Journal of Heredity 2003:94(4)
© 2003 The American Genetic Association 94:352-355
Brief Communication |
Association of the Yellow Leaf (y10) Mutant to Soybean Chromosome 3
From the Department of Crop Sciences, University of Illinois, Urbana-Champaign, Urbana, IL 61801.
Address correspondence to Ram J. Singh, Department of Crop Sciences, National Soybean Research Center, 1101 W. Peabody Drive, Urbana, IL 61801, or e-mail: r-singh{at}uiuc.edu.
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Abstract |
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At least 19 single recessive gene yellow leaf mutants and one duplicate recessive gene mutant have been described in soybean. This study was conducted to associate a yellow leaf mutant, y10, with a specific soybean chromosome by using primary trisomics (2n = 41). Seven soybean primary trisomics were hybridized as female parent with genetic stock strain, T161, carrying y10. F1 disomic and primary trisomic plants were identified cytologically. One disomic (control) and all primary trisomic plants were allowed to self-pollinate and F2 populations were classified for green versus yellow leaf mutant. The F2 population of Triplo 3 segregated in a 17:1 ratio, while a disomic (3:1) ratio was observed with Triplo 8-, 17-, 18-, and 20-derived F2 populations, suggesting that the y10 locus is on chromosome 3. The y10 locus was examined with four simple sequence repeat (SSR) markers (Satt584, Sat_033, Satt387, and Satt022) from molecular linkage group (MLG) N and y10 was found linked with Satt022. Therefore we confirmed the association of MLG N with chromosome 3. The possible association of y10 with Triplo 16 and Triplo 19 are discussed.
Soybean [Glycine max (L.) Merr., 2n = 40] contains more than 250 classical mutants, distributed on 20 classical linkage groups (CLGs) (Palmer and Shoemaker 1998). However, most of the mutants have not been associated with specific soybean chromosomes (Xu et al. 2000). Primary trisomics (2n = 2x + 1) are an invaluable cytogenetic tool to associate genes and classical and molecular linkage groups to specific chromosomes (Singh 2003). Twenty possible soybean primary trisomics (2n = 2x + 1 = 41) have been tentatively identified morphologically and cytologically (Xu et al. 2000). Primary trisomics have been used to locate a few genes to their respective chromosomes in soybean. Honeycutt et al. (1990) assigned v2 (variegated leaf) to Trisomic A (Triplo 5). Hedges and Palmer (1991) placed an isozyme marker, dia1 (diaphorase), to Trisomic D (Triplo 4). Xu et al. (2000) located eu1 (urease null), lx1 (lipoxygenase null), and p2 (puberulent) on Triplo 5, 13, and 20, respectively. Gardner et al. (2001) located Rps1-k (resistant to phytophthora root rot) on Triplo 3.
In soybean, at least 19 single recessive gene yellow leaf mutants and one duplicate recessive gene mutant have been described (Palmer et al. 2000). Some of these mutants have been assigned to specific CLGs, such as y9 to CLG 14 (Probst 1950; Thorson et al. 1989), y11 to CLG 6 (Mahama et al. 2002), y12 to CLG 1 (Kiang and Butt 1991), y13 to CLG 7 (Weiss 1970), y17 to CLG 14 (Devine 1998), and y23 to CLG 8 (Palmer et al. 1990). The objective of this study was to associate a yellow leaf mutant (y10) to a soybean chromosome by using primary trisomics and to map it with simple sequence repeat (SSR) markers.
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Materials and Methods |
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Primary trisomics in soybean were isolated, identified, and produced in the genetic background of soybean cv. Clark 63 by using a backcross procedure (Xu et al. 2000). All the primary trisomics carry the Y10 locus that conditions normal chlorophyll development. The genetic type T161 carries the y10 allele that conditions yellow leaf has weak growth at the early vegetative stage, recovers later, and is an excellent male parent. Seed of T161 was obtained from Dr. Randall Nelson (USDA-ARS, Urbana, IL). Primary trisomics 3, 8, 16, 17, 18, 19, and 20 were used as female parent to cross with T161. Primary trisomic and disomic plants were identified cytologically in the F1. One disomic and all primary trisomic plants were saved and allowed to grow and self-pollinate in the greenhouse. The F2 and F3 seeds were planted in the greenhouse and after the seedlings were established, individual plants were tagged, numbered, and classified for green versus yellow leaf phenotypes. Chromosome number was determined microscopically for all F2 recessive homozygotes of Triplo 19 x T161.
Twenty-two SSR markers from MLG N were screened against Triplo 3 and T161. Only four SSR markers showed polymorphisms. Ninety-six F2 plants derived from trisomic F1 plants of Triplo 3 x T161 were examined with SSR markers (Satt584, Sat_033, Satt387, and Satt022). DNA was isolated according to Walbot (1988). Polymerase chain reactions (PCRs) were undertaken in 10 µl volumes containing 30–45 ng of template DNA, 1.5 pmol of each primer, 0.2 mM dNTPs (Pharmacia Biotech Inc., Piscataway, NJ), 1.5 mM MgCl2, 1x PCR buffer, and 0.5 U Taq polymerase (Gibco BRL Life Technologies Inc., Gaithersburg, MD). Temperature cycling was performed in an MJ Research PTC 100 thermal controller. The amplification profile was set to run at 94°C for 3 min followed by six cycles of denaturing at 94°C for 30 s, annealing from 55°C to 50°C decreasing 1°C each cycle for 30 s, and 72°C for 1 min. The final cycle (94°C for 30 s, 50°C for 30 s, and 72°C for 1 min) was repeated 35 times. Amplification products were detected by 6% denaturing polyacrylamide gel electrophoresis and silver staining.
The trisomic ratio (17:1) in F2 suggested that the gene was located on the extra chromosome. The genotype of the F1 trisomic plant was expected to be Y10Y10y10. The trisomic ratio (17:1) was based on a 50% female transmission rate of the extra chromosome from the primary trisomic F1s with duplex genotype (Singh 2003). For the SSR markers, the segregation ratio in the F2 population was tested for goodness-of-fit to a genotypic disomic ratio (1:2:1) and to a trisomic ratio (6:11:1). The trisomic ratio (6:11:1) was based on a 50% transmission rate of the extra chromosome. A phenotypic ratio of 12.5:1 (genotypic ratio 5:7.5:1) was expected with a female transmission rate of 33.3% of the extra chromosome (Singh 2003).
The linkage relationships between y10 and SSR markers were determined according to the method of Satovic et al. (1998). The recombination fraction was estimated based on 50 F2 individuals. Genotypes (homozygous or heterozygous) were recorded according to F3 segregation ratios. The loci were considered in coupling phase and the F1 trisomic genotype was expected to be AB/AB/ab. The transmission rate of the extra chromosome in the female parent was considered 50%.
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Results and Discussion |
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The observed numbers of green and yellow leaf (y10) seedlings in the F2 populations are shown in Table 1. The F2 segregation data for Triplo 8, 17, 18, and 20 fit the expected disomic ratio (3:1) for a single recessive gene. The F2 populations from Triplo 16 x T161 and Triplo 19 x T161 differed from 3:1 and 17:1. The possibility of associating y10 with Triplo 16 and Triplo 19 will be discussed later. In the cross of Triplo 3 x T161, the ratio of green:yellow leaf was 194:8, which deviates from a 3:1 ratio and fits a 17:1 ratio. This suggested that locus y10 was located on chromosome 3.
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Since MLG N has been associated with chromosome 3 (Zou et al. in press), y10 should be mapped on MLG N. A total of 22 SSR markers, situated on MLG N, were tested for polymorphism against Triplo 3 and T161. Only four SSR markers, Satt584, Sat_033, Satt387, and Satt022, showed polymorphism and these four SSR markers were used to examine 96 F2 plants derived from Triplo 3 x T161 (Table 2). All the SSR markers showed the trisomic ratio (6:11:1), suggesting that these markers are located on chromosome 3. The present results confirm the association of MLG N with chromosome 3 (Zou et al. in press). Only Satt022 is linked with y10 with a recombination fraction of r = 0.192 (LOD = 3.422) (Table 3). The exact location of y10 on MLG N could not be determined because of the lack of polymorphic SSR markers in this region. Only 18.1% of SSR markers showed polymorphism between Triplo 3 and T161. Demirbas et al. (2001) also reported a low percentage of polymorphisms for SSR markers. In their effort to identify SSR markers linked to the soybean Rps genes for phytophthora resistance, 16.7–30.7% of markers showed polymorphism in the mapping populations.
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In this study, the trisomic ratio (17:1) is based on a 50% female transmission rate. Usually the actual transmission rate is lower than 50%, and a 12.5:1 ratio is recorded when female transmission of the extra chromosome is 33.3%. In addition, if the gene is located on a telotrisomic, the ratio will be 7:1. As discussed earlier, the F2 population from Triplo 16 x T161 and Triplo 19 x T161 showed a ratio of 9:1 (156:17) and 6:1 (297:47) instead of a disomic ratio (3:1) (Table 1). We counted 47 recessive homozygous F2 plants, derived from Triplo 19 x T161, and these plants were diploid. This raised the question: Is the y10 locus also located on Triplo 16 and Triplo 19? With SSR markers and trisomic analysis, Triplo 19 has been associated with MLG L, and Triplo 16 and Triplo 19 carry the same extra chromosome (Zou JJ, unpublished data). We tested SSR markers in the F2 progenies from Triplo 16 x G. soja (PI 407287), Triplo 19 x G. soja (PI 407287), and Triplo 3 x G. soja (PI 407287) (Table 4). All markers from MLG N showed a trisomic ratio with Triplo 3 and a disomic ratio with Triplo 16 (Triplo 19). Meanwhile, all markers from MLG L showed a trisomic ratio with Triplo 16 (Triplo 19) and a disomic ratio with Triplo 3. These results not only confirmed the association between MLG N and Triplo 3, and the association between MLG L and Triplo 16 (Triplo 19), but also indicated that Triplo 3 and Triplo 16 (Triplo 19) carried a different extra chromosome. Since y10 was located on chromosome 3 and linked with an SSR marker (Satt022) from MLG N, it is doubtful that y10 is also located on the extra chromosome of Triplo 16 (Triplo 19). Heterozygosity of the loci, population size, or other unknown factors might cause the segregation deviation of Triplo 16 and Triplo 19 from the disomic ratio. The spontaneous occurrence of telotrisomics, secondary trisomics, and tertiary trisomics in the progenies of primary trisomics is common (Singh 2003). Triplo 16 and Triplo 19 need to be reexamined for chromosome aberrations in the extra chromosome.
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In this study, we directly located y10 to the soybean chromosome 3 with primary trisomics and further tagged it with SSR marker Satt022 from MLG N. The linkage between y10 and other classical genes (L2, Rps1, Rps7, Hm, and Rpg4), which have been associated with MLG N (Cregan et al. 1999), still needs to be tested.
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Acknowledgments |
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We wish to thank Dr. Zlato Satovic (Faculty of Agriculture, Zagreb, Croatia) for suggestions in estimating the linkage in trisomic inheritance. We also wish to thank Dr. Perry B. Cregan (USDA-ARS, Beltsville, MD) for providing us with the SSR primers. This work was supported in part by C-FAR grant 1-5-95411.
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Footnotes |
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Corresponding Editor: Halina T. Knap
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References |
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