Mining of resistance gene analogue specific SSR markers (RGA-eSSRs) in pomegranate and their validation

Abstract

Abstract Pomegranate has been a highly nutritious and medicinally valued fruit for ages. Pomegranate cultivation is severely threatened by multiple biotic stresses, including bacterial blight and wilt, the major bottlenecks. Identifying candidate genes and markers linked to such genes is necessary to accelerate host resistance breeding. 132 resistance gene analogues (RGAs) specific simple sequence repeats (RGA-eSSRs) located on the coding regions of 103 RGAs were mined from 958 pomegranates RGAs. An average of 58.56 SSRs/Mb of RGA genes was observed with a relative density of 1170.84 bp of SSR motifs per Mb of RGA. Tri-nucleotide repeats were highly abundant in the RGA-eSSRs, followed by hexanucleotide repeats. The high density of RGAs was noticed on the RLK and CNL genes with an average of 3.34 and 3.0 SSRs per RGA, respectively. The abundance of RGA-eSSRs on each chromosome was correlated with the number of RGAs, with chromosome 08 bearing the highest RGA-eSSRs. Primers were designed for 121 of the 132 RGA-SSR and validated by both in silico and in vitro PCR. RGA-eSSRs exhibited high transferability with most markers amplified in four of the five genotypes tested under in silico PCR and 100% amplification in biological validation with eight genotypes. Pomegranate RGA-eSSRs also exhibited high polymorphism with an average polymorphic information content of 0.5. Discovered RGA-eSSRs are useful in the genetic mapping of biotic stress genes and diversity analysis.