Abstract
BackgroundPearl millet [Pennisetum glaucum (L.) R. Br.] is a staple food and fodder crop of marginal agricultural lands of sub-Saharan Africa and the Indian subcontinent. It is also a summer forage crop in the southern USA, Australia and Latin America, and is the preferred mulch in Brazilian no-till soybean production systems. Use of molecular marker technology for pearl millet genetic improvement has been limited. Progress is hampered by insufficient numbers of PCR-compatible co-dominant markers that can be used readily in applied breeding programmes. Therefore, we sought to develop additional SSR markers for the pearl millet research community.ResultsA set of new pearl millet SSR markers were developed using available sequence information from 3520 expressed sequence tags (ESTs). After clustering, unigene sequences (2175 singlets and 317 contigs) were searched for the presence of SSRs. We detected 164 sequences containing SSRs (at least 14 bases in length), with a density of one per 1.75 kb of EST sequence. Di-nucleotide repeats were the most abundant followed by tri-nucleotide repeats. Ninety primer pairs were designed and tested for their ability to detect polymorphism across a panel of 11 pairs of pearl millet mapping population parental lines. Clear amplification products were obtained for 58 primer pairs. Of these, 15 were monomorphic across the panel. A subset of 21 polymorphic EST-SSRs and 6 recently developed genomic SSR markers were mapped using existing mapping populations. Linkage map positions of these EST-SSR were compared by homology search with mapped rice genomic sequences on the basis of pearl millet-rice synteny. Most new EST-SSR markers mapped to distal regions of linkage groups, often to previous gaps in these linkage maps. These new EST-SSRs are now are used by ICRISAT in pearl millet diversity assessment and marker-aided breeding programs.ConclusionThis study has demonstrated the potential of EST-derived SSR primer pairs in pearl millet. As reported for other crops, EST-derived SSRs provide a cost-saving marker development option in pearl millet. Resources developed in this study have added a sizeable number of useful SSRs to the existing repertoire of circa 100 genomic SSRs that were previously available to pearl millet researchers.
Highlights
The use of molecular marker technology for the genetic improvement of pearl millet has made some headway, and pearl millet has been elevated to the status of a molecular crop through a series of collaborative projects involving the John Innes Centre (JIC), ICRISAT and their partners supported by the Plant Sciences Research Programme of the UK's Department for International Development (DFID) [1], and the Generation Challenge Programme (GCP) of the Consultative Group on International Agricultural Research (CGIAR)
It should be noted that the frequency and density estimates in this study might not reflect the exact picture for pearl millet considering the limited number of expressed sequence tags (ESTs) sequences analysed
The frequency of SSR-containing ESTs was quite high in this initial sample of the expressed portion of the pearl millet genome, so it appears that this approach can add reasonable numbers of SSRs to the existing pearl millet SSR collection at very modest cost provided that 1) the sequence information is available freely as a result of other research programs, 2) care is taken to minimize redundancy, and 3) primer synthesis and testing is limited to only sequences flanking the most highly repeated di, tri, and tetra-nucleotide motifs
Summary
More than 600 molecular markers have been created and mapped for pearl millet, a consensus linkage map has been produced [3], and quantitative trait loci (QTL) for disease resistance [4,5,6,7,8], drought tolerance [9,10,11], flowering time and grain and stover yield [12], and ruminant nutritional quality of straw [13] have been mapped These genetic tools for marker-assisted breeding of pearl millet are in place and available for anyone to use in improving pearl millet hybrids and to extend the economic lifespan of elite hybrid parental lines. These markers are not considered suitable for large-scale genotyping applications in an applied plant breeding program
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