Identification and Functional Characterization of Two Major Loci Associated with Resistance against Brown Planthoppers (Nilaparvata lugens (Stål)) Derived from Oryza nivara

Abstract

The brown planthopper (BPH) is a highly destructive pest of rice, causing significant economic losses in various regions of South and Southeast Asia. Researchers have made promising strides in developing resistance against BPH in rice. Introgression line RPBio4918-230S, derived from Oryza nivara, has shown consistent resistance to BPH at both the seedling and adult stages of rice plants. Segregation analysis has revealed that this resistance is governed by two recessive loci, known as bph39(t) and bph40(t), contributing to 21% and 22% of the phenotypic variance, respectively. We later mapped the genes using a backcross population derived from a cross between Swarna and RPBio4918-230S. We identified specific marker loci, namely RM8213, RM5953, and R4M17, on chromosome 4, flanking the bph39(t) and bph40(t) loci. Furthermore, quantitative expression analysis of candidate genes situated between the RM8213 and R4M17 markers was conducted. It was observed that eight genes exhibited up-regulation in RPBio4918-230S and down-regulation in Swarna after BPH infestation. One gene of particular interest, a serine/threonine-protein kinase receptor (STPKR), showed significant up-regulation in RPBio4918-230S. In-depth sequencing of the susceptible and resistant alleles of STPKR from Swarna and RPBio4918-230S, respectively, revealed numerous single nucleotide polymorphisms (SNPs) and insertion-deletion (InDel) mutations, both in the coding and regulatory regions of the gene. Notably, six of these mutations resulted in amino acid substitutions in the coding region of STPKR (R5K, I38L, S120N, T319A, T320S, and F348S) when compared to Swarna and the reference sequence of Nipponbare. Further validation of these mutations in a set of highly resistant and susceptible backcross inbred lines confirmed the candidacy of the STPKR gene with respect to BPH resistance controlled by bph39(t) and bph40(t). Functional markers specific for STPKR have been developed and validated and can be used for accelerated transfer of the resistant locus to elite rice cultivars.

Keywords: Oryza nivara; STPKR gene; brown planthopper (BPH); rice.

Figure 1

Illustration of the molecular linkage map of SSR markers associated with the loci responsible for BPH resistance, along with their respective positions, generated using IciMapping V4.1.

Figure 2

Presentation of the QTL likelihood curve depicting the LOD score for BPH damage score (DS). These QTLs were identified on chromosomes using QTL IciMapping version 4.1. The graph displays the LOD score curve along with the additive effects of the identified QTLs. DS = damage score.

Figure 3

Depiction of the condition of plants after 3 days of BPH infestation. Notably, the nymphs on the susceptible parent, Swarna, have progressed to the third and fourth instar stages. In contrast, the nymphs on the resistant parent remain in the second instar stage due to inadequate feeding.

Figure 4

Illustration of the differential expression patterns of genes as determined with qRT-PCR. It depicts the up-regulated and down-regulated genes among the commonly identified differentially expressed genes (DEGs) in RPbio4918-230S (RP) and Swarna (SW). Notably, there is a higher prevalence of up-regulated DEGs in RPbio4918-230S among the common set.

Figure 5

Protein sequence alignment of STPKR gene for RPbio4918-230(S) and Swarna.