Fine mapping of a major terminal drought tolerance QTL in pearl millet

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Pearl millet [Pennisetum glaucum (L.) R. Br.] is the sixth most important global cereal crop (after rice, wheat, maize, barley and sorghum) grown as a rainfed grain and fodder crop in the hottest, driest regions of sub-Saharan Africa and the Indian subcontinent. It produces nutritious grain and is a major human food for people living in the semi-arid, low input, dryland agriculture regions of Africa and South Asia. Terminal drought stress is a major constraint to pearl millet productivity and its yield stability. A major QTL for terminal drought tolerance (DT) has been identified and validated on pearl millet on linkage group 2 (LG2) using two independent marker assisted selection programmes. However, the confidence interval for this QTL is still large (circa 30 cM) thus precluding its applied use in marker-assisted selection programmes. Fine mapping of this major DT-QTL, therefore, has been undertaken in pearl millet to improve its genetic resolution and to identify underlying candidate genes. Highly specialised genetic stocks (QTL-NILs, a high resolution cross (HRC) and an inbred germplasm association panel), and genomic resources (gene sequences, gene-based markers and comparative genomics information) were generated and employed for fine mapping of this major DT-QTL. The HRC was developed by crossing two near-isogenic lines, ICMR 01029 (developed in the background of H 77/833-2 by transferring drought tolerance QTL interval on LG 2) and ICMR 01004 (developed in the background of H 77/833-2 by transferring downy mildew resistant QTLs on LG 1 and 4). Around 2500 F2 individuals of HRC were genotyped with 6 SSR markers covering the DT-QTL region on LG2 and 160 most informative recombinants were selected. Similarly, a germplasm association panel of 300 inbred lines, representing a core set obtained from a larger germplasm of 1000 diverse pearl millet breeding lines and accessions (including landraces, elite cultivars and mapping population parents) collected from pearl millet growing areas in Africa and Asia has been assembled. To saturate LG2 with gene-based markers and to identify candidate genes underlying this QTL, we utilized published information of synteny between pearl millet LG2 and rice chromosomes 2S, 3L, 6S and 10L. The genomic sequences of 100 selected genes within the rice BACs from each of the four syntenic rice chromosomes (between rice markers C1246 and C630 on 2S, C136 and RZ624 on 3L, PSR 490 and C235 on 6S and between R2447 and C1361 on 10L) were retrieved using the TIGR Rice database to design the primers. This exercise resulted in saturation of the DT-QTL region on linkage group (LG) 2 with 18 new candidate gene-based single nucleotide polymorphism (SNP) and conserved intron spanning primers (CISP) markers. Of these, 10 candidate genes have been reported to play important roles in regulation [transcription factors like zinc finger CCCH type, MADS-box], signal transduction (serine/threonine protein kinase, protein phosphatase 1 regulatory subunit SDS22), energy and carbon metabolism (genes for photosynthesis, photorespiration and oxidation such as PSI reaction center subunit III, chlorophyll a/b binding protein, alanine glyoxylate aminotransferase, acyl CoA oxidase), purine and pyrimidine nucleotide biosynthesis (uridylate kinase), and lipid biosynthesis (acetyl-CoA carboxylase) under drought stress. Genotyping of HRC with these gene-based markers and phenotyping under irrigated control and two terminal drought stress environments (late and early stress) reduced the mapping interval of this QTL by approximately 20 cM. To further reduce the mapping interval of this QTL, and to identify functional markers/favourable alleles associated with this QTL, we currently are genotyping pearl millet inbred germplasm association panel for these candidate genes and also phenotyping it for drought response under terminal drought stress conditions.