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 (preNILs) 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 (F 10 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.

Expression Profile of Defense Genes in Rice Lines Pyramided with Resistance Genes Against Bacterial Blight, Fungal Blast and Insect Gall Midge

“…However, in the present study this gene was not induced in rice line RPNF05. The target gene Gm4 in this line displayed 2 to 4 fold upward induction in stem tissue at 24 hai as earlier characterized (Divya et al 2015 ). This induction was not affected by simultaneous infection of the plant with BB pathogen.…”

“…Against the Asian rice gall midge 11 genes have been reported including one recessive; of which eight have been mapped and three genes cloned (Bentur et al 2016 ). Among these Gm1, gm3 , Gm4 and Gm8 are effective against most of the seven prevailing biotypes in India (Bentur et al 2011 ) and linked markers have been reported (Sundaram 2007 ; Dutta et al 2014 ; Sama et al 2014 ; Divya et al 2015 ). There have been several successful attempts to introgress and pyramid these genes through marker assisted selection in elite genetic backgrounds.…”

Current understanding of the genomic, genetic, and molecular control of insect resistance in rice

“…gm3 was mapped to a 560 kb region in RP2068-18-3-5 and a gene encoding an NB-ARC protein was tentatively linked to gall midge resistance (Sama et al 2014). GM4 from Abhaya was finely mapped to a 300 kb region; this region includes a candidate gene encoding a leucine-rich repeat (LRR) protein (Divya et al 2015). GM8 from the indica rice variety Aganni was mapped to a 430 kb region containing a gene encoding a proline-rich protein (Divya et al 2018).…”

“…A series of near-isogenic lines (NILs) with a single BPH resistance gene/QTL, including BPH3, bph4, BPH6, BPH9, BPH10, BPH14, BPH15, BPH17, BPH18, BPH20, BPH21, BPH24, BPH26, BPH32, qBPH3, and qBPH4, were developed in the background of the susceptible cultivar 9311 and IR24, respectively (Qiu et al 2010;Xiao et al 2016;. Furthermore, the NILs with a single GM (GM4, GM11), WBPH (Ovc, qOVA-1-3, qOVA-4, qOVA-5-1, and qOVA-5-2), and GRH (GRH1, GRH2, GRH4, GRH5, GRH6, and qGRH4) resistance genes were also developed (Yamasaki et al 2003;Fujita et al 2010b;Himabindu et al 2010;Divya et al 2015). Unfortunately, such plants bearing single resistance genes lost their efficacy in just a few years as the insect populations rapidly adapted or evolved to overcome the resistance (Jena and Kim 2010).…”

Hemipteran and dipteran pests: Effectors and plant host immune regulators

“…A number of loci conferring resistance to aphids, planthoppers and gall midges have been genetically identified and mapped (Smith and Clement 2012;Yasala et al 2012;Fujita et al 2013;Kamphuis et al 2013;Harris et al 2015). While only a small number of these resistance (R) genes have been cloned and functionally tested for their roles in resistance, several R loci span genes with NLR domains (Zhang 2007;Kamphuis et al 2013;Sama et al 2014;Divya et al 2015). To date, R genes to phloem-feeding hemipteran insects have been isolated from rice, tomato and melon.…”