Characterization and analysis of simple sequence repeat (SSR) loci in seashore paspalum (Paspalum vaginatum Swartz)

A size-fractionated TaqI genomic library of seashore paspalum (Paspalum vaginatum Swartz) was screened for the presence of (GA) _n and (CA) _n simple sequence repeats (SSRs). A total of 54 clones with a positive signal were detected among 13,000 clones screened. Forty-seven clones having repeats of n 3 were identified, of which 85% were perfect, 13% were imperfect and 2% were compound repeat sequences. Five of ten primer pairs synthesized to amplify selected loci resulted in a product in the expected size range and were subsequently used to examine SSR polymorphisms among 46 ecotypes of P. vaginatum. The number of alleles resolved on agarose or polyacrylamide gels were similar and ranged from 6 to 16 with an average of 14 per locus. Phenetic analysis of SSR polymorphisms revealed genetic relationships among the P. vaginatum ecotypes that were in general agreement with relationships determined previously by RAPD analysis of the same plant materials. Further screening of the genomic library did not identify (AT) _n , trimeric or tetrameric repeats. Hybridization of an (ATT)₈ oligonucleotide probe to genomic DNA isolated from I. batatas, E. coli, Citrullis lanatus and P. vaginatum suggested that the P. vaginatum genome contained significantly fewer ATT repeats than either the I. batatas or C. lanatus genome.     

Characterization of genetic identities and relationships of Brassica oleracea L. via a random amplified polymorphic DNA assay

Summary Effective conservation and the use of plant genetic resources are essential for future agricultural progress. Critical to this conservation effort is the development of genetic markers which not only distinguish individuals and accessions but also reflect the inherent variation and genetic relationships among collection holdings. We have examined the applicability of the random amplified polymorphic DNA (RAPD) assay for quick, cost-effective, and reliable use in addressing these needs in relation to collection organization and management. Twenty-five decamer oligonucleotide primers were screened individually with a test array composed of individuals representing a range of genetic relationships in Brassica oleracea L. (vegetable and forage cole crops). Over 140 reproducible, polymorphic fragments were generated for study. Each individual of the test array exhibited a unique molecular genotype and composites specific for accessions and botanical varieties could be established. An analysis of similarity based on amplified DNA fragments reflected the known genetic relationships among the selected entries. These results demonstrated that RAPD markers can be of great value in gene bank management for purposes of identification, measurement of variation, and establishment of genetic similarity at the intraspecific level.     

Genetic and genomic resources of sorghum to connect genotype with phenotype in contrasting environments

With the recent development of genomic resources and high‐throughput phenotyping platforms, the 21st century is primed for major breakthroughs in the discovery, understanding and utilization of plant genetic variation. Significant advances in agriculture remain at the forefront to increase crop production and quality to satisfy the global food demand in a changing climate all while reducing the environmental impacts of the world's food production. Sorghum, a resilient C₄ grain and grass important for food and energy production, is being extensively dissected genetically and phenomically to help connect the relationship between genetic and phenotypic variation. Unlike genetically modified crops such as corn or soybean, sorghum improvement has relied heavily on public research; thus, many of the genetic resources serve a dual purpose for both academic and commercial pursuits. Genetic and genomic resources not only provide the foundation to identify and understand the genes underlying variation, but also serve as novel sources of genetic and phenotypic diversity in plant breeding programs. To better disseminate the collective information of this community, we discuss: (i) the genomic resources of sorghum that are at the disposal of the research community; (ii) the suite of sorghum traits as potential targets for increasing productivity in contrasting environments; and (iii) the prospective approaches and technologies that will help to dissect the genotype–phenotype relationship as well as those that will apply foundational knowledge for sorghum improvement.     

Meta-analysis identifies pleiotropic loci controlling phenotypic trade-offs in sorghum

Community association populations are composed of phenotypically and genetically diverse accessions. Once these populations are genotyped, the resulting marker data can be reused by different groups investigating the genetic basis of different traits. Because the same genotypes are observed and scored for a wide range of traits in different environments, these populations represent a unique resource to investigate pleiotropy. Here, we assembled a set of 234 separate trait datasets for the Sorghum Association Panel, a group of 406 sorghum genotypes widely employed by the sorghum genetics community. Comparison of genome-wide association studies (GWAS) conducted with two independently generated marker sets for this population demonstrate that existing genetic marker sets do not saturate the genome and likely capture only 35–43% of potentially detectable loci controlling variation for traits scored in this population. While limited evidence for pleiotropy was apparent in cross-GWAS comparisons, a multivariate adaptive shrinkage approach recovered both known pleiotropic effects of existing loci and new pleiotropic effects, particularly significant impacts of known dwarfing genes on root architecture. In addition, we identified new loci with pleiotropic effects consistent with known trade-offs in sorghum development. These results demonstrate the potential for mining existing trait datasets from widely used community association populations to enable new discoveries from existing trait datasets as new, denser genetic marker datasets are generated for existing community association populations.     

Microsatellite identification and characterization in peanut (<Emphasis Type="Italic">A. hypogaea</Emphasis> L.)

A major constraint to the application of biotechnology to the improvement of the allotetraploid peanut, or groundnut (Arachis hypogaea L.), has been the paucity of polymorphism among germplasm lines using biochemical (seed proteins, isozymes) and DNA markers (RFLPs and RAPDs). Six sequence-tagged microsatellite (STMS) markers were previously available that revealed polymorphism in cultivated peanut. Here, we identify and characterize 110 STMS markers that reveal genetic variation in a diverse array of 24 peanut landraces. The simple-sequence repeats (SSRs) were identified with a probe of two 27,648-clone genomic libraries: one constructed using PstI and the other using Sau3AI/BamHI. The most frequent, repeat motifs identified were ATT and GA, which represented 29% and 28%, respectively, of all SSRs identified. These were followed by AT, CTT, and GT. Of the amplifiable primers, 81% of ATT and 70.8% of GA repeats were polymorphic in the cultivated peanut test array. The repeat motif AT showed the maximum number of alleles per locus (5.7). Motifs ATT, GT, and GA had a mean number of alleles per locus of 4.8, 3.8, and 3.6, respectively. The high mean number of alleles per polymorphic locus, combined with their relative frequency in the genome and amenability to probing, make ATT and GA the most useful and appropriate motifs to target to generate further SSR markers for peanut.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by J.S. Heslop-Harrison     

Population structure and its effect on haplotype diversity and linkage disequilibrium surrounding the xa5 locus of rice (Oryza sativa L.)

To assess the usefulness of linkage disequilibrium mapping in an autogamous, domesticated species, we have characterized linkage disequilibrium in the candidate region for xa5, a recessive gene conferring race-specific resistance to bacterial blight in rice. This trait and locus have good mapping information, a tractable phenotype, and available sequence data, but no cloned gene. We sampled 13 short segments from the 70-kb candidate region in 114 accessions of Oryza sativa. Five additional segments were sequenced from the adjacent 45-kb region in resistant accessions to estimate the distance at which linkage disequilibrium decays. The data show significant linkage disequilibrium between sites 100 kb apart. The presence of the xa5 resistant reaction in two ecotypes and in accessions with different haplotypes in the candidate region may indicate multiple origins or genetic heterogeneity for resistance. In addition, genetic differentiation between ecotypes emphasizes the need for controlling for population structure in the design of linkage disequilibrium studies in rice.     

Solid-phase PCR in microwells: effects of linker length and composition on tethering, hybridization, and extension

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells are suitable for enzymatic detection of immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer. The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.     

Microsatellites in Zea - variability,patterns of mutations,and use for evolutionary studies

To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees. Received: 10 February 2001 / Accepted: 21 May 2001     

Characterization of RFLP probe sequences for gene discovery and SSR development in Sorghum bicolor (L.) Moench

In this study, we collected and analyzed DNA sequence data for 789 previously mapped RFLP probes from Sorghum bicolor (L.) Moench. DNA sequences, comprising 894 non-redundant contigs and end sequences, were searched against three GenBank databases, nucleotide (nt), protein (nr) and EST (dbEST), using BLAST algorithms. Matching ESTs were also searched against nt and nr. Translated DNA sequences were then searched against the conserved domain database (CDD) to determine if functional domains/motifs were congruent with the proteins identified in previous searches. More than half (500/894 or 56%) of the query sequences had significant matches in at least one of the GenBank searches. Overall, proteins identified for 148 sequences (17%) were consistent among all searches, of which 66 sequences (7%) contained congruent coding domains. The RFLP probe sequences were also evaluated for the presence of simple sequence repeats (SSRs) and 60 SSRs were developed and assayed in an array of sorghum germplasm comprising inbreds, landraces and wild relatives. Overall, these SSR loci had lower levels of polymorphism ( D = 0.46, averaged over 51 polymorphic loci) compared with sorghum SSRs that were isolated by library hybridization screens ( D = 0.69, averaged over 38 polymorphic loci). This result was probably due to the relatively small proportion of di-nucleotide repeat-containing markers (42% of the total SSR loci) obtained from the DNA sequence data. These di-nucleotide markers also contained shorter repeat motifs than those isolated from genomic libraries. Based on BLAST results, 24 SSRs (40%) were located within, or near, previously annotated or hypothetical genes. We determined the location of 19 of these SSRs relative to putative coding regions. In general, SSRs located in coding regions were less polymorphic ( D = 0.07, averaged over three loci) than those from gene flanking regions, UTRs and introns ( D = 0.49, averaged over 16 loci). The sequence information and SSR loci generated through this study will be valuable for application to sorghum genetics and improvement, including gene discovery, marker-assisted selection, diversity and pedigree analyses, comparative mapping and evolutionary genetic studies.     

Simple sequence repeat marker diversity in cassava landraces: genetic diversity and differentiation in an asexually propagated crop

Cassava (Manihot esculenta) is an allogamous, vegetatively propagated, Neotropical crop that is also widely grown in tropical Africa and Southeast Asia. To elucidate genetic diversity and differentiation in the crop's primary and secondary centers of diversity, and the forces shaping them, SSR marker variation was assessed at 67 loci in 283 accessions of cassava landraces from Africa (Tanzania and Nigeria) and the Neotropics (Brazil, Colombia, Peru, Venezuela, Guatemala, Mexico and Argentina). Average gene diversity (i.e., genetic diversity) was high in all countries, with an average heterozygosity of 0.5358 ± 0.1184. Although the highest was found in Brazilian and Colombian accessions, genetic diversity in Neotropical and African materials is comparable. Despite the low level of differentiation [F_st(theta) = 0.091 ± 0.005] found among country samples, sufficient genetic distance (1-proportion of shared alleles) existed between individual genotypes to separate African from Neotropical accessions and to reveal a more pronounced substructure in the African landraces. Forces shaping differences in allele frequency at SSR loci and possibly counterbalancing successive founder effects involve probably spontaneous recombination, as assessed by parent-offspring relationships, and farmer-selection for adaptation.Communicated by H.C. Becker