embryo culture studies. Genetics 32: 945-960.
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SHERIDAN, W. F. and M. G. NEUFFER, 1981 Maize mutants altered in embryo
development, pp. 137-156, in Levels of Genetic Control in Development, edited by S.
SUBTELNEY and U. ABBOTT. New York.
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Supplementary Materials Figure Legends
Figure 1. – When fertilization of egg and polar nuclei of a c2 tester plant (A) is
accomplished by sperm of a pollen grain carrying normal chromosomes and the recessive
c2 allele (B), then a concordant kernel results with both the embryo and the endosperm
being colorless (C).
Figure 2.– When an embryo sac containing a normal chromosome 1 and a normal
chromosome 4 (bearing the recessive c2 allele) receives a hyperploid and a hypoploid
sperm from a pollen grain carrying a B-1-4 and 1-B chromosome and 4-1 translocated
chromosome, the dosage of chromosome segments and genetic markers in the endosperm
and embryo resulting from fertilization by the hyperploid and hypoploid sperm will vary,
depending on which of the two sperm fertilizes the egg and which fertilizes the polar
nuclei. (A) In the case of the polar nuclei fusing with the hyperploid sperm the resulting
endosperm will be hyperploid for the region on 1S from 0.05 distal to 0.87 and
hyperploid for the region on 4L from 0.42 distal to 1.00 (the end of the chromosome).
However, the distal most regions of chromosome arms 1S and 4L will differ in dosage;
for 1S, the region from 0.87 distal to 1.00 will be 3X in dosage while for 4L, the region
from 0.42 distal to 1.00 will be 4X in dosage. (B) The reciprocal dosage ratios will
occur in the endosperm resulting from fusion of the hypoploid sperm with the polar
nuclei. Here the endosperm will be hypoploid for the 1S region from .05 distal to 0.87
and hypoploid for the 4L region from 0.42 distal to 1.00. However, the distal most
regions of chromosomes arms 1S and 4L will differ in dosage because the 1S region from
0.87 distal to 1.00 will be 3X in dosage (as in the endosperm shown in A), but the 4L
region from 0.42 distal to 1.00 will be 2X in dosage. (C) When the hyperploid sperm
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fertilizes the egg, the resulting embryo will be hyperploid for the 1S region from 0.05
distal to 0.87 and hyperploid for the 4L region from 0.42 distal to 1.00. But, as in the
case for the endosperm, the distal most regions of chromosome arms 1S and 4L will
differ; for 1S, the region from 0.87 distal to 1.00 will be 2X in dosage, while for 4L, the
region from 0.42 distal to 1.00 will be 3X in dosage. (D) The reciprocal dosage ratios
will occur in the embryo resulting when the hypoploid sperm fertilizes the egg. This
embryo will be hypoploid for the 1S region from 0.05 distal to 0.87 and hypoploid for the
4L region from 0.42 distal to 1.00. But the distal most regions of chromosome arms 1S
and 4L will differ; for 1S the region from 0.87 distal to 1.00 will be 2X in dosage, while
for 4L, the region from 0.42 distal to 1.00 will be 1X in dosage.
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