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Journal of Heredity Advance Access originally published online on July 12, 2006
Journal of Heredity 2006 97(4):423-427; doi:10.1093/jhered/esl015
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© The American Genetic Association. 2006. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.

Genetic Analysis of 4 New Mutants at the Unstable k2 Mdh1-n y20 Chromosomal Region in Soybean

Min Xu, and Reid G. Palmer

From the Department of Agronomy and Interdepartmental Genetics Graduate Program, Iowa State University, Ames, IA 50011 (Xu); and US Department of Agriculture–Agricultural Research Service, Corn Insects and Crop Genetics Research Unit, Department of Agronomy, and Interdepartmental Genetics Graduate Program, Iowa State University, Ames, IA 50011 (Palmer)

Address correspondence to R. G. Palmer at the address above, or e-mail: rpalmer{at}iastate.edu.

In soybean (Glycine max (L.) Merr.), a chromosomal region defined by 3 closely linked loci, k2 (tan-saddle seed coat), Mdh1-n (malate dehydrogenase 1 null), and y20 (yellow foliage), is highly mutable. A total of 31 mutants have been reported from this region. In this study, a mutation with tan-saddle seed coat was found from bulk-harvested seed of cultivar Kenwood. Genetic analysis established that this tan-saddle seed coat mutation is allelic to the k2 locus and inherited as a recessive gene. Simple sequence repeat analysis showed that this mutant is not a contaminant from other existing k2 mutants. The mutant was named Kenwood-k2. To test for genetic instability at the k2 Mdh1-n y20 chromosomal region, Kenwood-k2 was crossed reciprocally with cultivars Harosoy and Williams. No new mutants were found in F2 families. In the genetic instability tests of T239 (k2) with cultivar Williams, 3 new mutants with yellow foliage (y20) and malate dehydrogenase 1 null (Mdh1-n) were identified. In the genetic instability tests of T261 (k2 Mdh1-n) with cultivar Williams, no new mutants were found. The Kenwood-k2 and the 3 yellow-foliage, malate dehydrogenase 1–null mutants provide additional genetic materials to study chromosomal aberrations in this mutable/unstable chromosomal region.

Several mutable or unstable loci have been described in soybean (Glycine max (L.) Merr.). They include Y18-m, cyt-Y3 (T284M), w4-m, wp-m, and r-m (for review, see Palmer and others 2004). These unstable loci conditioned variegated phenotypes. Different from these unstable loci, the unstable k2 Mdh1-n y20 chromosomal region does not condition mutable or variegated phenotypes. It is mutable because it is a "hotspot" for generating mutations (Chen and Palmer 1998).

The unstable k2 Mdh1-n y20 chromosomal region was defined by 3 closely linked loci, k2, Mdh1-n, and y20. The k2 locus conditions a tan-saddle pattern on the seed coat. Plants homozygous for a null allele, Mdh1-n, do not show malate dehydrogenase 1 activity on starch-gel electrophoresis (Hedges and Palmer 1992). The y20 locus conditions yellowish-green leaves.

So far, 31 mutants have been reported from the k2 Mdh1-n y20 chromosomal region (Xu and Palmer 2005a). A total of 25 y20 mutants have been described; 18 are associated with k2, but all 25 are associated with Mdh1-n. No k2k2 Mdh1Mdh1 y20y20 plants have been identified. For example, tan-saddle mutant seed were identified in a bulk harvest of cultivar Harosoy, precluding the identity of the original plants. The self-pollinated progeny of some tan-saddle seed were true breeding and were green plants (T239), and some were true breeding and were yellowish-green foliage at seedling stage. The latter plants also were identified as malate dehydrogenase null (Mdh1-n) and added to the US Department of Agriculture–Agricultural Research Service Soybean Genetic Type Collection as T253 (k2 Mdh1-n y20). It was suspected that a transposon was responsible for the mutant phenotypes (Palmer 1984; Palmer and others 1989). Genetic Type T261 (k2 Mdh1-n) was found as a spontaneous mutation in cultivar Mandarin (Ottawa). The Clark-k2 (L67-3843) tan saddle was found after seed X-irradiation of cultivar Clark.

Imsande and others (2001), using Southern blot analysis, reported that the Mdh1-n mutants examined (T253, T317, T323, and T324) were the result of deletions. The null phenotype correlated with the deletion of specific genomic restriction fragments that encoded the Mdh1 gene. The k2, Mdh1-n, and y20 loci were mapped on molecular linkage group H by using 5 mapping populations (Xu and Palmer 2005b). Three simple sequence repeat (SSR) markers that were closely linked to the k2 Mdh1-n y20 chromosomal region were identified that corresponded to deleted chromosome segments in T261 (Mdh1-n y20).

Chen and Palmer (1998) reported that tan-saddle mutant T239 was effective in generating Mdh1-n y20 mutants in certain cross-combinations. This "instability" experiment gave about 2.6% Mdh1-n mutants (10 out of 383 F2 families) from crosses of T239 with parents of w4-m and Y18-m, which were proposed to contain an active transposable element in their genomes. The control population of 833 F2 families from crosses of T239 with cultivar Harosoy gave no mutants. Genetic Type T261 (k2 Mdh1-n) crossed with parents of w4-m and Y18-m gave about 0.3% y20 mutants (1 mutant out of 323 F2 families). Tan-saddle mutant Clark-k2 (L67-3483) crossed with parents of w4-m and Y18-m gave no mutants in 273 F2 families. Chen and Palmer (1998) have proposed that the instability at the k2 Mdh1-n y20 chromosomal region was due to transposon activity or special chromosome structures that could generate chromosomal rearrangements such as deletions.

In 1993, tan-saddle mutant seed were found in a bulk harvest of cultivar Kenwood. Our objectives were 1) to determine the inheritance and allelism of this new tan-saddle mutant in Kenwood and 2) to test for instability of Kenwood-k2, T239, and T261 in crosses with cultivars Harosoy and Williams.


   Materials and Methods
Top
 Materials and Methods
Results and Discussion
 References
 
The soybean lines, genotypes, and phenotypes used in the inheritance, allelism, and instability studies are given in Table 1.


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  		Table 1. Soybean lines used in the instability studies

 
Origin and Inheritance of Kenwood-k2
Tan-saddle seed were found in a bulk harvest of cultivar Kenwood (Cianzio and others 1990) in 1993 at the Bruner Farm near Ames, IA. The plant with the most intense tan saddle was single-plant threshed in 1994. Planting, selecting for intense tan saddle, and harvesting self-pollinated seed were continued for the next 3 years.

The Kenwood-k2 plant with the most intense tan saddle was crossed as male parent with cultivar Harosoy (Weiss and Stevenson 1955). The F1 seed were advanced to the F2 at the University of Puerto Rico/Iowa State University soybean nursery near Isabela, Puerto Rico. F2 and F3 seed were planted at the Bruner Farm for seed coat color evaluation.

Allelism and Genetic Instability Studies with Kenwood-k2
For the allelism test, the Kenwood-k2 plant with the most intense tan saddle was crossed as male parent to tan-saddle Clark-k2 (L67-3483), which arose from X-irradiation of cultivar Clark (Johnson 1958).

To test for instability at the k2 Mdh1-n y20 chromosomal region, cultivars Harosoy and Williams (Bernard and Lindahl 1972) were crossed reciprocally with Kenwood-k2. The F1 seed from both tests were advanced to the F2 at Isabela, Puerto Rico. F2 and F3 seed were planted at the Bruner Farm for evaluation of the presence/absence of tan-saddle seed and to look for seedling/adult plant traits in the genetic instability study. The seedling traits would include chimeric-foliage, yellow-foliage, multileaflet plants, etc. The adult plant traits would include partial male and/or female sterility, complete male and female sterility, dwarfs, etc. Four male-sterile, female-fertile mutants were identified by Chen and Palmer (1996) in a test for instability and genetically characterized by Palmer (2000).

Origin Test of Kenwood-k2 with SSR Analysis
SSR analysis was conducted as previously described (Xu and Palmer 2005a). Six soybean lines, including Kenwood, Kenwood-k2, Harosoy, T239, Clark, and Clark-k2, were evaluated with 100 SSR markers randomly selected from the 20 molecular linkage groups constructed by Song and others (2004), which represented the 20 chromosomes of the soybean genome, 5 from each molecular linkage group, respectively.

Test of Instability from Reciprocal Crosses of T239 and T261 with cultivar Williams
To test for instability at the k2 Mdh1-n y20 chromosomal region, Genetic Types T239 and T261 were crossed reciprocally with cultivar Williams. The F1 seed were advanced to the F2 at Isabela, Puerto Rico. F2 and F3 seed were planted at the Bruner Farm for evaluation of the presence/absence of tan-saddle seed and to look for seedling/adult plant traits as mentioned in the allelism and genetic instability studies with Kenwood-k2.

Genetic Evaluation of Mutants
From the test for instability, 3 F2 families from crosses involving T239 and Williams were identified that segregated about 3 green:1 yellow viable plant. Green and yellow F2 plants were threshed individually and evaluated as plant-progeny rows.

Self-pollinated seed from the original yellow plants within each entry were evaluated for malate dehydrogenase according to the procedure of Cardy and Beversdorf (1984).

For inheritance studies, yellow plants from each of the 3 entries were crossed reciprocally with cultivar Harosoy. Seed generation advance and data evaluation were similar to the instability study with T239 and T261.

For allelism tests, yellow plants from each of the 3 entries were crossed with T325 (Mdh1-n y20). A small piece of cotyledon was taken from each F1 and F2 seed. The samples were evaluated for malate dehydrogenase. The F1 and F2 plants were classified for plant color at the Bruner Farm.


   Results and Discussion
Top
 Materials and Methods
 Results and Discussion
References
 
The Mutation of Tan-Saddle Seed Coat Found in Kenwood Was Conditioned by a New k2 Allele
During the inheritance experiment of Kenwood-k2, 3 hybrid seeds were obtained from the cross of Harosoy with Kenwood-k2. The 3 F2 progenies each segregated about 3 yellow seed coats:1 tan-saddle seed coat (Table 2), which suggested that the mutation with tan-saddle seed coat found in cultivar Kenwood was conditioned by a single gene and that it was recessive to wild-type yellow seed coat.


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  		Table 2. Inheritance of tan-saddle seed coat from the cross of Harosoy with Kenwood-k2 (F2 data)

 
In soybean, existing tan-saddle seed coat mutations are conditioned by a recessive k2 locus (for review, see Palmer and others 2004). To test if this mutant found in Kenwood was conditioned by the k2 locus, tan-saddle Clark-k2 was crossed with Kenwood-k2. The F1 seed were advanced to the F2, and all F2 plants were tan-saddle seed. Twenty F2 plants were evaluated as F3 families the following summer at the Bruner Farm. Ten F3 plants from each of the 20 progeny rows were evaluated for seed coat color pattern. All plants were tan-saddle seed. This result established that tan-saddle seed coat mutations of these 2 lines were allelic. They both were conditioned by the k2 allele.

One hundred SSR markers representing the 20 chromosomes (20 linkage groups) of the soybean genome were used to determine if Kenwood-k2 was a mutation in cultivar Kenwood. Results showed that no polymorphisms were detected between Kenwood-k2 and Kenwood by these 100 markers, but about 25% polymorphisms were found between Kenwood-k2 and T239 and between Kenwood-k2 and Clark-k2. This suggested that the Kenwood-k2 was not a contamination from other lines with k2 alleles but a new mutation derived from cultivar Kenwood.

Compared with T239, Kenwood-k2 Was Stable
Harosoy and Williams were crossed reciprocally with Kenwood-k2 to test for instability at the k2 Mdh1-n y20 chromosomal region. A total of 1705 F2 progeny rows were evaluated 6 times from emergence to maturity, among which 440 progeny rows were from reciprocal crosses of Harosoy and Kenwood-k2 and 1265 progeny rows were from reciprocal crosses of Williams and Kenwood-k2 (Table 3). The only variant observed was one foliage chimeric plant from the Kenwood-k2 with Williams combination. Progenies of this plant were wild type, and no variation was observed.


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  		Table 3. Test for instability of Kenwood-k2 with cultivars Harosoy and Williams

 
Williams was crossed reciprocally with T239 and T261 to produce 216 F2 families (Table 4). Three of 119 F2 progenies from the reciprocal crosses with Williams and T239 were observed to segregate about 3 green:1 yellow plant (Table 5), which is about 2.6% frequency. This was consistent with the previous results from crossing T239 with the parents of w4-m and Y18-m (Chen and Palmer 1998). Green and yellow plants were threshed individually, and progeny was tested. The F2 green plants gave about 1 all-green:2 segregating (green and yellow) progenies (Table 5). Within segregating progenies, the ratio was about 3 green:1 yellow plant (Table 5). The yellow F2 plants were true breeding.


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  		Table 4. Test for instability of T239 and T261 with cultivar Williams

 


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  		Table 5. Inheritance of the 3 yellow mutants found in the instability test from reciprocal crosses of T239 with cultivar Williams

 
No mutations were found from the reciprocal crosses of Williams and T261. The reason could be that too few F2 families were evaluated. According to previous results, the mutation rate of T261 in crosses with parents of w4-m and Y18-m was about 0.3% (Chen and Palmer 1998), but only 97 F2 families were evaluated in this study (Table 4).

The k2 alleles in T239, T261, and Kenwood-k2 arose spontaneously and independently. Compared with T239, the k2 allele in Kenwood-k2 in crosses with cultivars Harosoy and Williams did not induce mutations. The mutant-generating abilities of the k2 alleles in Kenwood-k2, T239, and T261 are different even though they condition similar tan-saddle phenotypes.

Genetic Characterization of the Yellow Plants Found in Reciprocal Crosses of T239 with cultivar Williams
Yellow plants were crossed reciprocally with cultivar Harosoy. The F1 plant color from all crosses was green, which suggested that the yellow phenotype was the result of a nuclear mutation. The F2 data and F2:3 family data were in agreement with a single recessive gene inheritance pattern (Table 6). The yellow F2 plants were true breeding.


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  		Table 6. Inheritance of the 3 yellow mutants found in the instability test from reciprocal crosses of T239 with cultivar Williams

 
For allelism tests, the yellow plants were crossed with T325, a Mdh1-n y20 mutant identified in a gene-tagging study with w4-m (Hedges and Palmer 1992). The parents, F1, and a random sample of F2 seed were tested for malate dehydrogenase and evaluated for plant color. The parents, F1, and F2 plants were all yellow foliage (y20), and the seeds were malate dehydrogenase null (Mdh1-n) (Table 7). The 3 independently derived mutants are alleles of Mdh1-n y20.


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  		Table 7. Allelism tests of the 3 yellow-foliage and malate dehydrogenase 1–null F2 plants

 
In soybean, the k2 locus was tightly linked to the Mdh1-n and y20 loci and resides in a chromosomal segment that is a hotspot for mutation (Chen and Palmer 1998). The mutations of Mdh1-n and y20 are suspected to correspond to a chromosomal segment deletion. The instability of the k2 Mdh1-n y20 chromosomal region could be due to an inactive transposable element or chromosomal rearrangements such as duplications or inversions residing in or nearby this region that could lead to deletions in this region (Chen and Palmer 1998).

As we know, the insertion and excision of transposable elements would generate rearrangements of the sequences flanking their insertion sites, including deletions, inversions, and duplications. Sometimes the deletion size could be very large. For example, the Tam3 transposable element has induced a deletion with size more than 20 kbp at the niv locus in Antirrhinum majus (for review, see Martin and Lister 1989).

This study identified a new tan-saddle mutant that occurred spontaneously in cultivar Kenwood. This is the first k2 mutant identified in cultivar Kenwood. Two Mdh1-n y20 mutants were identified in the cultivar Williams with T239 (k2) cross, and one Mdh1-n y20 mutant was identified in the T239 with cultivar Williams cross. The results of the instability study with T239 and cultivar Williams gave about 2.5% Mdh1-n y20 mutants (3 out of 119 F2 families). This is similar to the T239 crosses (10 out of 383 F2 families) reported by Chen and Palmer (1998). Thus, 35 mutants are known for the k2 Mdh1-n y20 chromosomal region in soybean. No mutants were found in reciprocal crosses of cultivar Williams with Kenwood-k2 or in reciprocal crosses of Williams with T261 (k2 Mdh1-n).

Molecular studies are necessary to elucidate the basic of this chromosomal instability. The new mutants found or generated in this study would provide additional genetic materials for studying the chromosomal aberrations in this chromosomal region.


   Acknowledgments
 
We thank Dr. K. S. Lewers, US Department of Agriculture–Agriculture Research Service (USDA-ARS), for providing the Kenwood-k2 seeds. This is a joint contribution of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 3769, and the USDA-ARS, Corn Insects and Crop Genetics Research Unit and was supported by the Hatch Act and the State of Iowa. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by Iowa State University or the USDA, and the use of the name by Iowa State University or the USDA implies no approval of the product to the exclusion of others that may also be suitable.


   Footnotes
 
Corresponding Editor: Halina Knap

Received October 10, 2005
Accepted March 22, 2006


   References
Top
 Materials and Methods
 Results and Discussion
 References
 

    Bernard RL and Lindahl DA. (1972) Registration of Williams soybean. Crop Sci 12:716.[Free Full Text]

    Cardy BJ and Beversdorf WD. (1984) Identification of soybean cultivars using isozyme electrophoresis. Seed Sci Technol 12:943–54.

    Chen XF and Palmer RG. (1996) Isolation of four independent mono genically inherited male-sterile mutants in soybean. Soybean Genet Newsl 23:134–9.

    Chen XF and Palmer RG. (1998) Instability at the k2 Mdh1-n y20 chromosomal region in soybean. Mol Gen Genet 260:309–18.[CrossRef][Web of Science][Medline]

    Cianzio SR, Schultz SP, Voss BK, Fehr WR. (1990) Registration of ‘Kenwood’ soybean. Crop Sci 30:1162.[Free Full Text]

    Hedges BR and Palmer RG. (1992) Inheritance of malate dehydrogenase nulls in soybean. Biochem Genet 30:491–502.[CrossRef][Web of Science][Medline]

    Imsande J, Pittig J, Palmer RG, Wimmer C, Gietl C. (2001) Independent spontaneous mitochondrial malate dehydrogenase null mutants in soybean are the result of deletions. J Hered 92:333–8.[Abstract/Free Full Text]

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