Development and mapping of microsatellite (SSR) markers in wheat.

Microsatellite DNA markers are consistently found to be more informative than other classes of markers in hexaploid wheat. The objectives of this research were to develop new primers flanking wheat microsatellites and to position the associated loci on the wheat genome map by genetic linkage mapping in the ITMI W7984 x Opata85 recombinant inbred line (RIL) population and/or by physical mapping with cytogenetic stocks. We observed that the efficiency of marker development could be increased in wheat by creating libraries from sheared rather than enzyme-digested DNA fragments for microsatellite screening, by focusing on microsatellites with the [ATT/TAA]n motif, and by adding an untemplated G-C clamp to the 5'-end of primers. A total of 540 microsatellite-flanking primer pairs were developed, tested, and annotated from random genomic libraries. Primer pairs and associated loci were assigned identifiers prefixed with BARC (the acronym for the USDA-ARS Beltsville Agricultural Research Center) or Xbarc, respectively. A subset of 315 primer sets was used to map 347 loci. One hundred and twenty-five loci were localized by physical mapping alone. Of the 222 loci mapped with the ITMI population, 126 were also physically mapped. Considering all mapped loci, 126, 125, and 96 mapped to the A, B, and D genomes, respectively. Twenty-three of the new loci were positioned in gaps larger than 10 cM in the map based on pre-existing markers, and 14 mapped to the ends of chromosomes. The length of the linkage map was extended by 80.7 cM. Map positions were consistent for 111 of the 126 loci positioned by both genetic and physical mapping. The majority of the 15 discrepancies between genetic and physical mapping involved chromosome group 5.

Single-nucleotide polymorphisms in soybean.

Single-nucleotide polymorphisms (SNPs) provide an abundant source of DNA polymorphisms in a number of eukaryotic species. Information on the frequency, nature, and distribution of SNPs in plant genomes is limited. Thus, our objectives were (1) to determine SNP frequency in coding and noncoding soybean (Glycine max L. Merr.) DNA sequence amplified from genomic DNA using PCR primers designed to complete genes, cDNAs, and random genomic sequence; (2) to characterize haplotype variation in these sequences; and (3) to provide initial estimates of linkage disequilibrium (LD) in soybean. Approximately 28.7 kbp of coding sequence, 37.9 kbp of noncoding perigenic DNA, and 9.7 kbp of random noncoding genomic DNA were sequenced in each of 25 diverse soybean genotypes. Over the >76 kbp, mean nucleotide diversity expressed as Watterson's theta was 0.00097. Nucleotide diversity was 0.00053 and 0.00111 in coding and in noncoding perigenic DNA, respectively, lower than estimates in the autogamous model species Arabidopsis thaliana. Haplotype analysis of SNP-containing fragments revealed a deficiency of haplotypes vs. the number that would be anticipated at linkage equilibrium. In 49 fragments with three or more SNPs, five haplotypes were present in one fragment while four or less were present in the remaining 48, thereby supporting the suggestion of relatively limited genetic variation in cultivated soybean. Squared allele-frequency correlations (r(2)) among haplotypes at 54 loci with two or more SNPs indicated low genome-wide LD. The low level of LD and the limited haplotype diversity suggested that the genome of any given soybean accession is a mosaic of three or four haplotypes. To facilitate SNP discovery and the development of a transcript map, subsets of four to six diverse genotypes, whose sequence analysis would permit the discovery of at least 75% of all SNPs present in the 25 genotypes as well as 90% of the common (frequency >0.10) SNPs, were identified.

Characterization of trinucleotide SSR motifs in wheat.

Length differences among trinucleotide-based microsatellite alleles can be more easily detected and frequently produce fewer "stutter bands" as compared to dinucleotide-based microsatellite markers. Our objective was to determine which trinucleotide motif(s) would be the most-polymorphic and abundant source of trinucleotide microsatellite markers in wheat ( Triticum aestivumL.). Four genomic libraries of cultivar 'Chinese Spring' were screened with nine trinucleotide probes. Based on the screening of 28550 clones, the occurrences of (CTT/GAA) (n), (GGA/CCT) (n), (TAA/ATT) (n), (CAA/GTT) (n), (GGT/CCA) (n), (CAT/GTA) (n), (CGA/GCT) (n), (CTA/GAT) (n), and (CGT/GCA) (n) repeats were estimated to be 5.4x10(4), 3.5x10(4), 3.2x10(4), 1.2x10(4), 6.3x10(3), 4.9x10(3), 4.5x10(3), 4.5x10(3) and 3.6x10(3), i.e., once every 293 kbp, 456 kbp, 500 kbp, 1.3 Mbp, 2.6 Mbp, 3.2 Mbp, 3.6 Mbp, 3.6 Mbp and 4.5 Mbp in the wheat genome, respectively. Of 236 clones selected for sequencing, 38 (93%) (TAA/ATT) (n), 30 (43%) (CTT/GAA) (n), 16 (59%) (CAA/GTT) (n), 3 (27%) (CAT/GTA) (n) and 2 (4%) (GGA/CCT) (n) clones contained microsatellites with eight or more perfect repeats. From these data, 29, 27 and 16 PCR primer sets were designed and tested to the (TAA/ATT) (n), (CTT/GAA) (n) and (CAA/GTT) (n) microsatellites, respectively. A total of 12 (41.4%) primers designed to (TAA/ATT) (n), four (14.8%) to (CTT/GAA) (n), and two (12.5%) to (CAA/GTT) (n) resulted in polymorphic markers. The results indicated that (TAA/ATT) (n) microsatellites would provide the most-abundant and the most-polymorphic source of trinucleotide microsatellite markers in wheat.