Identification of quantitative trait loci controlling soybean seed protein and oil content.

Soybean is a major source of seed protein and oil globally with an average composition of 40% protein and 20% oil in the seed. The goal of this study was to identify quantitative trait loci (QTL) conferring seed protein and oil content utilizing a population constructed by crossing an above average protein content line, PI 399084 to another line that had a low protein content value, PI 507429, both from the USDA soybean germplasm collection. The recombinant inbred line (RIL) population, PI 507429 x PI 399084, was evaluated in two replications over four years (2018-2021); the seeds were analyzed for seed protein and oil content using near-infrared reflectance spectroscopy. The recombinant inbred lines and the two parents were re-sequenced using genotyping by sequencing. A total of 12,761 molecular markers, which came from genotyping by sequencing, the SoySNP6k BeadChip and selected simple sequence repeat (SSR) markers from known protein QTL chromosomal regions were used for mapping. One QTL was identified on chromosome 2 explaining up to 56.8% of the variation for seed protein content and up to 43% for seed oil content. Another QTL identified on chromosome 15 explained up to 27.2% of the variation for seed protein and up to 41% of the variation for seed oil content. The protein and oil QTLs of this study and their associated molecular markers will be useful in breeding to improve nutritional quality in soybean.

Network Inference of Transcriptional Regulation in Germinating Low Phytic Acid Soybean Seeds.

The low phytic acid (lpa) trait in soybeans can be conferred by loss-of-function mutations in genes encoding myo-inositol phosphate synthase and two epistatically interacting genes encoding multidrug-resistance protein ATP-binding cassette (ABC) transporters. However, perturbations in phytic acid biosynthesis are associated with poor seed vigor. Since the benefits of the lpa trait, in terms of end-use quality and sustainability, far outweigh the negatives associated with poor seed performance, a fuller understanding of the molecular basis behind the negatives will assist crop breeders and engineers in producing variates with lpa and better germination rate. The gene regulatory network (GRN) for developing low and normal phytic acid soybean seeds was previously constructed, with genes modulating a variety of processes pertinent to phytic acid metabolism and seed viability being identified. In this study, a comparative time series analysis of low and normal phytic acid soybeans was carried out to investigate the transcriptional regulatory elements governing the transitional dynamics from dry seed to germinated seed. GRNs were reverse engineered from time series transcriptomic data of three distinct genotypic subsets composed of lpa soybean lines and their normal phytic acid sibling lines. Using a robust unsupervised network inference scheme, putative regulatory interactions were inferred for each subset of genotypes. These interactions were further validated by published regulatory interactions found in Arabidopsis thaliana and motif sequence analysis. Results indicate that lpa seeds have increased sensitivity to stress, which could be due to changes in phytic acid levels, disrupted inositol phosphate signaling, disrupted phosphate ion (Pi) homeostasis, and altered myo-inositol metabolism. Putative regulatory interactions were identified for the latter two processes. Changes in abscisic acid (ABA) signaling candidate transcription factors (TFs) putatively regulating genes in this process were identified as well. Analysis of the GRNs reveal altered regulation in processes that may be affecting the germination of lpa soybean seeds. Therefore, this work contributes to the ongoing effort to elucidate molecular mechanisms underlying altered seed viability, germination and field emergence of lpa crops, understanding of which is necessary in order to mitigate these problems.

Identification of Quantitative Disease Resistance Loci Toward Four Pythium Species in Soybean.

In this study, four recombinant inbred line (RIL) soybean populations were screened for their response to infection by Pythium sylvaticum, Pythium irregulare, Pythium oopapillum, and Pythium torulosum. The parents, PI 424237A, PI 424237B, PI 408097, and PI 408029, had higher levels of resistance to these species in a preliminary screening and were crossed with "Williams," a susceptible cultivar. A modified seed rot assay was used to evaluate RIL populations for their response to specific Pythium species selected for a particular population based on preliminary screenings. Over 2500 single-nucleotide polymorphism (SNP) markers were used to construct chromosomal maps to identify regions associated with resistance to Pythium species. Several minor and large effect quantitative disease resistance loci (QDRL) were identified including one large effect QDRL on chromosome 8 in the population of PI 408097 × Williams. It was identified by two different disease reaction traits in P. sylvaticum, P. irregulare, and P. torulosum. Another large effect QDRL was identified on chromosome 6 in the population of PI 408029 × Williams, and conferred resistance to P. sylvaticum and P. irregulare. These large effect QDRL will contribute toward the development of improved soybean cultivars with higher levels of resistance to these common soil-borne pathogens.

Mining germplasm panels and phenotypic datasets to identify loci for resistance to Phytophthora sojae in soybean.

Phytophthora sojae causes Phytophthora root and stem rot of soybean and has been primarily managed through deployment of qualitative Resistance to P. sojae genes (Rps genes). The effectiveness of each individual or combination of Rps gene(s) depends on the diversity and pathotypes of the P. sojae populations present. Due to the complex nature of P. sojae populations, identification of more novel Rps genes is needed. In this study, phenotypic data from previous studies of 16 panels of plant introductions (PIs) were analyzed. Panels 1 and 2 consisted of 448 Glycine max and 520 G. soja, which had been evaluated for Rps gene response with a combination of P. sojae isolates. Panels 3 and 4 consisted of 429 and 460 G. max PIs, respectively, which had been evaluated using individual P. sojae isolates with complex virulence pathotypes. Finally, Panels 5-16 (376 G. max PIs) consisted of data deposited in the USDA Soybean Germplasm Collection from evaluations with 12 races of P. sojae. Using these panels, genome-wide association (GWA) analyses were carried out by combining phenotypic and SoySNP50K genotypic data. GWA models identified two, two, six, and seven novel Rps loci with Panels 1, 2, 3, and 4, respectively. A total of 58 novel Rps loci were identified using Panels 5-16. Genetic and phenotypic dissection of these loci may lead to the characterization of novel Rps genes that can be effectively deployed in new soybean cultivars against diverse P. sojae populations.

A transcriptional regulatory network of Rsv3-mediated extreme resistance against Soybean mosaic virus.

Resistance genes are an effective means for disease control in plants. They predominantly function by inducing a hypersensitive reaction, which results in localized cell death restricting pathogen spread. Some resistance genes elicit an atypical response, termed extreme resistance, where resistance is not associated with a hypersensitive reaction and its standard defense responses. Unlike hypersensitive reaction, the molecular regulatory mechanism(s) underlying extreme resistance is largely unexplored. One of the few known, naturally occurring, instances of extreme resistance is resistance derived from the soybean Rsv3 gene, which confers resistance against the most virulent Soybean mosaic virus strains. To discern the regulatory mechanism underlying Rsv3-mediated extreme resistance, we generated a gene regulatory network using transcriptomic data from time course comparisons of Soybean mosaic virus-G7-inoculated resistant (L29, Rsv3-genotype) and susceptible (Williams82, rsv3-genotype) soybean cultivars. Our results show Rsv3 begins mounting a defense by 6 hpi via a complex phytohormone network, where abscisic acid, cytokinin, jasmonic acid, and salicylic acid pathways are suppressed. We identified putative regulatory interactions between transcription factors and genes in phytohormone regulatory pathways, which is consistent with the demonstrated involvement of these pathways in Rsv3-mediated resistance. One such transcription factor identified as a putative transcriptional regulator was MYC2 encoded by Glyma.07G051500. Known as a master regulator of abscisic acid and jasmonic acid signaling, MYC2 specifically recognizes the G-box motif ("CACGTG"), which was significantly enriched in our data among differentially expressed genes implicated in abscisic acid- and jasmonic acid-related activities. This suggests an important role for Glyma.07G051500 in abscisic acid- and jasmonic acid-derived defense signaling in Rsv3. Resultantly, the findings from our network offer insights into genes and biological pathways underlying the molecular defense mechanism of Rsv3-mediated extreme resistance against Soybean mosaic virus. The computational pipeline used to reconstruct the gene regulatory network in this study is freely available at https://github.com/LiLabAtVT/rsv3-network.

Soybean mosaic virus: a successful potyvirus with a wide distribution but restricted natural host range.

TAXONOMY: Soybean mosaic virus (SMV) is a species within the genus Potyvirus, family Potyviridae, which includes almost one-quarter of all known plant RNA viruses affecting agriculturally important plants. The Potyvirus genus is the largest of all genera of plant RNA viruses with 160 species. PARTICLE: The filamentous particles of SMV, typical of potyviruses, are about 7500 Å long and 120 Å in diameter with a central hole of about 15 Å in diameter. Coat protein residues are arranged in helices of about 34 Å pitch having slightly less than nine subunits per turn. GENOME: The SMV genome consists of a single-stranded, positive-sense, polyadenylated RNA of approximately 9.6 kb with a virus-encoded protein (VPg) linked at the 5' terminus. The genomic RNA contains a single large open reading frame (ORF). The polypeptide produced from the large ORF is processed proteolytically by three viral-encoded proteinases to yield about 10 functional proteins. A small ORF, partially overlapping the P3 cistron, pipo, is encoded as a fusion protein in the N-terminus of P3 (P3N + PIPO). BIOLOGICAL PROPERTIES: SMV's host range is restricted mostly to two plant species of a single genus: Glycine max (cultivated soybean) and G. soja (wild soybean). SMV is transmitted by aphids non-persistently and by seeds. The variability of SMV is recognized by reactions on cultivars with dominant resistance (R) genes. Recessive resistance genes are not known. GEOGRAPHICAL DISTRIBUTION AND ECONOMIC IMPORTANCE: As a consequence of its seed transmissibility, SMV is present in all soybean-growing areas of the world. SMV infections can reduce significantly seed quantity and quality (e.g. mottled seed coats, reduced seed size and viability, and altered chemical composition). CONTROL: The most effective means of managing losses from SMV are the planting of virus-free seeds and cultivars containing single or multiple R genes. KEY ATTRACTIONS: The interactions of SMV with soybean genotypes containing different dominant R genes and an understanding of the functional role(s) of SMV-encoded proteins in virulence, transmission and pathogenicity have been investigated intensively. The SMV-soybean pathosystem has become an excellent model for the examination of the genetics and genomics of a uniquely complex gene-for-gene resistance model in a crop of worldwide importance.

Inference of Transcription Regulatory Network in Low Phytic Acid Soybean Seeds.

A dominant loss of function mutation in myo-inositol phosphate synthase (MIPS) gene and recessive loss of function mutations in two multidrug resistant protein type-ABC transporter genes not only reduce the seed phytic acid levels in soybean, but also affect the pathways associated with seed development, ultimately resulting in low emergence. To understand the regulatory mechanisms and identify key genes that intervene in the seed development process in low phytic acid crops, we performed computational inference of gene regulatory networks in low and normal phytic acid soybeans using a time course transcriptomic data and multiple network inference algorithms. We identified a set of putative candidate transcription factors and their regulatory interactions with genes that have functions in myo-inositol biosynthesis, auxin-ABA signaling, and seed dormancy. We evaluated the performance of our unsupervised network inference method by comparing the predicted regulatory network with published regulatory interactions in Arabidopsis. Some contrasting regulatory interactions were observed in low phytic acid mutants compared to non-mutant lines. These findings provide important hypotheses on expression regulation of myo-inositol metabolism and phytohormone signaling in developing low phytic acid soybeans. The computational pipeline used for unsupervised network learning in this study is provided as open source software and is freely available at https://lilabatvt.github.io/LPANetwork/.

Candidate Gene Sequence Analyses toward Identifying -Type Resistance to.

is one of three genetic loci conferring strain-specific resistance to (SMV). The locus has been mapped to a 154-kb region on chromosome 14, containing a cluster of five nucleotide-binding leucine-rich repeat (NB-LRR) resistance genes. High sequence similarity between the candidate genes challenges fine mapping of the locus. Among the five, Glyma14g38533 showed the highest transcript abundance in 1 to 3 h of SMV-G7 inoculation. Comparative sequence analyses were conducted with the five candidate NB-LRR genes from susceptible (-type) soybean [ (L.) Merr.] cultivar Williams 82, resistant (-type) cultivar Hwangkeum, and resistant lines L29 and RRR. Sequence comparisons revealed that Glyma14g38533 had far more polymorphisms than the other candidate genes. Interestingly, Glyma14g38533 gene from -type lines exhibited 150 single-nucleotide polymorphism (SNP and six insertion-deletion (InDel) markers relative to -type line, Furthermore, the polymorphisms identified in three -type lines were highly conserved. Several polymorphisms were validated in 18 -type resistant and six -type susceptible lines and were found associated with their disease response. The majority of the polymorphisms were located in LRR domain encoding region, which is involved in pathogen recognition via protein-protein interactions. These findings associating Glyma14g38533 with -type resistance to SMV suggest it is the most likely candidate gene for .

Identification of haplotypes at the Rsv4 genomic region in soybean associated with durable resistance to soybean mosaic virus.

KEY MESSAGE: Discovery of new germplasm sources and identification of haplotypes for the durable Soybean mosaic virus resistance gene, Rsv 4, provide novel resources for map-based cloning and genetic improvement efforts in soybean. The Soybean mosaic virus (SMV) resistance locus Rsv4 is of interest because it provides a durable type of resistance in soybean [Glycine max (L.) Merr.]. To better understand its molecular basis, we used a population of 309 BC3F2 individuals to fine-map Rsv4 to a ~120 kb interval and leveraged this genetic information in a second study to identify accessions 'Haman' and 'Ilpumgeomjeong' as new sources of Rsv4. These two accessions along with three other Rsv4 and 14 rsv4 accessions were used to examine the patterns of nucleotide diversity at the Rsv4 region based on high-depth resequencing data. Through a targeted association analysis of these 19 accessions within the ~120 kb interval, a cluster of four intergenic single-nucleotide polymorphisms (SNPs) was found to perfectly associate with SMV resistance. Interestingly, this ~120 kb interval did not contain any genes similar to previously characterized dominant disease resistance genes. Therefore, a haplotype analysis was used to further resolve the association signal to a ~94 kb region, which also resulted in the identification of at least two Rsv4 haplotypes. A haplotype phylogenetic analysis of this region suggests that the Rsv4 locus in G. max is recently introgressed from G. soja. This integrated study provides a strong foundation for efforts focused on the cloning of this durable virus resistance gene and marker-assisted selection of Rsv4-mediated SMV resistance in soybean breeding programs.

The HC-Pro and P3 cistrons of an avirulent Soybean mosaic virus are recognized by different resistance genes at the complex Rsv1 locus.

The complex Rsv1 locus in soybean plant introduction (PI) 'PI96983' confers extreme resistance (ER) against Soybean mosaic virus (SMV) strain N but not SMV-G7 and SMV-G7d. Both the SMV helper-component proteinase (HC-Pro) and P3 cistrons can serve as avirulence factors recognized by Rsv1. To understand the genetics underlying recognition of the two cistrons, we have utilized two soybean lines (L800 and L943) derived from crosses between PI96983 (Rsv1) and Lee68 (rsv1) with distinct recombination events within the Rsv1 locus. L800 contains a single PI96983-derived member (3gG2) of an Rsv1-associated subfamily of nucleotide-binding leucine-rich repeat (NB-LRR) genes. In contrast, although L943 lacks 3gG2, it contains a suite of five other NB-LRR genes belonging to the same family. L800 confers ER against SMV-N whereas L943 allows limited replication at the inoculation site. SMV-N-derived chimeras containing HC-Pro from SMV-G7 or SMV-G7d gained virulence on L943 but not on L800 whereas those with P3 replacement gained virulence on L800 but not on L943. In reciprocal experiments, SMV-G7- and SMV-G7d-derived chimeras with HC-Pro replacement from SMV-N lost virulence on L943 but retained virulence on L800 whereas those with P3 replacement lost virulence on L800 while remaining virulent on L943. These data demonstrate that distinct resistance genes at the Rsv1 locus, likely belonging to the NB-LRR class, mediate recognition of HC-Pro and P3.

Evolution of a complex disease resistance gene cluster in diploid Phaseolus and tetraploid Glycine.

We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.

Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique.

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species.

Importance of epistasis as the genetic basis of heterosis in an elite rice hybrid.

The genetic basis of heterosis was investigated in an elite rice hybrid by using a molecular linkage map with 150 segregating loci covering the entire rice genome. Data for yield and three traits that were components of yield were collected over 2 years from replicated field trials of 250 F(2:3) families. Genotypic variations explained from about 50% to more than 80% of the total variation. Interactions between genotypes and years were small compared with the main effects. A total of 32 quantitative trait loci (QTLs) were detected for the four traits; 12 were observed in both years and the remaining 20 were detected in only one year. Overdominance was observed for most of the QTLs for yield and also for a few QTLs for the component traits. Correlations between marker heterozygosity and trait expression were low, indicating that the overall heterozygosity made little contribution to heterosis. Digenic interactions, including additive by additive, additive by dominance, and dominance by dominance, were frequent and widespread in this population. The interactions involved large numbers of marker loci, most of which individually were not detectable on single-locus basis; many interactions among loci were detected in both years. The results provide strong evidence that epistasis plays a major role as the genetic basis of heterosis.

Copia-like retrotransposons in rice: sequence heterogeneity, species distribution and chromosomal locations.

Degenerated oligonucleotide primers were used to amplify, clone, and analyze sequence heterogeneity and chromosomal distribution of 23 PCR fragments corresponding to the reverse transcriptase domain of copia-like retrotransposons in rice. Of the 23 fragments 22 could be aligned by their deduced amino acid sequences and were divided into 6 groups according to the phylogenetic and Southern blot analyses. Amino acid sequence differences among the 22 aligned fragments ranged from 1 to 64%. Southern blot analysis of 10 rice accessions including indica, japonica and common wild rice, using these 23 fragments as probes, showed that copia-like retrotransposons were present in moderate to high copy numbers in all the rice genome although the exact copy number cannot be determined. The major difference revealed by southern analysis is a differentiation between the four indica varieties as one group and the four japonica varieties and the two wild rice accessions as another group. Polymorphisms were also detected among the indica and japonica varieties by major bands and repeatable minor bands. Five hybridization bands were mapped to chromosomes 3, 4, 8, and 9, respectively. All the five bands were inherited in a dominant Mendelian fashion and were not allelic with each other, indicating that the same element did not reside on the same location in different rice accessions. No transcript of the copia-like reverse transcriptase was detected on northern blot. The results suggest that the sequence heterogeneity and distributional variability of retrotransposons may be one of contributory factors causing genetic diversity in rice.

Amplified fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis.

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F2 segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.

Molecular marker heterozygosity and hybrid performance in indica and japonica rice.

An essential assumption underlying markerbased prediction of hybrid performance is a strong linear correlation between molecular marker heterozygosity and hybrid performance or heterosis. This study was intended to investigate the extent of the correlations between molecular marker heterozygosity and hybrid performance in crosses involving two sets of rice materials, 9 indica and 11 japonica varieties. These materials represent a broad spectrum of the cultivated rice gene pool including landraces, primitive cultivars, historically important cultivars, modern elite cultivars and parents of superior hybrids. Varieties within each set were intermated in all possible nonreciprocal pairs resulting in 36 crosses in the indica set and 55 in the japonica set. The F1s and their parents, 111 entries in total, were examined for performance of seven traits in a replicated field trial. The parents were surveyed for polymorphisms using 96 RFLP and ten SSR markers selected at regular intervals from a published molecular marker linkage map. Molecular marker genotypes of the F1 hybrids were deduced from the parental genotypes. The analysis showed that, with very few exceptions, correlations in the indica dataset were higher than in that of their japonica counterparts. Among the seven traits analyzed, plant height showed the highest correlation between heterozygosity and hybrid performance and heteorsis in both indica and japonica datasets. Correlations were low to intermediate between hybrid performance and heterozygosity (both general and specific) in yield and yield component traits in both indica and japonica sets, and also low to intermediate between specific heterozygosity and heterosis in the indica set, whereas very little correlation was detected between heterosis and heterozygosity (either general or specific) in the japonica set. In comparison to the results from our previous studies, we concluded that the relationship between molecular marker heterozygosity and heterosis is variable, depending on the genetic materials used in the study, the diversity of rice germplasms and the complexity of the genetic basis of heterosis.

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.

Using bulked extremes and recessive class to map genes for photoperiod-sensitive genic male sterility in rice.

Photoperiod-sensitive genic male sterile (PS-GMS) rice has a number of desirable characteristics for hybrid rice production. In this study we made use of a published rice genetic linkage map to determine the locations of PSGMS genes and we have characterized the effects of these genes on sterility by using molecular markers. A two-step approach was designed for mapping the genes: (i) identifying possible PSGMS gene-containing chromosome regions with bulked DNA from extreme fertile and extreme sterile plants of a very large F2 population and (ii) determining the map locations of the genes in extreme sterile individuals. We show that this mapping method is much more cost effective and statistically efficient than using a random sample of an F2 population. We identified two chromosomal regions each containing a PSGMS locus, one designated pms1 on chromosome 7 and one designated pms2 on chromosome 3. The existence of these two loci was confirmed by a large sample assay and with data on rationing progenies of the F2 plants. A marker-based analysis shows that the effect of pms1 is 2-3 times larger than that of pms2 and that dominance is almost complete at both loci. Implications in the breeding of PSGMS rice lines are discussed.

Extraordinarily polymorphic microsatellite DNA in barley: species diversity, chromosomal locations, and population dynamics.

This study was undertaken to assess the extent of genetic variation in barley simple sequence repeats (SSRs) and to study the evolutionary dynamics of SSR alleles. SSR polymorphisms were resolved by the polymerase chain reaction with four pairs of primers. In total, 71 variants were observed in a sample of 207 accessions of wild and cultivated barley. Analyses of wheat-barley addition lines and barley doubled haploids identified these variants (alleles) with four loci, each located on a different chromosome. The numbers of alleles detected at a locus corresponded to the number of nucleotide repeats in the microsatellite sequences. The numbers of alleles at two loci were 28 and 37; to our knowledge these are the largest numbers of alleles for single Mendelian loci reported in plants. Three alleles were resolved by each of the other two loci. Allelic diversity was greater in wild than in cultivated barley and surveys of two generations (F8 and F53) of Composite Cross II, an experimental population of cultivated barley, showed that few of the alleles present in the 28 parents survived into generation F53, whereas some infrequent alleles reached high frequencies. Such changes in frequency indicate that the chromosomal segments marked by the SSR alleles are under the influence of natural selection. The SSR variants allow specific DNA sequences to be followed through generations. Thus, the great resolving power of SSR assays may provide clues regarding the precise targets of natural and man-directed selection.

RFLP markers linked to the durable stem rust resistance gene Rpg1 in barley.

The gene, Rpg1, conferring stable resistance in barley to the wheat stem rust pathogen (Puccinia graminis f. sp. tritici) was mapped using two doubled haploid populations. Rpg1 mapped to the extreme subteleomeric region of barley chromosome 1P 0.3 and 1.1 cM proximal from the molecular markers ABG704 and plastocyanin (Plc), respectively, and 2.2 cM distal from MWG036B. The closest marker, ABG704, was sequenced and PCR-based markers were developed.