Identification of a new resistance locus against the rice gall midge, Orseolia oryzae (Wood-Mason), in Thai rice landrace MN62M

The rice gall midge (RGM), Orseolia oryzae (Wood-Mason), is one of the most destructive insect pests of rice, and it causes significant yield losses annually in Asian countries. The development of resistant rice varieties is considered as the most effective and economical approach for maintaining yield stability by controlling RGM. Identification of resistance genes will help in marker-assisted selection (MAS) to pyramid the resistance genes and develop a durable resistance variety against RGM in areas with frequent outbreaks. A mitochondrial gene, cytochrome C oxidase I (COI), was used to analyze the genetic diversity among Thai RGM populations. The phylogenetic tree indicated that the Thai RGM populations were homogeneously distributed throughout the country, except for some populations in central and northeast Thailand that probably became isolated from the main population. The reactions of the resistant rice varieties carrying different resistance genes revealed different RGM biotypes in Thailand. The Thai rice landrace MN62M showed resistance to all RGM populations used in this study. We identified a novel genetic locus for resistance to RGM, designated as GM12 , on the short arm of rice chromosome 2. The locus was identified using linkage analysis in 144 F 2 plants derived from a cross between susceptible cultivar KDML105 and RGM-resistant cultivar MN62M with single nucleotide polymorphism (SNP) markers and F 2:3 phenotype. The locus was confirmed and mapped using SNP and simple sequence repeat (SSR) markers surrounding the target chromosomal location. Finally, the locus was mapped between two flanking markers, RM6800 and

al. 2015). Existence and emergence of new virulent RGM biotypes have been reported, and the resistance genes in several RGM-resistant rice varieties were ineffective against such biotypes (Bentur et al. 2016). To identify the biotype of Thai populations, a differential set of resistant rice varieties with known resistance genes needs to be developed. Therefore, germplasm screening and identification of resistance genes in Thai rice germplasm are necessary for classifying the RGM biotypes in Thai populations.
Exploitation of host plant resistance to RGM is an economical and environmentfriendly approach to manage the pest (Khush 1977). Breeding resistant rice varieties is an economically acceptable method for managing RGM. To date, 11 major resistance genes, designated GM1-11, that confer resistance to RGM populations have been identified, mostly in South Asia (Bentur et  The reaction of 13 resistant rice varieties carrying nine differential RGM resistance genes revealed four distinct RGM biotypes from different parts of Thailand (Table 1).
The parental varieties, KDML105 and MN62M, F 1 , and derived F 2:3 families were evaluated for RGM resistance in the greenhouse during the wet season of 2018 (Additional file 1: Table S1). The average damage to KDML105 and MN62M was 9 (100% plant with gall) and 0 (no plant with gall), indicating susceptible and resistant reactions to RGM, respectively. The frequency distribution of the damage severity (percentage of plants infested with galls) in 300 F 2:3 families from the KDML105 × MN62M population is shown in Fig. 2  To confirm the chromosomal location of the GM12 locus, SSR markers covering the target locus were selected and used for the parental polymorphism survey. Of the 25 SSR markers, only three markers showed polymorphism between the parents.
Then, SSR and SNP markers on the short arm of chromosome 2 were used to construct the linkage map and perform QTL analysis. The new RGM resistance gene in MN62M was located on chromosome 2 between SSR and SNP markers RM6800 and S2_419160, respectively (Fig. 4). To the best of our knowledge, no RGM resistance gene has been reported on rice chromosome 2, suggesting that the gene in MN62M is probably a novel gene.

Candidate genes associated with RGM resistance
Genes within the target region were identified by taking into account the physical location of the interval markers located at 310-420 kb on the short arm of rice chromosome 2. The total number of genes identified within the regions (Additional file 3:  The new RGM-resistance locus identified in this study provides SNP and SSR markers (Additional file 4: Figure S2)     QTL maps of the RGM resistance phenotype from the F 2:3 family was analyzed using MapQTL 6.0 (Van Ooijen 2004). The MQM mapping method was used to detect significant associations between the phenotypic trait and marker data set.
Significant LOD thresholds were determined using the permutation test at 1,000 permutations per trait. The PVE by a single QTL was estimated based on the population variance found within the progeny by maximum likelihood estimation.
In silico analysis of putative candidate genes in the targeted region was performed with the available sequence annotation database http://rapdb.dna.affrc.go.jp. The location and function of these genes were noted to identify the putative candidate for gall midge resistance.        Mapping of the rice gall midge resistance gene (black bar) on the SNP and SSR linkage map for ric

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download.  Figure S1.docx Figure S2.docx