Cloning of a gene encoding LRR protein and its validation as candidate gall midge resistance gene, Gm4, in rice

Abstract

The Asian rice gall midge, Orseolia oryzae, is an important pest of rice. Gm4, a major rice gall midge resistance gene in the indica rice variety Abhaya has been fine mapped within 0.3 Mb region with the flanking markers RM22551 and RM22562. Among the 70 putative candidate genes identified in the reference Nipponbare rice genome in this region, two genes coding for leucine rich repeat (LRR) proteins were shortlisted for further analysis. Polymorphisms was observed between the parents improved samba masuri (ISM) (susceptible parent) and Abhaya (resistant parent) and the recombinant inbred line (RIL)-derived near isogenic lines (pre-NILs) 482R (resistant line) and 489S (susceptible line) with one pair of the markers targeting the candidate genes. Polymorphism co-segregated with the trait in a larger mapping population consisting of 40 resistant and 40 susceptible RILs (F10 generation). A major segment of this gene (LOC_Os08g09670.1), covering one of the two exons, was cloned and sequenced from both parents as well as from the two pre-NILs 482R and 489S. Data revealed a large deletion in the Abhaya fragment as compared to that of ISM. At least five amino acid substitutions were detected, between the alleles, that are likely to influence the protein function. Expression analysis of this putative candidate gene through reverse transcription real time PCR showed more than two fold increase in its expression levels in the resistant parent Abhaya and resistant line 482R at 24 h after gall midge infestation when compared with the uninfested controls. These results suggested the LRR coding gene to be the candidate for Gm4. In addition, the current work also identified a functional marker (LRR-del) for the detection of the gene for use in marker-assisted introgression of Gm4.