R. M. Biyashev

A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome

A map of the barley genome consisting of 295 loci was constructed. These loci include 152 cDNA restriction fragment length polymorphism (RFLP), 114 genomic DNA RFLP, 14 random amplified polymorphic DNA (RAPD), five isozyme, two morphological, one disease resistance and seven specific amplicon polymorphism (SAP) markers. The RFLP-identified loci include 63 that were detected using cloned known function genes as probes. The map covers 1,250 centiMorgans (cM) with a 4.2 cM average distance between markers. The genetic lengths of the chromosomes range from 124 to 223 cM and are in approximate agreement with their physical lengths. The centromeres were localized to within a few markers on all of the barley chromosomes except chromosome 5. Telomeric regions were mapped for the short (plus) arms of chromosomes 1, 2 and 3 and the long (minus) arm of chromosomes 7.

Development of simple sequence repeat DNA markers and their integration into a barley linkage map

Simple sequence repeats (SSRs), or microsatellites, are a new class of PCR-based DNA markers for genetic mapping. The objectives of the present study were to develop SSR markers for barley and to integrate them into an existing barley linkage map. DNA sequences containing SSRs were isolated from a barley genomic library and from public databases. It is estimated that the barley genome contains one (GA)n repeat every 330 kb and one (CA)n repeat every 620 kb. A total of 45 SSRs were identified and mapped to seven barley chromosomes using doubled-haploid lines and/or wheat-barley addition-line assays. Segregation analysis for 39 of these SSRs identified 40 loci. These 40 markers were placed on a barley linkage map with respect to 160 restriction fragment length polymorphism (RFLP) and other markers. The results of this study demonstrate the value of SSRs as markers in genetic studies and breeding research in barley.

Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice

Genetic polymorphisms of ten microsatellite DNA loci were examined among 238 accessions of landraces and cultivars that represent a significant portion of the distribution range for both indica and japonica groups of cultivated rice. In all, 93 alleles were identified with these ten markers. The number of alleles varied from a low of 3 or 4 at each of four loci, to an intermediate value of 9–14 at five loci, and to an extra-ordinarily high 25 at one locus. The numbers of alleles per locus are much larger than those detected using other types of markers. The number of alleles detected at a locus is significantly correlated with the number of simple sequence repeats in the targeted microsatellite DNA. Indica rice has about 14% more alleles than japonica rice, and such allele number differences are more pronounced in landraces than in cultivars. The indica-japonica differentiation component accounted for about 10% of the diversity in the total sample, and twice as much differentiation was detected in cultivars as in landraces. About two-thirds as many alleles were observed in cultivars as in landraces; another two-thirds of the alleles in the cultivar group were found in modern elite cultivars or parents of hybrid rice. The majority of the simple sequence repeat (SSR) alleles that were present in high or intermediate frequencies in landraces ultimately survived into modern elite cultivars and hybrids. The greater resolving power and the efficient production of massive amounts of SSR data may be particularly useful for germplasm assessment and evolutionary studies of crop plants.

Analysis of the barley and rice genomes by comparative RFLP linkage mapping

Comparative genetic mapping of rice and barley, both major crop species with extensive genetic resources, offers the possibility of uniting two well-established and characterized genetic systems. In the present study, we screened 229 molecular markers and utilized 110 polymorphic orthologous loci to construct comparative maps of the rice and barley genomes. While extensive chromosomal rearrangements, including inversions and intrachromosomal translocations, differentiate the rice and barley genomes, several syntenous chromosomes are evident. Indeed, several chromosomes and chromosome arms appear to share nearly identical gene content and gene order. Seventeen regions of conserved organization were detected, spanning 287 cM (24%) and 321 cM (31%) of the rice and barley genomes, respectively. The results also indicate that most (72%) of the single-copy sequences in barley are also single copy in rice, suggesting that the large barley genome arose by unequal crossing over and amplification of repetitive DNA sequences and not by the duplication of single-copy sequences. Combining these results with those previously reported for comparative analyses of rice and wheat identified nine putatively syntenous chromosomes among barley, wheat and rice. The high degree of gene-order conservation as detected by comparative mapping has astonishing implications for interpreting genetic information among species and for elucidating chromosome evolution and speciation.

Diversity and evolution of a non-TIR-NBS sequence family that clusters to a chromosomal ’’hotspot” for disease resistance genes in soybean

Abstract In soybean, genes controlling resistance to numerous diseases have been shown to cluster to regions on several chromosomes. One such vital chromosomal region is on the soybean molecular linkage group (MLG) F flanked by the RFLP markers K644 and B212. Here, genes controlling resistance to bacterial blight, Phytophthora root rot, and several viral diseases, as well as QTLs conditioning resistance to corn earworm, root knot nematode, and white mold have been mapped. We have previously identified two classes (b and j) of disease resistance-related nucleotide binding site (NBS) sequences that localize to this cluster of genes. Using both cDNA and genomic analyses, we have studied one multi-gene family of sequences representing the previously reported class j NBS of soybean. This class of NBS resembles the RPS2-like NBS sequences. RPS2 and similar resistance genes are referred to as non-TIR because they do not encode motifs homologous to the Toll-Interleukin-1 region (TIR). By designing PCR primers that specifically target these non-TIR-NBS encoding sequences, we have amplified at least six class j sequence members from soybean. In addition, we have conducted genomic and cDNA library screenings to identify additional class j members. In all, we have characterized 12 class j NBS sequence members. These members have been mapped within a 2-cM region of the soybean F linkage group. We have also identified homoeologous chromosomal regions on linkage groups A2 and E that contain class j NBS sequences. A BLAST search of the GenBank database has shown that non-TIR NBS sequences are present across the legume family. We have compared these non-TIR sequences from other legumes with the soybean clones to assess the level of diversity within this class of disease resistance-related sequences.

Genetic variants in root architecture-related genes in a Glycine soja accession, a potential resource to improve cultivated soybean

BackgroundRoot system architecture is important for water acquisition and nutrient acquisition for all crops. In soybean breeding programs, wild soybean alleles have been used successfully to enhance yield and seed composition traits, but have never been investigated to improve root system architecture. Therefore, in this study, high-density single-feature polymorphic markers and simple sequence repeats were used to map quantitative trait loci (QTLs) governing root system architecture in an inter-specific soybean mapping population developed from a cross between Glycine max and Glycine soja.ResultsWild and cultivated soybean both contributed alleles towards significant additive large effect QTLs on chromosome 6 and 7 for a longer total root length and root distribution, respectively. Epistatic effect QTLs were also identified for taproot length, average diameter, and root distribution. These root traits will influence the water and nutrient uptake in soybean. Two cell division-related genes (D type cyclin and auxin efflux carrier protein) with insertion/deletion variations might contribute to the shorter root phenotypes observed in G. soja compared with cultivated soybean. Based on the location of the QTLs and sequence information from a second G. soja accession, three genes (slow anion channel associated 1 like, Auxin responsive NEDD8-activating complex and peroxidase), each with a non-synonymous single nucleotide polymorphism mutation were identified, which may also contribute to changes in root architecture in the cultivated soybean. In addition, Apoptosis inhibitor 5-like on chromosome 7 and slow anion channel associated 1-like on chromosome 15 had epistatic interactions for taproot length QTLs in soybean.ConclusionRare alleles from a G. soja accession are expected to enhance our understanding of the genetic components involved in root architecture traits, and could be combined to improve root system and drought adaptation in soybean.

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F1 hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F1s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.

Genome-wide transcriptome analyses of developing seeds from low and normal phytic acid soybean lines

BackgroundLow phytic acid (lpa) crops are potentially eco-friendly alternative to conventional normal phytic acid (PA) crops, improving mineral bioavailability in monogastric animals as well as decreasing phosphate pollution. The lpa crops developed to date carry mutations that are directly or indirectly associated with PA biosynthesis and accumulation during seed development. These lpa crops typically exhibit altered carbohydrate profiles, increased free phosphate, and lower seedling emergence, the latter of which reduces overall crop yield, hence limiting their large-scale cultivation. Improving lpa crop yield requires an understanding of the downstream effects of the lpa genotype on seed development. Towards that end, we present a comprehensive comparison of gene-expression profiles between lpa and normal PA soybean lines (Glycine max) at five stages of seed development using RNA-Seq approaches. The lpa line used in this study carries single point mutations in a myo-inositol phosphate synthase gene along with two multidrug-resistance protein ABC transporter genes.ResultsRNA sequencing data of lpa and normal PA soybean lines from five seed-developmental stages (total of 30 libraries) were used for differential expression and functional enrichment analyses. A total of 4235 differentially expressed genes, including 512-transcription factor genes were identified. Eighteen biological processes such as apoptosis, glucan metabolism, cellular transport, photosynthesis and 9 transcription factor families including WRKY, CAMTA3 and SNF2 were enriched during seed development. Genes associated with apoptosis, glucan metabolism, and cellular transport showed enhanced expression in early stages of lpa seed development, while those associated with photosynthesis showed decreased expression in late developmental stages. The results suggest that lpa-causing mutations play a role in inducing and suppressing plant defense responses during early and late stages of seed development, respectively.ConclusionsThis study provides a global perspective of transcriptomal changes during soybean seed development in an lpa mutant. The mutants are characterized by earlier expression of genes associated with cell wall biosynthesis and a decrease in photosynthetic genes in late stages. The biological processes and transcription factors identified in this study are signatures of lpa-causing mutations.

A Method for Combining Isolates of Phytophthora sojae to Screen for Novel Sources of Resistance to Phytophthora Stem and Root Rot in Soybean

Soybean cultivars with specific single resistance genes (Rps) are grown to reduce yield loss due to Phytophthora stem and root rot caused by the oomycete pathogen Phytophthora sojae. To identify novel Rps loci, soybean lines are often screened several times, each time with an isolate of P. sojae that differs in virulence on various Rps genes. The goal of this study was to determine whether several isolates of P. sojae that differ in virulence on Rps genes could be combined into a single source of inoculum and used to screen soybean lines for novel Rps genes. A set of 14 soybean differential lines, each carrying a specific Rps gene, was inoculated with three isolates of P. sojae, which differed in virulence on 6 to 10 Rps genes, individually or in a 1:1:1 mixture. Inoculum containing the 1:1:1 mixture of isolates was virulent on 13 Rps genes. The mixed-inoculum method was used to screen 1,019 soybean accessions in a blind assay for novel sources of resistance. In all, 17% of Glycine max accessions and 11% of G. soja accessions were resistant (≤30% dead plants), suggesting that these accessions may carry a novel Rps gene or genes. Advantages of combining isolates into a single source of inoculum include reduced cost, ability to screen soybean germplasm with inoculum virulent on all known Rps genes, and ease of identifying novel sources of resistance. This study is a precursor to identifying novel sources of resistance to P. sojae in soybean using RXLR effectors.

Metabolite Profiling of Soybean Seed Extracts from Near-Isogenic Low and Normal Phytate Lines Using Orthogonal Separation Strategies

Untargeted metabolomic profiling using liquid chromatography-mass spectrometry (LC-MS) was applied to lipid-depleted methanolic extracts of soybean seeds utilizing orthogonal chromatographic separations (reversed-phase and hydrophilic interaction) in both positive and negative ionization modes. Four near-isogenic lines (NILs) differing in mutations for two genes encoding highly homologous multidrug resistant proteins (MRPs) were evaluated. The double mutant exhibited a low phytate phenotype, whereas the other three NILs, the two single mutants and the wild type, did not. Principal component analysis (PCA) of the four LC-MS data sets fully separated the low phytate line from the other three. While the levels of neutral oligosaccharides were the same for all lines, there were significant metabolite differences residing in the levels of malonyl isoflavones, soyasaponins, and arginine. Two methanol-soluble polypeptides were also found as differing in abundance levels, one of which was identified as the allergen Gly m 1.