Fine mapping the soybean aphid resistance gene Rag1 in soybean

The soybean aphid (Aphis glycines Matsumura) is an important soybean [Glycine max (L.) Merr.] pest in North America. The dominant aphid resistance gene Rag1 was previously mapped from the cultivar ‘Dowling’ to a 12 cM marker interval on soybean chromosome 7 (formerly linkage group M). The development of additional genetic markers mapping closer to Rag1 was needed to accurately position the gene to improve the effectiveness of marker-assisted selection (MAS) and to eventually clone it. The objectives of this study were to identify single nucleotide polymorphisms (SNPs) near Rag1 and to position these SNPs relative to Rag1. To generate a fine map of the Rag1 interval, 824 BC₄F₂ and 1,000 BC₄F₃ plants segregating for the gene were screened with markers flanking Rag1. Plants with recombination events close to the gene were tested with SNPs identified in previous studies along with new SNPs identified from the preliminary Williams 82 draft soybean genome shotgun sequence using direct re-sequencing and gene-scanning melt-curve analysis. Progeny of these recombinant plants were evaluated for aphid resistance. These efforts resulted in the mapping of Rag1 between the two SNP markers 46169.7 and 21A, which corresponds to a physical distance on the Williams 82 8× draft assembly (Glyma1.01) of 115 kilobase pair (kb). Several candidate genes for Rag1 are present within the 115-kb interval. The markers identified in this study that are closely linked to Rag1 will be a useful resource in MAS for this important aphid resistance gene.     

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.     

Fine mapping of the Pc locus of Sorghum bicolor, a gene controlling the reaction to a fungal pathogen and its host-selective toxin

Milo disease in sorghum is caused by isolates of the soil-borne fungus Periconia circinata that produce PC-toxin. Susceptibility to milo disease is conditioned by a single, semi-dominant gene, termed Pc. The susceptible allele (Pc) converts to a resistant form (pc) spontaneously at a gametic frequency of 10⁻³ to 10⁻⁴. A high-density genetic map was constructed around the Pc locus using DNA markers, allowing the Pc gene to be delimited to a 0.9 cM region on the short arm of sorghum chromosome 9. Physically, the Pc-region was covered by a single BAC clone. Sequence analysis of this BAC revealed twelve gene candidates. Several of the predicted genes in the region are homologous to disease resistance loci, including one NBS-LRR resistance gene analogue that is present in multiple tandem copies. Analysis of pc isolines derived from Pc/Pc sorghum suggests that one or more members of this NBS-LRR gene family are the Pc genes that condition susceptibility.     

Fine mapping of the soybean aphid-resistance gene Rag2 in soybean PI 200538

The discovery of biotype diversity of soybean aphid (SA: Aphis glycines Matsumura) in North America emphasizes the necessity to identify new aphid-resistance genes. The soybean [Glycine max (L.) Merr.] plant introduction (PI) 200538 is a promising source of SA resistance because it shows a high level of resistance to a SA biotype that can overcome the SA-resistance gene Rag1 from ‘Dowling’. The SA-resistance gene Rag2 was previously mapped from PI 200538 to a 10-cM marker interval on soybean chromosome 13 [formerly linkage group (LG) F]. The objective of this study was to fine map Rag2. This fine mapping was carried out using lines derived from 5,783 F₂ plants at different levels of backcrossing that were screened with flanking genetic markers for the presence of recombination in the Rag2 interval. Fifteen single nucleotide polymorphism (SNP) markers and two dominant polymerase chain reaction-based markers near Rag2 were developed by re-sequencing target intervals and sequence-tagged sites. These efforts resulted in the mapping of Rag2 to a 54-kb interval on the Williams 82 8× assembly (Glyma1). This Williams 82 interval contains seven predicted genes, which includes one nucleotide-binding site-leucine-rich repeat gene. SNP marker and candidate gene information identified in this study will be an important resource in marker-assisted selection for aphid resistance and for cloning the gene.     

Fine mapping of a major quantitative trait locus that regulates pod shattering in soybean

Legumes represent the second most important family of crop plants, accounting for ~27 % of the world’s crop production. While some legumes are grown as forages or vegetables, most crop legumes are grown for harvesting their nutritious seeds. The legume seeds are contained in the pod, which is composed of a single seed-bearing carpel that, when matures, splits open along two seams, a process called pod dehiscence or pod shattering. Pod shattering before or during harvest causes yield losses of grain legumes. Moreover, the dominant shattering trait of the wild progenitors is a limiting factor for efficient introgression of value-added traits into elite breeding lines. Knowledge of the genetic mechanisms underlying pod shattering will facilitate breeding of shattering-resistant varieties, expedite introgression of agronomically favorable traits from wild species to elite breeding lines, and enrich our understanding of the evolution of seed dispersal and crop domestication in diverse crop species. Here we report fine mapping of a major quantitative trait locus (designated as qPDH1) that regulates pod shattering in soybean (Glycine max). A combination of linkage and association mapping allowed us to delimit the qPDH1 locus within a 47-kb region on chromosome 16. The data reported here will facilitate positional cloning of the underlying gene and the development of breeder-friendly genetic markers for marker-assisted selection in soybean.     

Fine physical mapping of the rice stripe resistance gene locus, Stvb-i

The Stvb-i gene confers stripe disease resistance to rice. For positional cloning, we constructed a physical map spanning 1.8-cM distance between flanking markers, consisting of 18 bacterial artificial chromosome (BAC) clones, around the Stvb-i locus on rice chromosome 11. The 18 clones were isolated by screening a BAC library derived from a japonica cultivar, Shimokita, with three Stvb-i-linked RFLP markers and DraI-digested DNAs of a yeast artificial chromosome (YAC) clone. The results of Southern hybridization and restriction enzyme analyses indicated that these BAC clones are contiguous and cover about a 700-kb region containing the Stvb-i allele. Utilizing end and internal fragments of the BAC insert DNAs, 33 molecular markers were generated within a small chromosomal region including the Stvb-i locus. Genotyping analysis with these markers for a resistant cultivar and four nearby recombinants selected from 120 F₂ individuals indicated that Stvb-i is contained within an approximately 286-kb region covered with two overlapping BAC clones. Received: 25 August 1999 / Accepted: 16 November 1999     

Fine mapping of qPAA8, a gene controlling panicle apical development in rice

In rice, one detrimental factor influencing single panicle yield is the frequent occurrence of panicle apical abortion (PAA) under unfavorable climatic conditions. Until now, no detailed genetic information has been available to avoid PAA in rice breeding. Here, we show that the occurrence of PAA is associated with the accumulation of excess hydrogen peroxide. Quantitative trait loci (QTLs) mapping for PAA in an F(2) population derived from the cross of L-05261 (PAA line) × IRAT129 (non-PAA variety) identified seven QTLs over a logarithm of the odd (LOD) threshold of 2.5, explaining approximately 50.1% of phenotypic variance for PAA in total. Five of the QTLs with an increased effect from L-05261, were designated as qPAA3-1, qPAA3-2, qPAA4, qPAA5 and qPAA8, and accounted for 6.8%, 5.9%, 4.2%, 13.0% and 12.2% of phenotypic variance, respectively. We found that the PAA in the early heading plants was mainly controlled by qPAA8. Subsequently, using the sub-populations specific for qPAA8 based on marker-assisted selection, we further narrowed qPAA8 to a 37.6-kb interval delimited by markers RM22475 and 8-In112. These results are beneficial for PAA gene clone.     

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.     

Fine mapping of the Co-4 locus of common bean reveals a resistance gene candidate, COK-4, that encodes for a protein kinase

The SAS13 SCAR marker, tightly linked with the Co-4 ² gene segregating in a population of 1018 F₂ individual plants, was used as a starting point for cloning gene sequences associated with the Co-4 locus that conditions resistance to anthracnose caused by the fungal pathogen Colletotrichum lindemuthianum in common bean (Phaseolus vulgaris). A contig developed from genomic clones flanking the marker region revealed a 1110-bp open reading frame, named COK-4. The predicted COK-4 protein contains a serine-threonine kinase domain highly similar to the protein encoded by the Pto gene in tomato, but with a highly hydrophobic membrane-spanning region. COK-4 homologs were cloned and sequenced from different bean cultivars. Single nucleotide polymorphisms were found between the homologous sequences and were confirmed with three restriction enzymes. Restriction patterns among three bean cultivars known to possess different alleles at the Co-4 locus, SEL 1308 (Co-4 ² ), TO (Co-4) and Black Magic (co-4), were polymorphic. Absolute co-segregation between COK-4 restriction patterns and the disease phenotype was observed in 96 F₃ families. More than one copy of the COK-4 gene homolog exists in the bean genome as demonstrated by Southern analysis. These results suggest that COK-4 is part of the Co-4 locus conditioning resistance to C. lindemuthianum in bean. Received: 22 June 2000 / Accepted: 20 November 2000     

Fine mapping a self-fertility locus in perennial ryegrass

Key message

A self-fertility locus was fine mapped to a 1.6 cM region on linkage group 5 in a perennial ryegrass population. This locus was the main determinant of pollen self-compatibility.

Abstract

In grasses, self-incompatibility (SI) is characterized by a two-loci gametophytic (S and Z) mechanism acting together in the recognition and inhibition of self-pollen. Mutations affecting the expression of SI have been reported in a few grass species. In perennial ryegrass (Lolium perenne L.), a mutation independent from S and Z, and mapping on linkage group 5 (LG 5), was previously reported to produce self-fertile plants. Here, we describe fine mapping of the self-fertility (SF) gene in a perennial ryegrass population and determine whether there is any effect of other genomic regions on the pollen compatibility. The phenotypic segregation of SF showed a bimodal distribution with one mean at 49% pollen compatibility and the other at 91%. Marker-trait association analysis showed that only markers on LG 5 were significantly associated with the trait. A single gene model explained 82% of the observed variability and no effects of the other regions were detected. Using segregation and linkage analysis, the SF locus was located to a 1.6 cM region on LG 5. The flanking marker sequences were aligned to rice and Brachypodium distachyon reference genomes to estimate the physical distance. We provide markers tightly linked to SF that can be used for introgression of this trait into advanced breeding germplasm. Moreover, our results represent a further step towards the identification of the SF gene in LG 5.

     

The Rj4 allele in soybean represses nodulation by chlorosis-inducing bradyrhizobia classified as DNA homology group II by antibiotic resistance profiles

Summary To determine the relationship between nodulation restriction by the Rj4 allele of soybean, rhizobitoxine-induced chlorosis, and taxonomic grouping of bradyrhizobia, 119 bradyrhizobial isolates were tested in Leonard jar culture for nodulation response and chlorosis induction. In addition to strain USDA 61, the strain originally reported as defining the Rj4 response, eight other isolates (i.e., USDA 62, 83, 94, 238, 252, 259, 260, and 340) were discovered to elicit the nodulation interdiction of the Rj4 allele. Only 16% of all the bradyrhizobial strains tested induced chlorosis, but seven of the nine strains (78%) interdicted by the Rj4 allele were chlorosis-inducing strains. Furthermore, in tests for antibiotic resistance profile, eight of the nine interdicted strains (89%) were classed in DNA homology group II. This evidence suggests that the Rj4 allele has a positive value to the host plant in shielding it from nodulation by certain chlorosis-inducing bradyrhizobia of a DNA homology group with impaired efficiency of nitrogen fixation with soybean.     

Fine genetic mapping of a locus controlling short internode length in melon (Cucumis melo L.)

Compact and dwarfing vining habits in melon (Cucumis melo L.; 2n = 2x = 24) may have commercial importance since they can contribute to the promotion of concentrated fruit set and can be planted in higher plant densities than standard vining types. A study was designed to determine the genetics of dwarfism associated with a diminutive (short internodes) melon mutant line PNU-D1 (C. melo ssp. cantalupensis). PNU-D1 was crossed with inbred wild-type melon line PNU-WT1 (C. melo ssp. agrestis), and resultant F₁ progeny were then self-pollinated to produce an F₂ population that segregated as dwarf and vining plant types. Primary stem length of F₂ progeny assessed under greenhouse conditions indicated that a single recessive gene, designated mdw1, controlled dwarfism in this population. To identify the chromosomal location associated with mdw1, an simple sequence repeat (SSR)-based genetic linkage map was constructed using 94 F₂ progeny. Using 76 SSR markers positioned on 15 linkage groups spanning 462.84 cM, the location of mdw1 was localized to Chromosome 7. Using the putative dwarfing-associated genes, fine genetic mapping of the mdw1 genomic region was facilitated with 1,194 F₂ progeny that defined the genetic distance between mdw1 and cytokinin oxidase gene, a candidate gene for compact growth habit (cp) in cucumber, to be 1.7 cM. The candidate gene ERECTA (serin/threonine kinase) and UBI (ubiquitin) were also mapped to genomic regions flanking mdw1 at distances of 0.6 and 1.2 cM, respectively.     

Genetic allelism and linkage tests of a soybean gene,Rfg1, controlling nodulation with Rhizobium fredii strain USDA 205

A soybean gene, Rfg1, controlling nodulation with strain USDA 205, the type strain for the fast-growing species Rhizobium fredii, was tested for allelism with the Rj4 gene. The Rj4 gene conditions ineffective nodulation primarily with certain strains of the slow-growing soybean microsymbiont, Bradyrhizobium elkanii. The F2 seeds of the cross of the cultivars Peking, carrying the alleles rfg1, Rj4, i (controlling inhibition of seed coat color) and W1 (controlling flower color), and Kent, carrying the alleles Rfg1, rj4, i-i and w1, were evaluated for nodulation response with strain USDA 205 by planting surface disinfested seeds in sterilized vermiculite in growth trays and inoculating with a stationary phase broth culture of strain USDA 205 at planting. Plants were classified for nodulation response visually after four weeks growth and transplanted to the field for F3 seed production. Flower color, purple (W1) vs white (w1), was determined in the field. The allele present at the i locus was determined by classification of F3 seed coat color. The F3 seeds were planted in growth trays and inoculated with strain USDA 61 of Bradyrhizobium elkanii to determine the genotype for the Rj4 locus. The Rfg1 and Rj4 genes were determined to be located at separate loci. Chi-square analysis for linkage indicated that Rfg1 segregated independently of the Rj4, I and W1 loci.     

Fine mapping of the diabetes-susceptibility locus, IDDM4, on chromosome 11q13.

Genomewide linkage studies of type 1 diabetes (or insulin-dependent diabetes mellitus [IDDM]) indicate that several unlinked susceptibility loci can explain the clustering of the disease in families. One such locus has been mapped to chromosome 11q13 (IDDM4). In the present report we have analyzed 707 affected sib pairs, obtaining a peak multipoint maximum LOD score (MLS) of 2.7 (lambda(s)=1.09) with linkage (MLS>=0.7) extending over a 15-cM region. The problem is, therefore, to fine map the locus to permit structural analysis of positional candidate genes. In a two-stage approach, we first scanned the 15-cM linked region for increased or decreased transmission, from heterozygous parents to affected siblings in 340 families, of the three most common alleles of each of 12 microsatellite loci. One of the 36 alleles showed decreased transmission (50% expected, 45.1% observed [P=.02, corrected P=.72]) at marker D11S1917. Analysis of an additional 1,702 families provided further support for negative transmission (48%) of D11S1917 allele 3 to affected offspring and positive transmission (55%) to unaffected siblings (test of heterogeneity P=3x10-4, corrected P=. 01]). A second polymorphic marker, H0570polyA, was isolated from a cosmid clone containing D11S1917, and genotyping of 2,042 families revealed strong linkage disequilibrium between the two markers (15 kb apart), with a specific haplotype, D11S1917*03-H0570polyA*02, showing decreased transmission (46.4%) to affected offspring and increased transmission (56.6%) to unaffected siblings (test of heterogeneity P=1.5x10-6, corrected P=4.3x10-4). These results not only provide sufficient justification for analysis of the gene content of the D11S1917 region for positional candidates but also show that, in the mapping of genes for common multifactorial diseases, analysis of both affected and unaffected siblings is of value and that both predisposing and nonpredisposing alleles should be anticipated.     

AFLP mapping of soybean maturity gene E4

Days to flowering and maturity are controlled by genes E1-E7 and J in soybean. Previous studies revealed that E1-E5 and E7 influence tolerances to low-temperature-induced seed coat browning in different directions at various intensities. The E4 locus is useful for the development of early maturing cultivars with chilling tolerance because the recessive allele conditions both the early-maturing habit and chilling tolerance. This study was conducted to obtain a fine map of E4 by amplified fragment length polymorphism (AFLP) analysis using a F(8:9) family segregating for E4 that was developed from a cross between photoperiod-insensitive Japanese landraces, Sakamotowase (E4) and Miharudaizu (e4). AFLP analysis using a total of 4096 primer pairs detected 20 polymorphic markers between near-isogenic lines for E4. Linkage mapping incorporated 16 AFLP markers into a previously constructed genetic map around E4 in linkage group I. Eight AFLP markers were localized to unfilled areas between E4 and the closest markers identified previously. Two AFLP markers flanking E4, e48m41-8 and e18m38-8, were mapped at positions 0.6 and 5.4 cM apart from E4, respectively. They were dominant and in cis arrangement with the recessive allele (e4) conditioning the photoperiod insensitivity and chilling tolerance. These markers can be used in developing more precise markers for fine mapping and marker-assisted selection and in isolating the underlying gene via genome walking approaches.     

Fine mapping of a male sterility gene, vr1, on chromosome 4 in rice

xs1 is a male sterile rice mutant derived from a spontaneous mutation. Pollen development in the xs1 mutant proceeds normally until the vacuolation stage, at which time xs1 pollen fails to vacuolate and no viable pollen is produced. Genetic analysis indicates that the xs1 mutant phenotype is controlled by a single recessive gene, designated vacuolation retardation 1 (vr1), which was mapped to rice chromosome 4. In order to fine-map the vr1 locus, two large mapping populations were generated and several SSR and InDel markers were developed from publicly available rice genomic sequences. By employing a strategy of chromosome-walking, the vr1 gene was finally located within a genetic interval of 0.27 cM, flanked by the markers FID30 and FS15, with distances of 0.11 and 0.16 cM, respectively, and co-segregating with the marker FC4-2. Based on the japonica rice genome sequence, the vr1 locus is estimated to cover a 48-kb region containing eight putative genes. Our results will facilitate the cloning and functional characterization of the vr1 gene.