A putative candidate for the recessive gall midge resistance gene gm3 in rice identified and validated

Abstract

We report here tagging and fine-mapping of gm3 gene, development of a functional marker for it and its use in marker-assisted selection. The recessive rice gall midge resistance gene, gm3 identified in the rice breeding line RP2068-18-3-5 confers resistance against five of the seven Indian biotypes of the Asian rice gall midge Orseolia oryzae. We report here tagging and fine-mapping of gm3 gene, development of a functional marker for it and demonstrated its use in marker-assisted selection (MAS). A mapping population consisting of 302 F10 recombinant inbred lines derived from the cross TN1 (susceptible)/RP2068-18-3-5, was screened against gall midge biotype 4 (GMB4) and analyzed with a set of 89 polymorphic SSR markers distributed uniformly across the rice genome. Two SSR markers, RM17480 and gm3SSR4, located on chromosome 4L displayed high degree of co-segregation with the trait phenotype and flanked the gene. In silico analysis of the genomic region spanning these two markers contained 62 putatively expressed genes, including a gene encoding an NB-ARC (NBS-LRR) domain containing protein. A fragment of this gene was amplified with the designed marker, NBcloning 0.9 Kb from the two susceptible TN1, Improved Samba Mahsuri (B95-1) and two resistant cultivars, RP 2068-18-3-5 and Phalguna (with Gm2 gene). The amplicons were observed to be polymorphic between the susceptible and resistant genotypes and hence were cloned and sequenced. A new primer, gm3del3, which was designed based on sequence polymorphism, amplified fragments with distinct size polymorphism among RP2068-18-3-5, Phalguna and TN1 and B95-1 and displayed no recombination in the entire mapping population. Expression of the candidate NB-ARC gene in the susceptible TN1 and the resistant RP2068-18-3-5 plants following infestation with GMB4 was analyzed, through real-time reverse transcription PCR. Results showed twofold enhanced expression in RP2068-18-3-5 plants, but not in TN1 plants, 120 h after infestation. Amino acid sequence and structure analysis of the proteins coded by different alleles of gm3 gene showed deletion of eight amino acids due to an early stop codon in RP2068-18-3-5 resulting in a change in the functional domain of the protein. The gm3del3 was used as a functional marker for introgression of gm3 gene into the genetic background of the elite bacterial blight resistant cultivar Improved Samba Mahsuri (B95-1) through MAS.

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

“…type resistance. Of these genes, a NB-ARC coding gene has been earlier identified and functionally validated as the candidate gene for gm3 (Sama et al 2014). The present report is the second putative gall midge resistance gene to be cloned and validated.…”

“…The functional allele of the recessive gene gm3 coding for NB-ARC domain containing protein, lacked a complete exon of 320 bp in the resistant rice variety RP2068-18-3-5 when compared with the allele found in the susceptible rice variety TN1. In addition, a 92 bp region was also deleted in RP2068-18-3-5 when compared with the third allele in the rice variety Phalguna carrying Gm2 (Sama et al 2014). Likewise, in the NBS-LRR-domain-containing protein coding for Pi54 lacked a 144 bp segment in an exon in the functional allele (Ramkumar et al 2011).…”

“…Breeding and cultivation of resistant rice varieties is the main approach to contain this pest. So far 11 gall midge resistance genes have been reported and nine of these have been mapped (Yasala et al 2012;Sama et al 2014). Seven distinct biotypes of the pest have been characterized based on the reaction of gene differential rice varieties (Vijaya Lakshmi et al 2006).…”

Markers Assisted Selection for Pyramiding of Gallmidge Resistance Genes in Kavya, a Popular Rice Variety

“…The three improved line (namely WGL-1068) displayed high resistance with (score '0'), which were scored under IRRI-SES scale as highly resistant. Though, in the majority of the studies, advanced lines had been screened in the glass house for resistance against this pest (Nanda et al, 2010;Sama et al, 2014 andDivya et al, 2015), there are reports where in the lines had been screened in field (Mohan et al, 1997;Vijayalakshmi et al, 2006) against gall midge.…”

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.…”