Fine mapping of a resistance gene to bacterial leaf pustule in soybean

Soybean bacterial leaf pustule (BLP) is a prevalent disease caused by Xanthomonas axonopodis pv. glycines. Fine mapping of the BLP resistant gene, rxp, is needed to select BLP resistant soybean cultivars by marker-assisted selection (MAS). We used a total of 227 recombinant inbred lines (RILs) derived from a cross between ‘Taekwangkong’ (BLP susceptible) and ‘Danbaekkong’ (BLP resistant) for rxp fine mapping and two different sets of near isogenic lines (NILs) from Hwangkeumkong × SS2-2 and Taekwangkong × SS2-2 were used for confirmation. Using sequences between Satt372 and Satt486 flanking rxp from soybean genome sequences, eight simple sequence repeats (SSR) and two single nucleotide polymorphism (SNP) markers were newly developed in a 6.2-cM interval. Linkage mapping with the RILs and NILs allowed us to map the rxp region with high resolution. The genetic order of all markers was completely consistent with their physical order. QTL analysis by comparison of the BLP phenotyping data with all markers showed rxp was located between SNUSSR17_9 and SNUSNP17_12. Gene annotation analysis of the 33 kb region between SNUSSR17_9 and SNUSNP17_12 suggested three predicted genes, two of which could be candidate genes of BLP resistance: membrane protein and zinc finger protein. Candidate genes showed high similarity with their paralogous genes, which were located on the duplicated regions obtaining BLP resistance QTLs. High-resolution map in rxp region with eight SSR and two SNP markers will be useful for not only MAS of BLP resistance but also characterization of rxp.     

Molecular mapping of the genomic region conferring resistance to soybean stem canker in Hutcheson soybean

Genetic resistance to soybean stem canker, caused by the fungus Diaporthe phaseolorum var. meridionalis (Dpm), is controlled by five major, dominant, nonallelic genes Rdm1 to Rdm5. A genomic region containing the Rdm4 and Rdm5 genes was first described in Hutcheson soybean, where they were found to confer specific resistance to Argentinean physiological races of Dpm. Here, we report the genetic mapping of Rdm4 and Rdm5 loci using two pheno- and genotypically characterized F_2:3 populations derived from Hutcheson cultivar. The mapping populations were screened with amplified fragment length polymorphism (AFLP) markers using bulk segregant analysis, and with simple sequence repeat (SSR) markers. Linkage analysis indicated that the Rdm4 and Rdm5 resistance loci were located in a genomic region collinear with the molecular linkage group (MLG) A2 (chromosome 8) of the soybean genetic map. The linkage group contains two SSR markers, Sat_162 and Satt233, flanking the Rdm4 and Rdm5 loci. These SSR will be useful to increase the efficiency of selection in breeding programs aimed to incorporate Rdm4 and Rdm5 genes into soybean elite germplasm.     

Genetic mapping of QTLs conditioning soybean sprout yield and quality

Soybean sprouts have been used as a food in the Orient since ancient times. In this study, 92 restriction fragment length polymorphism (RFLP) loci and two morphological markers (W1 and T) were used to identify quantitative trait loci (QTLs) associated with soybean sprout-related traits in 100 F₂-derived lines from the cross of ’Pureunkong’×’Jinpumkong 2’. The genetic map consisted of 76 loci which covered about 756 cM and converged into 20 linkage groups. Eighteen markers remained unlinked. Phenotypic data were collected in 1996 and 1997 for hypocotyl length, percentage of abnormal seedlings, and sprout yield 6 days after germination at 20°C. Hypocotyl length was determined as the average length from the point of initiation of the first secondary root to the point of attachment of the cotyledons. The number of decayed seeds and seedlings, plus the number of stunted seedlings (less than 2-cm growth), was recorded a s abnormal seedlings. Seed weight was determined based on the 50-seed sample. Sprout yield was recorded as the total fresh weight of soybean sprouts produced from the 50-seed sample divided by the dry weight of the 50-seed sample. Four QTLs were associated with sprout yield in the combined analysis across 2 years. For the QTL linked to L154 on the Linkage Group (LG) G the positive allele was derived from Pureunkong (R ² = 0.19), whereas at the other three QTLs (A089 on LG B1, A668n on LG K and B046 on LG L) the positive alleles were from Jinpumkong 2. QTLs conditioning seed weight were linked to markers A802n (LG B1), A069 (LG E), Cr321 (LG F) and A235 (LG G). At these four markers, the Jinpumkong allele increased seed weight. Markers K011n on LG B1, W1 on LG F and A757 on LG L were linked to QTLs conditioning hypocotyl length; and Bng119, K455n and K418n to QTLs conditioning the abnormal seedlings. The QTLs conditioning sprout yield were in the same genomic locations as the QTLs for seed weight identified in this population or from previously published research, indicating that QTLs for sprout yield are genetically linked to seed-weight QTLs or else that seed-weight QTLs pleiotropically condition sprout yield. These data demonstrate that effective marker-assisted selection may be feasible for enhancing sprout yield in a soybean. The transgressive segregation of sprout yield, as well as the existence of two QTLs conditioning greater than 10% of the phenotypic variation in sprout yields provides an opportunity to select for progeny lines with a greater sprout yield than currently preferred cultivars such as Pureunkong. Received: 23 August 2000 / Accepted: 23 January 2001     

Molecular mapping of four ovule lethal mutants in soybean

We report genetic mapping of four soybean ovule lethal mutants, PS-1, PS-2, PS-3, and PS-4, which had been identified as female partial-sterile mutants from a gene-tagging study. The four mutants had been classified into two mutation classes: (1) PS-1—sporophytic mutation affects sporophytically expressed genes; and (2) PS-2, PS-3, and PS-4 mutants—female gametophyte-specific mutations affect gametophytically expressed genes and are transmitted through the male, but not the female gametes. Molecular mapping demonstrated that these four mutant genes and previously reported female-partial sterile gene, Fsp1, are located independently on soybean molecular linkage groups (MLG-) using SSR markers. PS-1, designated as Fsp2 and Genetic Type Collection number T364, is located between SSR markers Satt170 and Satt363 on MLG-C2 and linked by 13.9 cM and 12.1 cM, respectively. PS-2, designated as Fsp3 and Genetic Type Collection number T365H, is located between SSR markers Satt538 and Satt429 on MLG-A2 and linked by 13.3 cM and 25.4 cM, respectively. PS-3, designated as Fsp4 and Genetic Type Collection number T366H, is located on the terminus of MLG-F and linked to Sat 152 by 13.1 cM. PS-4, designated as Fsp5 and Genetic Type Collection number T367H, is located between SSR markers Satt324 and Satt138 on MLG-G and linked by 19.6 cM and 7.5 cM, respectively. SSR markers adjacent to Fsp3, Fsp4, and Fsp5 were distorted from a 1:2:1 ratio and fit a 1:1 ratio. The segregation distortions of SSR markers adjacent to Fsp3, Fsp4, and Fsp5 are in support of male, but not female transmission of the Fsp3, Fsp4, and Fsp5 gametes.This is a joint contribution of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 3769 and from the USDA, Agricultural Research Service, Corn Insects and Crop Genetics Research Unit, and supported by Hatch Act and 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.Communicated by J. Dvorak     

Molecular mapping of two loci that confer resistance to Asian rust in soybean

Asian soybean rust (ASR) is caused by the fungal pathogen Phakopsora pachyrhizi Sydow & Sydow. It was first identified in Brazil in 2001 and quickly infected soybean areas in several countries in South America. Primary efforts to combat this disease must involve the development of resistant cultivars. Four distinct genes that confer resistance against ASR have been reported: Rpp1, Rpp2, Rpp3, and Rpp4. However, no cultivar carrying any of those resistance loci has been released. The main objective of this study was to genetically map Rpp2 and Rpp4 resistance genes. Two F(2:3) populations, derived from the crosses between the resistant lines PI 230970 (Rpp2), PI 459025 (Rpp4) and the susceptible cultivar BRS 184, were used in this study. The mapping populations and parental lines were inoculated with a field isolate of P. pachyrhizi and evaluated for lesion type as resistant (RB lesions) or susceptible (TAN lesions). The mapping populations were screened with SSR markers, using the bulk segregant analysis (BSA) to expedite the identification of linked markers. Both resistance genes showed an expected segregation ratio for a dominant trait. This study allowed mapping Rpp2 and Rpp4 loci on the linkage groups J and G, respectively. The associated markers will be of great value on marker assisted selection for this trait.     

Molecular mapping of 36 soybean male-sterile, female-sterile mutants

Mutability of the w ( 4 ) flower color locus in soybean [Glycine max (L.) Merr.] is conditioned by an unstable allele designated w ( 4 ) -m. Germinal revertants, purple-flower plants, recovered among self-pollinated progeny of mutable flower plants were associated with the generation of necrotic root, chlorophyll-deficiency, and sterility mutations. Thirty-seven male-sterile, female-sterile mutant lines were generated from 37 independent reversion events at the w ( 4 ) -m locus. The first germinal revertant study had one male-sterile, female-sterile mutant (st8, T352), located on Molecular Linkage Group (MLG) J. The second study had 36 germinal-revertant derived sterility mutants descended from four mutable categories of w ( 4 ) -m. The mutable categories were 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. The objectives of the present study were to; (1) molecularly map the 36 male-sterile, female-sterile mutants, and to (2) compare map locations of these mutants with T352 (st8), identified from the first germinal revertant study. Thirty-three of 36 male-sterile, female-sterile mutations were derived from germinal reversions that were classified in the late excision categories. Thirty-five male-sterile mutants mapped to the st8 region on MLG J. The only exception mapped to MLG G. Most likely mutants were generated through insertion of a putative transposon that was excised from the w ( 4 ) locus. The location of 36 of 37 mutations to a single chromosomal region suggests preference for sequence-dependent insertion.     

A soybean mapping population specific to the early soybean production system.

The objective of this research was to create a soybean [Glycine max (L.) Merr] genetic resource in the form of a publicly available, well-characterized mapping population specific to maturity groups (MG) used in the early soybean production system. A total of 568 simple sequence repeat (SSR) markers were tested for polymorphism between soybean breeding line DS97-84-1 (MG IV) and germplasm line DT97-4290 (MG IV). A 90-genotype subset of an F2 population from a cross between these lines was evaluated for genetic linkage using 162 polymorphic SSRs, plant height, pod color (L2/l2), flower color (W1/w1) and stem termination (Dt1/dt1). A 1514 cM (Kosambi) genetic map covering 65% of the soybean genome based on 157 linked SSR markers was created. Comparison with the composite soybean genetic map was used to verify map order. Loci for pod color, flower color and stem termination fell in the expected position on the map indicating this is a normally segregating mapping population. Loci for height were identified on linkage groups C2, D1a, D1b, H, L, M and O. MG IV and V soybean genotypes are critical for the early soybean production system widely used in the midsouthern US. However, only two mapping populations have been reported in Soybase for MG IV and V genotypes. Additionally, the parents used in this cross are known to differ in their response to soybean cyst nematode and charcoal rot, which constitute two major pathology threats to Midsouth soybean production. The population and map reported herein represent an important genetic resource for the early soybean production system.     

Molecular mapping of soybean aphid resistance genes in PI 567541B

The soybean aphid (Aphis glycines Matsumura) is an important pest of soybean [Glycine max (L.) Merr.] in North America since it was first reported in 2000. PI 567541B is a newly discovered aphid resistance germplasm with early maturity characteristics. The objectives of this study were to map and validate the aphid resistance genes in PI 567541B using molecular markers. A mapping population of 228 F₃ derived lines was investigated for the aphid resistance in both field and greenhouse trials. Two quantitative trait loci (QTLs) controlling the aphid resistance were found using the composite interval mapping method. These two QTLs were localized on linkage groups (LGs) F and M. PI 567541B conferred resistant alleles at both loci. An additive × additive interaction between these two QTLs was identified using the multiple interval mapping method. These two QTLs combined with their interaction explained most of the phenotypic variation in both field and greenhouse trials. In general, the QTL on LG F had less effect than the one on LG M, especially in the greenhouse trial. These two QTLs were further validated using an independent population. The effects of these two QTLs were also confirmed using 50 advanced breeding lines, which were all derived from PI 567541B and had various genetic backgrounds. Hence, these two QTLs identified and validated in this study could be useful in improving soybean aphid resistance by marker-assisted selection.     

SSR mapping and confirmation of the QTL from PI96354 conditioning soybean resistance to southern root-knot nematode

Root-knot nematodes (Meloidogyne spp.) can cause severe yield loss of soybean [Glycine max (L.) Merr.] in the southern production region of the USA. Planting root-knot nematode-resistant cultivars is the most effective method of preventing yield loss. DNA marker-assisted breeding may accelerate the development of root-knot nematode-resistant cultivars. RFLP markers have previously been used to identify quantitative trait loci (QTLs) conferring resistance to southern root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood] (Mi) in a F_2:3 soybean population created by crossing the resistant PI96354 and the susceptible ’Bossier.’ A major QTL on linkage group (LG) O conditioning 31% of the variation in Mi gall number and a minor QTL on LG-G conditioning 14% of the gall variation were reported. With the development of SSR markers for soybean improvement, a higher level of mapping resolution and semi-automated detection has become possible. The objectives of this research were: (1) to increase the marker density in the genomic regions of the QTLs for Mi resistance on LG-O and LG-G with SSR markers; and (2) to confirm the effect of the QTLs in a second population and a different genetic background. With SSR markers, the QTL on LG-O was flanked by Satt492 and Satt358, and on LG-G by Satt012 and Satt505. Utilizing SSR markers flanking the two QTLs, marker-assisted selection was performed in a second F_2:3 population of PI96354× Bossier. Results confirmed the effectiveness of marker-assisted selection to predict the Mi phenotypes. By screening the BC₂F₂ population of Prichard (3)×G93–9009 we confirmed that selection for the minor QTL on LG-G with flanking SSR markers would enhance the resistance of lines containing the major QTL (which is most-likely Rmi1). Received: 29 September 2000 / Accepted: 17 April 2001     

Molecular linkage mapping and phylogeny of the chalcone synthase multigene family in soybean

Chalcone synthase (CHS), the key enzyme in the flavonoid biosynthesis pathway, is encoded by a multigene family, CHS1–CHS8 and dCHS1 in soybean. A tandem repeat of CHS1, CHS3 and CHS4, and dCHS1 that is believed to be located in the vicinity comprises the I locus that suppresses coloration of the seed coat. This study was conducted to determine the location of all CHS members by using PCR-based DNA markers. Primers were constructed based on varietal differences in either the nucleotide sequence of the 5-upstream region or the first intron of two cultivars, Misuzudaizu, with a yellow seed coat (II), and Moshidou Gong 503, with a brown seed coat (ii). One hundred and fifty recombinant inbred lines that originated from a cross between these two cultivars were used for linkage mapping together with 360 markers. Linkage mapping confirmed that CHS1, CHS3, CHS4, dCHS1, and the I locus are located at the same position in molecular linkage group (MLG) A2. CHS5 was mapped at a distance of 0.3 cM from the gene cluster. CHS2 and CHS6 were located in the middle region of MLGs A1 and K, respectively, while CHS7 and CHS8 were found at the distal end of MLGs D1a and B1, respectively. Phylogenetic analysis indicated that CHS1, CHS3, CHS4, and CHS5 are closely related, suggesting that gene duplication may have occurred repeatedly to form the I locus. In addition, CHS7 and CHS8 located at the distal end and CHS2, CHS6, and CHS members around the I locus located around the middle of the MLG are also related. Ancient tetraploidization and repeated duplication may be responsible for the evolution of the complex genetic loci of the CHS multigene family in soybean.     

Molecular mapping of two genes conferring resistance to Phytophthora sojae in a soybean landrace PI 567139B

Phytophthora root and stem rot (PRR), caused by the soil-borne oomycete pathogen Phytophthora sojae, is one of the most destructive diseases of soybean. PRR can be effectively controlled by race-specific genes conferring resistance to P. sojae (Rps). However, the Rps genes are usually non-durable, as populations of P. sojae are highly diverse and quick to adapt, and can be overcome 8–15 years after deployment. Thus, it is important to identify novel Rps genes for development of resistant soybean cultivars. PI 567139B is a soybean landrace carrying excellent resistance to nearly all predominant P. sojae races in Indiana. A mapping population consisting of 245 F₂ individuals and 403 F_2:3 families was developed from a cross between PI 567139B and the susceptible cultivar ‘Williams’, and used to dissect the resistance carried by PI 567139B. We found that the resistance in PI 567139B was conferred by two independent Rps genes, designated RpsUN1 and RpsUN2. The former was mapped to a 6.5 cM region between SSR markers Satt159 and BARCSOYSSR_03_0250 that spans the Rps1 locus on chromosome 3, while the latter was mapped to a 3.0 cM region between BARCSOYSSR_16_1275 and Sat_144, approximately 3.0–3.4 cM upstream of Rps2 on chromosome 16. According to the ‘Williams 82’ reference genome sequence, both regions are highly enriched with NBS-LRR genes. Marker assisted resistance spectrum analyses of these genes with 16 isolates of P. sojae, in combination with the mapping results, suggested that RpsUN1 was likely to be a novel allele at the Rps1 locus, while RpsUN2 was more likely to be a novel Rps gene.     

Molecular mapping of the fasciation mutation in soybean, Glycine max (Leguminosae)

The spontaneous fasciation mutation generates novel developmental diversity in cultivated soybean, Glycine max (L.) Merrill. An increased apical dominance in the mutant inhibits axillary buds, causes a branchless phenotype, and restricts reproduction to shoot apices. The fasciation mutation is encoded by a recessive (f) allele at a single locus. The mutation, despite its importance in soybean development, has no locus assignment on previously reported molecular maps of soybean. A population of 70 F(2) progeny was derived from a cross between 'Clark 63' and the fasciation mutant. More than 700 molecular markers (amplified restriction fragment length polymorphisms [AFLPs], random amplified polymorphic DNAs [RAPDs], restriction fragment length polymorphisms [RFLPs], and simple sequence repeats [SSRs]) were used in mapping of the fasciation phenotype. Twenty linkage groups (LGs) corresponding to the public soybean molecular map are represented on the Clark 63 × fasciation mutant molecular map that spans 3050 centimorgans (cM). The f locus was mapped on LG D1b+W and linked with two AFLPs and four SSR markers (Satt005, Satt141, Satt600, and Satt703). No linkage was found between the f locus and several cDNA polymorphic loci between the wild type and the mutant. The known map position of the f locus and demonstration of the mutant phenotype from early postembryonic throughout reproductive stages provide an excellent resource for investigations of molecular mechanisms affecting soybean ontogeny.     

Molecular mapping of the male-sterile, female-sterile mutant gene (st8) in soybean

Soybean male-sterile, female-sterile mutant genes have been identified by genetic and cytological studies. The St8 gene has been identified as an asynaptic mutation resulting in male and female sterility. This mutant gene was derived from a gene-tagging study using the soybean w4-mutable line. In this report we identified the genetic map position of st8 via restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers. The St8 gene mutation was located between RFLP marker E107 and SSR markers Satt132, Sct_065, and Satt414 on molecular linkage group J and linked to each by 7.8 cM and 3.4 cM, respectively.     

Molecular marker diversity of SCN-resistant sources in soybean.

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe; HG) is one of the most destructive pests of soybean (Glycine max (L.) Merr.) in the United States. Over 100 SCN-resistant accessions within the USDA Soybean Germplasm Collection have been identified, but little is known about the genetic diversity of this SCN-resistant germplasm. The objective of this research was to evaluate the genetic variation and determine the genetic relationships among SCN-resistant accessions. One hundred twenty-two genotypes were evaluated by 85 simple sequence repeat (SSR) markers from 20 linkage groups. Non-hierarchical (VARCLUS) and hierarchical (Ward's) clustering were combined with multidimensional scaling (MDS) to determine relationships among tested lines. The 85 SSR markers produced 566 allelic fragments with a mean polymorphic information content (PIC) value of 0.35. The 122 lines were grouped into 7 clusters by 2 different clustering methods and the MDS results consistently corresponded to the assigned clusters. Assigned clusters were dominated by genotypes that possess one or more unique SCN resistance genes and were associated with geographical origins. The results of analysis of molecular variance (AMOVA) showed that the variation differences among clusters and individual lines were significant, but the differences among individuals within clusters were not significant.     

Molecular biology of bacterial bioluminescence.

The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.     

Molecular mapping of soybean rust resistance in soybean accession PI 561356 and SNP haplotype analysis of the Rpp1 region in diverse germplasm

Soybean rust (SBR), caused by Phakopsora pachyrhizi Sydow, is one of the most economically important and destructive diseases of soybean [Glycine max (L.) Merr.] and the discovery of novel SBR resistance genes is needed because of virulence diversity in the pathogen. The objectives of this research were to map SBR resistance in plant introduction (PI) 561356 and to identify single nucleotide polymorphism (SNP) haplotypes within the region on soybean chromosome 18 where the SBR resistance gene Rpp1 maps. One-hundred F(2:3) lines derived from a cross between PI 561356 and the susceptible experimental line LD02-4485 were genotyped with genetic markers and phenotyped for resistance to P. pachyrhizi isolate ZM01-1. The segregation ratio of reddish brown versus tan lesion type in the population supported that resistance was controlled by a single dominant gene. The gene was mapped to a 1-cM region on soybean chromosome 18 corresponding to the same interval as Rpp1. A haplotype analysis of diverse germplasm across a 213-kb interval that included Rpp1 revealed 21 distinct haplotypes of which 4 were present among 5 SBR resistance sources that have a resistance gene in the Rpp1 region. Four major North American soybean ancestors belong to the same SNP haplotype as PI 561356 and seven belong to the same haplotype as PI 594538A, the Rpp1-b source. There were no North American soybean ancestors belonging to the SNP haplotypes found in PI 200492, the source of Rpp1, or PI 587886 and PI 587880A, additional sources with SBR resistance mapping to the Rpp1 region.