Ju-Kyung Yu

Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley

BackgroundEarlier comparative maps between the genomes of rice (Oryza sativa L.), barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were linkage maps based on cDNA-RFLP markers. The low number of polymorphic RFLP markers has limited the development of dense genetic maps in wheat and the number of available anchor points in comparative maps. Higher density comparative maps using PCR-based anchor markers are necessary to better estimate the conservation of colinearity among cereal genomes. The purposes of this study were to characterize the proportion of transcribed DNA sequences containing simple sequence repeats (SSR or microsatellites) by length and motif for wheat, barley and rice and to determine in-silico rice genome locations for primer sets developed for wheat and barley Expressed Sequence Tags.ResultsThe proportions of SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) and motifs varied with the length of the SSRs within and among the three species, with trinucleotide SSRs being the most frequent. Distributions of genomic microsatellites (gSSRs), EST-derived microsatellites (EST-SSRs), and transcribed regions in the contiguous sequence of rice chromosome 1 were highly correlated. More than 13,000 primer pairs were developed for use by the cereal research community as potential markers in wheat, barley and rice.ConclusionTrinucleotide SSRs were the most common type in each of the species; however, the relative proportions of SSR types and motifs differed among rice, wheat, and barley. Genomic microsatellites were found to be primarily located in gene-rich regions of the rice genome. Microsatellite markers derived from the use of non-redundant EST-SSRs are an economic and efficient alternative to RFLP for comparative mapping in cereals.

EST derived SSR markers for comparative mapping in wheat and rice

Structural and functional relationships between the genomes of hexaploid wheat (Triticum aestivum L.) (2n=6x=42) and rice (Oryza sativa L.) (2n=2x=24) were evaluated using linkage maps supplemented with simple sequence repeat (SSR) loci obtained from publicly available expressed sequence tags (ESTs). EST-SSR markers were developed using two main strategies to design primers for each gene: (1) primer design for multiple species based on supercluster analysis, and (2) species-specific primer design. Amplification was more consistent using the species-specific primer design for each gene. Forty-four percent of the primers designed specifically for wheat sequences were successful in amplifying DNA from both species. Existing genetic linkage maps were enhanced for the wheat and rice genomes using orthologous loci amplified with 58 EST-SSR markers obtained from both wheat and rice ESTs. The PCR-based anchor loci identified by these EST-SSR markers support previous patterns of conservation between wheat and rice genomes; however, there was a high frequency of interrupted colinearity. In addition, multiple loci amplified by these primers made the comparative analysis more difficult. Enhanced comparative maps of wheat and rice provide a useful tool for interpreting and transferring molecular, genetic, and breeding information between these two important species. These EST-SSR markers are particularly useful for constructing comparative framework maps for different species, because they amplify closely related genes to provide anchor points across species.

Large-scale, cost-effective screening of PCR products in marker-assisted selection applications

A simple, PCR-based method has been developed for the rapid genotyping of large numbers of samples. The method involves a alkaline extraction of DNA from plant tissue using a slight modification of the procedure of Wang et al. (Nucleic Acids Res 21:4153–4154, 1993). Template DNA is amplified using allelespecific associated primers (ASAPs) which, at stringent annealing temperatures, generate only a single DNA fragment and only in those individuals possessing the appropriate allele. This approach eliminates the need to separate amplified DNA fragments by electrophoresis. Instead, samples processing the appropriate allele are identified by direct staining of DNA with ethidium bromide. Total technician time required for extraction, amplification and detection of 96 samples is about 4 h, and this time requirement can be reduced by automation. Excluding labor, cost per sample is less than

Impact of plant breeding on genetic diversity of the Canadian hard red spring wheat germplasm as revealed by EST-derived SSR markers

0.40. The method is tested using the codominant isozyme marker, alcohol dehydrogenase (Adh-1) gene in pea (Pisum sativum), and applied to the screening of photoperiod genes in common bean (Phaseolus vulgaris L.).

Transfer of simple sequence repeat (SSR) markers from major cereal crops to minor grass species for germplasm characterization and evaluation

Genetic diversity changes in wheat germplasm have been studied using different molecular markers, but little is known about the impact of plant breeding on the transcribed segments of the wheat genome. The objective of this study was to assess diversity changes in 75 Canadian hard red wheat cultivars released from 1845 to 2004 using 37 EST-derived microsatellite (eSSR) markers. These markers were derived from at least 19 transcribed sequences with putative functions assigned and sampled 17 wheat chromosomes. A total of 138 eSSR alleles was detected, and their allelic frequencies ranged from 0.01 to 0.99 with an average of 0.41. Allelic counts were significantly reduced at three loci for cultivars released after 1990. Sixteen alleles at 14 loci in pre-1910 cultivars were lost in cultivars released after 1990. The lost alleles had frequencies ranging from 0.03 to 0.17 and averaging 0.07. Partitioning the eSSR variation showed the four ancestral families accounted for 14.7% of the variation, followed by the six breeding periods with 12.8% and the eight breeding programs with 5.8%. A genetic shift was observed in the cultivars released over the six breeding periods, reflecting the various breeding efforts. These results illustrate the impact of the Canadian wheat breeding on the transcriptional segments of the wheat genome. These findings, along with those from genomic SSR markers, suggest the Canadian wheat breeding programs have reduced genetic diversity in the hard red spring wheat.

QTL mapping of agronomic traits in tef [ Eragrostis tef (Zucc) Trotter]

A major challenge for the molecular characterization and evaluation of minor grass species germplasm is the lack of sufficient DNA markers. A set of 210 simple sequence repeat (SSR) markers developed from major cereal crops (self-pollinated wheat and rice, mainly self-pollinated sorghum and out-crossing maize) were evaluated for their transferability to minor grass species (finger millet, Eleusine coracana ; seashore paspalum, Paspalum vaginatum ; and bermudagrass, Cynodon dactylon ). In total, 412 cross-species polymorphic amplicons were identified. Over half of the primers generated reproducible cross-species or cross-genus amplicons. The transfer rate of SSR markers was correlated with the phylogenetic relationship (or genetic relatedness) of these species. The average transfer rate of genomic SSR markers was different from the average transfer rate of expressed sequence tag (EST)-SSR markers. The level of polymorphism was significantly higher among species (67%) than within species (34%), and was related to the degree of out-crossing for each species. The level of polymorphism detected within species was 57% from self-incompatible species, 39% from out-crossing species and 20% from self-pollinated species. Genomic SSRs detected a higher level of polymorphism than EST-SSRs. The use of transferred polymorphic SSR markers for the characterization and evaluation of germplasm is discussed.

Development and mapping of EST-derived simple sequence repeat markers for hexaploid wheat

BackgroundTef [Eragrostis tef (Zucc.) Trotter] is the major cereal crop in Ethiopia. Tef is an allotetraploid with a base chromosome number of 10 (2n = 4× = 40) and a genome size of 730 Mbp. The goal of this study was to identify agronomically important quantitative trait loci (QTL) using recombinant inbred lines (RIL) derived from an inter-specific cross between E. tef and E. pilosa (30-5).ResultsTwenty-two yield-related and morphological traits were assessed across eight different locations in Ethiopia during the growing seasons of 1999 and 2000. Using composite interval mapping and a linkage map incorporating 192 loci, 99 QTLs were identified on 15 of the 21 linkage groups for 19 traits. Twelve QTLs on nine linkage groups were identified for grain yield. Clusters of more than five QTLs for various traits were identified on seven linkage groups. The largest cluster (10 QTLs) was identified on linkage group 8; eight of these QTLs were for yield or yield components, suggesting linkage or pleotrophic effects of loci. There were 15 two-way interactions of loci to detect potential epistasis identified and 75% of the interactions were derived from yield and shoot biomass. Thirty-one percent of the QTLs were observed in multiple environments; two yield QTLs were consistent across all agro-ecology zones. For 29.3% of the QTLs, the alleles from E. pilosa (30-5) had a beneficial effect.ConclusionThe extensive QTL data generated for tef in this study will provide a basis for initiating molecular breeding to improve agronomic traits in this staple food crop for the people of Ethiopia.