Nearly 900 SSRs (simple sequence repeats) were identified among 15,000 ESTs (expressed sequence tags) belonging to bread wheat ( Triticum aestivumL.). The SSRs were defined by their minimum length, which ranged from 14 to 21 bp. The maximum length ranged from 24 to 87 bp depending upon the length of the repeat unit itself (1-7 bp). The average density of SSRs was one SSR per 9.2 kb of EST sequence screened. The trinucleotide repeats were the most abundant SSRs detected. As a representative sample, 78 primer pairs were designed, which were also used to screen the dbEST entries for Hordeum vulgare and Triticum tauschii (donor of the D-genome of cultivated wheat) using a cut-off E (expectation) value of 0.01. On the basis of in silico analysis, up to 55.12% of the primer pairs exhibited transferability from Triticum to Hordeum, indicating that the sequences flanking the SSRs are not only conserved within a single genus but also between related genera in Poaceae. Primer pairs for the 78 SSRs were synthesized and used successfully for the study of (1) their transferability to 18 related wild species and five cereal species (barley, oat, rye, rice and maize); and (2) polymorphism between the parents of four mapping populations available with us. A subset of 20 EST-SSR primers was also used to assess genetic diversity in a collection of 52 elite exotic wheat genotypes. This was done with a view to compare their utility relative to other molecular markers (gSSRs, AFLPs, and SAMPL) previously used by us for the same purpose with the same set of 52 bread wheat genotypes. Although only a low level of polymorphism was detected, relative to that observed with genomic SSRs, the study suggested that EST-SSRs can be successfully used for a variety of purposes, and may actually prove superior to SSR markers extracted from genomic libraries for diversity estimation and transferability.

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QTL interval mapping for grain protein content (GPC) in bread wheat was conducted for the first time, using a framework map based on a mapping population, which was available in the form of 100 recombinant inbred lines (RILs). The data on GPC for QTL mapping was recorded by growing the RILs in five different environments representing three wheat growing locations from Northern India; one of these locations was repeated for 3 years. Distribution of GPC values followed normal distributions in all the environments, which could be explained by significant g x e interactions observed through analyses of variances, which also gave significant effects due to genotypes and environments. Thirteen (13) QTLs were identified in individual environments following three methods (single-marker analysis or SMA, simple interval mapping or SIM and composite interval mapping or CIM) and using LOD scores that ranged from 2.5 to 6.5. Threshold LOD scores (ranging from 3.05 to 3.57), worked out and used in each case, however, detected only seven of the above 13 QTLs. Only four (QGpc.ccsu-2B.1; QGpc.ccsu-2D.1; QGpc.ccsu-3D.1 and QGpc.ccsu-7A.1) of these QTLs were identified either in more than one location or following one more method other than CIM; another QTL (QGpc.ccsu-3D.2), which was identified using means for all the environments, was also considered to be important. These five QTLs have been recommended for marker-assisted selection (MAS). The QTLs identified as above were also validated using ten NILs derived from three crosses. Five of the ten NILs possessed 38 introgressed segments from 16 chromosomes and carried 42 of the 173 markers that were mapped. All the seven QTLs were associated with one or more of the markers carried by the above introgressed segments, thus validating the corresponding markers. More markers associated with many more QTLs to be identified should become available in the future by effective MAS for GPC improvement.

Barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS) is an effective strategy for rapid functional analysis of genes in wheat leaves, but its utility to transiently express genes, and silencing in other tissues including root, flower, and developing grains, has not been demonstrated in monocots. We monitored green fluorescent protein (GFP) expression to demonstrate the utility of BSMV as a transient expression vector and silenced genes in various wheat tissues to expand VIGS utility to characterize tissue-specific genes. An antisense construct designed for coronatine insensitive1 (COI1) showed an 85% decrease in COI1 transcript level in roots accompanied by a 26% reduction in root length. Similarly, silencing of seed-specific granule-bound starch synthase by antisense and hairpin constructs resulted in up to 82% reduction in amylose content of the developing grains. VIGS of meiosis-specific genes demonstrated by silencing wheat homologue of disrupted meiosis cDNA1 (DMC1) by an antisense construct resulted in a 75-80% reduction in DMC1 transcript level accompanied by an average of 37.2 univalents at metaphase I. The virus-based transient GFP expression was observed in the leaf, phloem, and root cortex at 10-17 days post-inoculation. A novel observation was made that 8-11% of the first selfed generation progeny showed VIGS inheritance and that this proportion increased to 53-72% in the second and to 90-100% in the third generations. No viral symptoms were observed in the progeny, making it possible to study agronomic traits by VIGS. VIGS inheritance is particularly useful to study genes expressing during seed germination or other stages of early plant growth.