Comparative analysis of sequences of mitochondrial genomes of wild abortive male sterile (WA-CMS) and male fertile lines of rice, development of functional markers for WA-CMS trait and their use in assessment of genetic purity of seeds of WA-CMS lines

Abstract

WA-CMS system based rice hybrids are widely adopted in many rice growing countries, including India. Even though it is well known that the trait is controlled by mitochondria, the genes underpinning the trait remain enigmatic. In the present study, a complete genome-wide comparative sequence analysis was performed using draft mitochondrial genomes of WA-CMS and male fertile lines in a step-wise manner, progressively covering 5–10 kb every time through BLASTN tool. The sequence polymorphisms identified in different mitochondrial regions were targeted to develop two different sets of dominant PCR-based markers, one consisting of six markers targeting WA-CMS mitochondria, the other set consisting of five markers targeting male fertile mitochondria in addition to development of a set of eight co-dominant PCR-based markers targeting both the genomes. When a set of candidate genes/ORFs reported earlier to be associated with WA-CMS trait in rice were analyzed through RT-PCR of RNA isolated from immature rice florets, it was observed that the chimeric ORF, WA352 is expressed only in WA-CMS line and hybrid (i.e. genotypes containing sterile mitochondria), indicating it’s candidacy for the WA-CMS trait. Targeting the functional nucleotide polymorphism between WA-CMS and maintainer mitochondria with respect to WA352, two dominant markers, one targeting sterile and another targeting fertile mitochondria were developed. In addition, a robust, co-dominant functional marker targeting the candidate gene was also developed and validated for its utility in identification of genetic impurities in seed lots of WA-CMS lines.