Mutation in a PHD-finger protein MS4 causes male sterility in soybean.

BACKGROUND: Male sterility has tremendous scientific and economic importance in hybrid seed production. Identification and characterization of a stable male sterility gene will be highly beneficial for making hybrid seed production economically feasible. In soybean, eleven male-sterile, female-fertile mutant lines (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, msMOS, and msp) have been identified and mapped onto various soybean chromosomes, however the causal genes responsible for male sterility are not isolated. The objective of this study was to identify and functionally characterize the gene responsible for the male sterility in the ms4 mutant. RESULTS: The ms4 locus was fine mapped to a 216 kb region, which contains 23 protein-coding genes including Glyma.02G243200, an ortholog of Arabidopsis MALE MEIOCYTE DEATH 1 (MMD1), which is a Plant Homeodomain (PHD) protein involved in male fertility. Isolation and sequencing of Glyma.02G243200 from the ms4 mutant line showed a single base insertion in the 3rd exon causing a premature stop codon resulting in truncated protein production. Phylogenetic analysis showed presence of a homolog protein (MS4_homolog) encoded by the Glyma.14G212300 gene. Both proteins were clustered within legume-specific clade of the phylogenetic tree and were likely the result of segmental duplication during the paleoploidization events in soybean. The comparative expression analysis of Ms4 and Ms4_homologs across the soybean developmental and reproductive stages showed significantly higher expression of Ms4 in early flowering (flower bud differentiation) stage than its homolog. The functional complementation of Arabidopsis mmd1 mutant with the soybean Ms4 gene produced normal stamens, successful tetrad formation, fertile pollens and viable seeds, whereas the Ms4_homolog was not able to restore male fertility. CONCLUSIONS: Overall, this is the first report, where map based cloning approach was employed to isolate and characterize a gene responsible for the male-sterile phenotype in soybean. Characterization of male sterility genes may facilitate the establishment of a stable male sterility system, highly desired for the viability of hybrid seed production in soybean. Additionally, translational genomics and genome editing technologies can be utilized to generate new male-sterile lines in other plant species.

The endogenous transposable element Tgm9 is suitable for generating knockout mutants for functional analyses of soybean genes and genetic improvement in soybean.

In soybean, variegated flowers can be caused by somatic excision of the CACTA-type transposable element Tgm9 from Intron 2 of the DFR2 gene encoding dihydroflavonol-4-reductase of the anthocyanin pigment biosynthetic pathway. DFR2 was mapped to the W4 locus, where the allele containing Tgm9 was termed w4-m. In this study we have demonstrated that previously identified morphological mutants (three chlorophyll deficient mutants, one male sterile-female fertile mutant, and three partial female sterile mutants) were caused by insertion of Tgm9 following its excision from DFR2. Analyses of Tgm9 insertion sites among 105 independent mutants demonstrated that Tgm9 hops to all 20 soybean chromosomes from its original location on Chromosome 17. Some genomic regions are prone to increased Tgm9-insertions. Tgm9 transposed over 25% of the time into exon or intron sequences. Tgm9 is therefore suitable for generating an indexed insertional mutant collection for functional analyses of most soybean genes. Furthermore, desirable Tgm9-induced stable knockout mutants can be utilized in generating improved traits for commercial soybean cultivars.

Soybean proteins GmTic110 and GmPsbP are crucial for chloroplast development and function.

We have identified a viable-yellow and a lethal-yellow chlorophyll-deficient mutant in soybean. Segregation patterns suggested single-gene recessive inheritance for each mutant. The viable- and lethal-yellow plants showed significant reduction of chlorophyll a and b. Photochemical energy conversion efficiency and photochemical reflectance index were reduced in the viable-yellow plants relative to the wildtype, whereas the lethal-yellow plants showed no electron transport activity. The viable-yellow plants displayed reduced thylakoid stacking, while the lethal-yellow plants exhibited failure of proplastid differentiation into normal chloroplasts with grana. Genetic analysis revealed recessive epistatic interaction between the viable- and the lethal-yellow genes. The viable-yellow gene was mapped to a 58kb region on chromosome 2 that contained seven predicted genes. A frame shift mutation, due to a single base deletion in Glyma.02g233700, resulted in an early stop codon. Glyma.02g233700 encodes a translocon in the inner membrane of chloroplast (GmTic110) that plays a critical role in plastid biogenesis. The lethal-yellow gene was mapped to an 83kb region on chromosome 3 that contained 13 predicted genes. Based on the annotated functions, we sequenced three potential candidate genes. A single base insertion in the second exon of Glyma.03G230300 resulted in a truncated protein. Glyma.03G230300 encodes for GmPsbP, an extrinsic protein of Photosystem II that is critical for oxygen evolution during photosynthesis. GmTic110 and GmPsbP displayed highly reduced expression in the viable- and lethal-yellow mutants, respectively. The yellow phenotypes in the viable- and lethal-yellow mutants were due to the loss of function of GmTic110 or GmPsbP resulting in photooxidative stress.

Transposon Tagging of a Male-Sterility, Female-Sterility Gene, St8, Revealed that the Meiotic MER3 DNA Helicase Activity Is Essential for Fertility in Soybean.

The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion from variegated to purple flower indicates excision of Tgm9, and its insertion at a new locus. Previously, we have identified a male-sterile, female-sterile mutant among the selfed progenies of a revertant plant carrying only purple flowers. Co-segregation between Tgm9 and the sterility phenotype suggested that the mutant was generated by insertion of Tgm9 at the St8 locus. The transposon was localized to exon 10 of Glyma.16G072300 that shows high identity to the MER3 DNA helicase involved in crossing over. Molecular analysis of fertile branches from two independent revertant plants confirmed precise excision of Tgm9 from the st8 allele, which restored fertility. In soybean, the gene is expressed in flower-buds, trifoliate leaves and stem. Phylogenetic analysis placed St8 in a clade with the Arabidopsis and rice MER3 suggesting that St8 is most likely the orthologous MER3 soybean gene. This study established the utility of Tgm9 in gene identification as well as in forward and reverse genetics studies.

Molecular mapping of five soybean genes involved in male-sterility, female-sterility.

In soybean, asynaptic and desynaptic mutants lead to abnormal meiosis and fertility reduction. Several male-sterile, female-sterile mutants have been identified and studied in soybean, however, some of these mutants have not been mapped to locations on soybean chromosomes. The objectives of this study were to molecularly map five male-sterile, female-sterile genes (st2, st4, st5, st6, and st7) in soybean and compare the map locations of these genes with already mapped sterility genes. Microsatellite markers were used in bulked segregant analyses to locate all five male-sterile, female-sterile genes to soybean chromosomes, and markers from the corresponding chromosomes were used on F2 populations to generate genetic linkage maps. The st2, st4, st5, st6, and st7 genes were located on molecular linkage group (MLG) B1 (chromosome 11), MLG D1a (chromosome 01), MLG F (chromosome 13), MLG B2 (chromosome 14), and D1b (chromosome 02), respectively. The st2, st4, st5, st6, and st7 genes were flanked to 10.3 (∼ 399 kb), 6.3 (∼ 164 kb), 3.9 (∼ 11.8 Mb), 11.0 (∼ 409 kb), and 5.3 cM (∼ 224 kb), and the flanked regions contained 57, 17, 362, 52, and 17 predicted genes, respectively. Future characterization of candidate genes should facilitate identification of the male- and female-fertility genes, which may provide vital insights on structure and function of genes involved in the reproductive pathway in soybean.

Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants.

The soybean [Glycine max (L.) Merr.] chlorophyll-deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a nonsynonymous nucleotide substitution in the third exon of a Mg-chelatase subunit gene (ChlI1a) on chromosome 13. This gene was selected as a candidate for a different yellow foliage mutant, T219H (Y11y11), that had been previously mapped to chromosome 13. Although the phenotypes of MinnGold and T219H are clearly distinct, sequencing of ChlI1a in T219H identified a different nonsynonymous mutation in the third exon, only six base pairs from the MinnGold mutation. This information, along with previously published allelic tests, were used to identify and clone a third yellow foliage mutation, CD-5, which was previously mapped to chromosome 15. This mutation was identified in the ChlI1b gene, a paralog of ChlI1a. Sequencing of the ChlI1b allele in CD-5 identified a nonsynonymous substitution in the third exon that confers an identical amino acid change as the T219H substitution at ChlI1a. Protein sequence alignments of the two Mg-chelatase subunits indicated that the sites of amino acid modification in MinnGold, T219H, and CD-5 are highly conserved among photosynthetic species. These results suggest that amino acid alterations in this critical domain may create competitive inhibitory interactions between the mutant and wild-type ChlI1a and ChlI1b proteins.

Molecular mapping of three male-sterile, female-fertile mutants and generation of a comprehensive map of all known male sterility genes in soybean.

In soybean, an environmentally stable male sterility system is vital for making hybrid seed production commercially viable. Eleven male-sterile, female-fertile mutants (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, msMOS, and msp) have been identified in soybean. Of these, eight (ms2, ms3, ms5, ms7, ms8, ms9, msMOS, and msp) have been mapped to soybean chromosomes. The objectives of this study were to (i) locate the ms1, ms4, and ms6 genes to soybean chromosomes; (ii) generate genetic linkage maps of the regions containing these genes; and (iii) develop a comprehensive map of all known male-sterile, female-fertile genes in soybean. The bulked segregant analysis technique was used to locate genes to soybean chromosomes. Microsatellite markers from the corresponding chromosomes were used on F2 populations to generate genetic linkage maps. The ms1 and ms6 genes were located on chromosome 13 (molecular linkage group F) and ms4 was present on chromosome 2 (molecular linkage group D1b). Molecular analyses revealed markers Satt516, BARCSOYSSR_02_1539, and AW186493 were located closest to ms1, ms4, and ms6, respectively. The ms1 and ms6 genes, although present on the same chromosome, were independently assorting with a genetic distance of 73.7 cM. Using information from this study and compiled information from previously published male sterility genes in soybean, a comprehensive genetic linkage map was generated. Eleven male sterility genes were present on seven soybean chromosomes. Four genes were present in two regions on chromosome 2 (molecular linkage group D1b) and two genes were present on chromosome 13 (molecular linkage group F).

Exploration of presence/absence variation and corresponding polymorphic markers in soybean genome.

This study was designed to reveal the genome-wide distribution of presence/absence variation (PAV) and to establish a database of polymorphic PAV markers in soybean. The 33 soybean whole-genome sequences were compared to each other with that of Williams 82 as a reference genome. A total of 33,127 PAVs were detected and 28,912 PAV markers with their primer sequences were designed as the database NJAUSoyPAV_1.0. The PAVs scattered on whole genome while only 518 (1.8%) overlapped with simple sequence repeats (SSRs) in BARCSOYSSR_1.0 database. In a random sample of 800 PAVs, 713 (89.13%) showed polymorphism among the 12 differential genotypes. Using 126 PAVs and 108 SSRs to test a Chinese soybean germplasm collection composed of 828 Glycine soja Sieb. et Zucc. and Glycine max (L.) Merr. accessions, the per locus allele number and its variation appeared less in PAVs than in SSRs. The distinctness among alleles/bands of PCR (polymerase chain reaction) products showed better in PAVs than in SSRs, potential in accurate marker-assisted allele selection. The association mapping results showed SSR + PAV was more powerful than any single marker systems. The NJAUSoyPAV_1.0 database has enriched the source of PCR markers, and may fit the materials with a range of per locus allele numbers, if jointly used with SSR markers.

A candidate male-fertility female-fertility gene tagged by the soybean endogenous transposon, Tgm9.

In soybean, the W4 gene encoding dihydroflavonol-4-reductase controls anthocyanin pigment biosynthesis in flowers. The mutant allele, w4-m, is characterized by variegated flowers and was evolved from the insertion of an endogenous transposable element, Tgm9, in intron II of the W4 gene. In the w4-m mutant line, reversion of the unstable allele from variegated to normal purple flower in revertants would indicate Tgm9's excision accompanied by its insertion into a second locus. We identified a male-sterile, female-sterile mutant from such germinal revertant bearing purple flowers. The objectives of our investigation were to map the sterility locus, identify candidate genes for the male-fertile, female-fertile phenotype, and then determine if sterility is associated with the insertion of Tgm9 in the sterility locus. We used bulked segregant analysis to map the locus to molecular linkage group J (chromosome 16). Fine mapping enabled us to flank the locus to a 62-kb region that contains only five predicted genes. One of the genes in that region, Glyma16g07850.1, codes for a helicase. A rice homolog of this gene has been shown to control crossing over and fertility phenotype. Thus, Glyma16g07850.1 is most likely the gene regulating the male and female fertility phenotype in soybean. DNA blot analysis of the segregating individuals for Tgm9 showed perfect association between sterility and the presence of the transposon. Most likely, the sterility mutation was caused by the insertion of Tgm9. The transposable element should facilitate identification of the male- and female-fertility gene. Characterization of the fertility gene will provide vital molecular insight on the reproductive biology of soybean and other plants.

Molecular Mapping of D1, D2 and ms5 Revealed Linkage between the Cotyledon Color Locus D2 and the Male-Sterile Locus ms5 in Soybean.

In soybean, genic male sterility can be utilized as a tool to develop hybrid seed. Several male-sterile, female-fertile mutants have been identified in soybean. The male-sterile, female-fertile ms5 mutant was selected after fast neutron irradiation. Male-sterility due to ms5 was associated with the "stay-green" cotyledon color mutation. The cotyledon color trait in soybean is controlled by two loci, D1 and D2. Association between cotyledon color and male-sterility can be instrumental in early phenotypic selection of sterility for hybrid seed production. The use of such selection methods saves time, money, and space, as fewer seeds need to be planted and screened for sterility. The objectives of this study were to compare anther development between male-fertile and male-sterile plants, to investigate the possible linkages among the Ms5, D1 and D2 loci, and to determine if any of the d1 or d2 mutations can be applied in hybrid seed production. The cytological analysis during anther development displayed optically clear, disintegrating microspores and enlarged, engorged pollen in the male-sterile, female-fertile ms5ms5 plants, a common characteristic of male-sterile mutants. The D1 locus was mapped to molecular linkage group (MLG) D1a and was flanked by Satt408 and BARCSOYSSR_01_1622. The ms5 and D2 loci were mapped to MLG B1 with a genetic distance ~12.8 cM between them. These results suggest that use of the d2 mutant in the selection of male-sterile line may attenuate the cost hybrid seed production in soybean.

Soybean susceptibility to manufactured nanomaterials with evidence for food quality and soil fertility interruption.

Based on previously published hydroponic plant, planktonic bacterial, and soil microbial community research, manufactured nanomaterial (MNM) environmental buildup could profoundly alter soil-based food crop quality and yield. However, thus far, no single study has at once examined the full implications, as no studies have involved growing plants to full maturity in MNM-contaminated field soil. We have done so for soybean, a major global commodity crop, using farm soil amended with two high-production metal oxide MNMs (nano-CeO(2) and -ZnO). The results provide a clear, but unfortunate, view of what could arise over the long term: (i) for nano-ZnO, component metal was taken up and distributed throughout edible plant tissues; (ii) for nano-CeO(2), plant growth and yield diminished, but also (iii) nitrogen fixation--a major ecosystem service of leguminous crops--was shut down at high nano-CeO(2) concentration. Juxtaposed against widespread land application of wastewater treatment biosolids to food crops, these findings forewarn of agriculturally associated human and environmental risks from the accelerating use of MNMs.

Segregation distortion in a region containing a male-sterility, female-sterility locus in soybean.

In diploid segregation, each alternative allele has a 50% chance of being passed on to the offspring. Mutations in genes involved in the process of meiotic division or early stages of reproductive cell development can affect allele frequency in the gametes. In addition, competition among gametes and differential survival rates of gametes can lead to segregation distortion. In a recent transformation study, a male-sterile, female-sterile (MSFS) mutant was identified in the soybean cultivar, Williams. The mutant in heterozygous condition segregated 3 fertile:1 sterile in the progeny confirming monogenic inheritance. To map the lesion, we generated an F(2) mapping population by crossing the mutant (in heterozygous condition) with Minsoy (PI 27890). The F(2) progeny showed strong segregation distortion against the MSFS phenotype. The objectives of our study were to molecularly map the gene responsible for sterility in the soybean genome, to determine if the MSFS gene is a result of T-DNA insertion during Agrobacterium-mediated transformation, and to map the region that showed distorted segregation. The fertility/sterility locus was mapped to molecular linkage group (MLG) D1a (chromosome Gm01) using bulked segregant analysis. The closest marker, Satt531, mapped 9.4cM from the gene. Cloning of insertion sites for T-DNA in the mutant plants revealed that there are two copies of T-DNA in the genome. Physical locations of these insertion sites do not correlate with the map location of the MSFS gene, suggesting that MSFS mutation may not be associated with T-DNA insertions. Segregation distortion was most extreme at or around the st_A06-2/6 locus suggesting that sterility and segregation distortion are tightly linked attributes. Our results cue that the distorted segregation may be due to a gamete elimination system.

Molecular mapping of 2 environmentally sensitive male-sterile mutants in soybean.

In soybean [Glycine max (L.) Merr.], manual cross-pollination to produce large quantities of hybrid seed is difficult and time consuming. Identification of an environmentally stable male-sterility system could make hybrid seed production commercially valuable. In soybean, 2 environmentally sensitive male-sterile, female-fertile mutants (ms8 and msp) have been identified. Inheritance studies showed that sterility in both mutants is inherited as a single gene. The objectives of this study were to 1) confirm that msp and ms8 are independent genes; 2) identify the soybean chromosomes that contain the msp and the ms8 genes using bulked segregant analyses (BSAs); and 3) make a genetic linkage map of the regions containing these genes. Mapping populations consisting of 176 F(2) plants for ms8 and 134 F(2) plants for msp were generated. BSA revealed that Sat_389 and Satt172 are closely associated markers with ms8 and msp, respectively. Map location of Sat_389 suggested that the ms8 gene is located on chromosome 7; molecular linkage group (MLG) M. Map location of Satt172 indicated that the msp gene is located on chromosome 2 (MLG Dlb). Genetic linkage maps developed using F(2) populations revealed that ms8 is flanked by a telomere and Sat_389 and msp is flanked by Sat_069 and GMES4176. The region between the telomere and Sat_389 is physically 160 Kb. Soybean sequence information revealed that there are 13 genes present in that region. Protein BLASTP analyses revealed that homologs of 3 of the 13 genes are known to a play role in cell division, suggesting putative candidates for ms8.

Fluorescence in situ hybridization-based karyotyping of soybean translocation lines.

Soybean (Glycine max [L.] Merr.) is a major crop species and, therefore, a major target of genomic and genetic research. However, in contrast to other plant species, relatively few chromosomal aberrations have been identified and characterized in soybean. This is due in part to the difficulty of cytogenetic analysis of its small, morphologically homogeneous chromosomes. The recent development of a fluorescence in situ hybridization -based karyotyping system for soybean has enabled our characterization of most of the chromosomal translocation lines identified to date. Utilizing genetic data from existing translocation studies in soybean, we identified the chromosomes and approximate breakpoints involved in five translocation lines.

Genetic analysis of genes controlling natural variation of seed coat and flower colors in soybean.

Soybean exhibits natural variation in flower and seed coat colors via the deposition of various anthocyanin pigments in the respective tissues. Although pigmentation in seeds or flowers has been well dissected at molecular level in several plant species, the genes controlling natural variation in anthocyanin traits in the soybean are not completely understood. To evaluate the genetic correlation between genetic loci and genes, 8 enzyme-encoding gene families and a transcription factor were localized in a soybean genome-wide genetic map. Among the seed coat color-controlling loci, the genetic location of the gene encoding for W1 was substantiated in the context of the current soybean molecular genetic map and O was postulated to correspond to anthocyanidin reductase. Among the genetic loci that regulate flower pigmentation, the genetic locations of the genes encoding for W1, W4, and Wp were identified, W3 was mapped on soybean linkage group B2 (chromosome 14), and W2 was postulated to correspond to an MYB transcription factor. Correlation studies between the developed markers and 3 color-controlling loci provided important empirical data that should prove useful in the design of marker-assisted breeding schemes as well as future association studies involving soybean.

Excision of an active CACTA-like transposable element from DFR2 causes variegated flowers in soybean [Glycine max (L.) Merr.].

Active endogenous transposable elements, useful tools for gene isolation, have not been reported from any legume species. An active transposable element was suggested to reside in the W4 locus that governs flower color in soybean. Through biochemical and molecular analyses of several revertants of the w4-m allele, we have shown that the W4 locus encodes dihydroflavonol-4-reductase 2 (DFR2). w4-m has arisen through insertion of Tgm9, a 20,548-bp CACTA-like transposable element, into the second intron of DFR2. Tgm9 showed high nucleic acid sequence identity to Tgmt*. Its 5' and 3' terminal inverted repeats start with conserved CACTA sequence. The 3' subterminal region is highly repetitive. Tgm9 carries TNP1- and TNP2-like transposase genes that are expressed in the mutable line, T322 (w4-m). The element excises at a high frequency from both somatic and germinal tissues. Following excision, reinsertions of Tgm9 into the DFR2 promoter generated novel stable alleles, w4-dp (dilute purple flowers) and w4-p (pale flowers). We hypothesize that the element is fractured during transposition, and truncated versions of the element in new insertion sites cause stable mutations. The highly active endogenous transposon, Tgm9, should facilitate genomics studies specifically that relate to legume biology.

The male sterility locus ms3 is present in a fertility controlling gene cluster in soybean.

Soybean [Glycine max (L.) Merr.] is self-pollinated. To produce large quantities of hybrid seed, insect-mediated cross-pollination is necessary. An efficient nuclear male-sterile system for hybrid seed production would benefit from molecular and/or phenotypic markers linked to male fertility/sterility loci to facilitate early identification of phenotypes. Nuclear male-sterile, female-fertile ms3 mutant is a single recessive gene and displays high outcrossed seed set with pollinators. Our objective was to map the ms3 locus. A segregating population of 150 F(2) plants from Minsoy (PI 27890) x T284H, Ms3ms3 (A00-68), was screened with 231 simple sequence repeat markers. The ms3 locus mapped to molecular linkage group (MLG) D1b (Gm02) and is flanked by markers Satt157 and Satt542, with a distance of 3.7 and 12.3 cM, respectively. Female-partial sterile-1 (Fsp1) and the Midwest Oilseed male-sterile (msMOS) mutants previously were located on MLG D1b. msMOS and Fsp1 are independent genes located very close to each other. All 3 genes are located in close proximity of Satt157. We believe that this is the first report of clustering of fertility-related genes in plants. Characterization of these closely linked genes may help in understanding the evolutionary relationship among them.

Positioning 3 qualitative trait loci on soybean molecular linkage group E.

In soybean (Glycine max [L.] Merr.), 3 qualitative trait loci (Pb, Y9, and Y17) are located on classical linkage group 14, which corresponds to molecular linkage group (MLG) E. The Pb locus conditions sharp/blunt pubescence tip; the y9 and y17 loci condition green/chlorotic foliage. The gene order is not known. Our objective was to determine the gene order on soybean MLG E of the Pb, Y9, and Y17 loci using previously mapped simple sequence repeat (SSR) markers. Allelism tests between y9 and y17 gave normal green foliage F(1) plants, indicating nonallelism. Our F(2) data from the allelism test could not distinguish between a 1:1 or a 9:7 ratio. The F(2:3) family segregation indicated a very close genetic linkage between the y9 and the y17 loci. Two molecular mapping populations were developed. Population-1 segregated for Pb and y9, and population-2 segregated for Pb and y17. The gene order on soybean MLG E, using SSR markers, was Pb, Y9, and Y17.

Allelism and molecular mapping of soybean necrotic root mutants.

Mutability of the w4 flower color locus in soybean, Glycine max (L.) Merr., is conditioned by an allele designated w4-m. Germinal revertants recovered among self-pollinated progeny of mutable plants have been associated with the generation of necrotic root mutations, chlorophyll-deficiency mutations, and sterility mutations. A total of 24 necrotic root mutant lines were generated from a total of 24 independent reversion events at the w4-m locus. The initial mutable population included 4 mutable categories for w4-m, designated (1) low frequency of early excisions, (2) low frequency of late excisions, (3) high frequency of early excisions, and (4) high frequency of late excisions. These mutable categories were based upon flower phenotype, i.e., somatic tissue. A total of 22 of 24 necrotic root mutations occurred from germinal reversions classified in the high frequency of excision categories. Of these 22 mutants, 14 came from early excisions and 8 came from late excisions. These necrotic root mutants were allelic to 6 previously identified necrotic root mutants derived from the study of germinal revertants, i.e., gene tagging studies, chemical mutagenesis, and "spontaneous" occurrences from genetic crosses. Thus, all 30 necrotic root mutants in soybean are allelic. An F2 mapping population from the cross of Minsoy (Rn1 Rn1) x T328 (rn1 rn1) was used to map the Rn1 locus using simple sequence repeat (SSR) markers. The Rn1 locus was located between Satt288 and Satt612 on molecular linkage group G.

Genetic analysis of 4 new mutants at the unstable k2 Mdh1-n y20 chromosomal region in soybean.

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.