Development of allele-specific SNP markers for PPR10 gene at Rf4 locus of Fertility Restorer Gene for Identification of Maintainer and Restorer lines
DOI:
https://doi.org/10.14456/gag.2017.6Keywords:
Allele-specific SNP marker, Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), Restorer of fertility (Rf) gene, Pentatricopeptide repeat (PPR) protein, Wild abortive- cytoplasmic male sterility (WA-CMS)Abstract
Cytoplasmic male sterility (CMS) -associated genes are located in mitochondrial genome whereas restorer of fertility (Rf) genes are located in nuclear genome. Two major restorer gene loci of Wild abortive-CMS (WA-CMS), Rf3 and Rf4, are required for the production of viable pollen. The Rf4 locus of indica rice cultivars were reported, which contains four RNA-binding pentatricopeptide repeat protein (PPR) encoding genes. Preliminary sequence analysis of reported PPR gene at Rf4 locus from maintainer and restorer lines revealed the difference in size of protein only PPR10 protein that resulted from single nucleotide polymorphism (SNP). In this study, PPR10 gene was isolated from four Thai rice cultivars by Polymerase Chain Reaction (PCR) of genomic DNA. The nucleotide sequences of cloned PPR10 gene from Khao Dawk Mali 105 and Nahng Mon S-4, Pathum Thani 1 and Suphan Buri 1 were analyzed by ClustalX program. One of fifteen SNPs at position 1,392 caused nonsense mutation and classified PPR10 protein into 2 groups by the length. Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) is fast, reliable and low cost. Then, four primers of ARMS-PCR were developed to use as allele-specific DNA marker, which is the first specific gene marker for Rf4 locus. The tetra- primer ARMS-PCR products of 5 maintainer cultivars and 4 restorer cultivars were G allele (416 bp) and T allele (242 bp), respectively. Therefore, tetra-primer ARMS-PCR could be allele specific PPR10 gene marker and used to identity maintainer and restorer Thai rice cultivars of WA-CMS. This allele-specific PPR10 gene markers will be used for germplasm genotyping and hybrid rice breeding program.
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