Multiomics-assisted characterization of rice-Yellow Stem Borer interaction provides genomic and mechanistic insights into stem borer resistance in rice.

KEY MESSAGE: By deploying a multi-omics approach, we unraveled the mechanisms that might help rice to combat Yellow Stem Borer infestation, thus providing insights and scope for developing YSB resistant rice varieties. Yellow Stem Borer (YSB), Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae), is a major pest of rice, that can lead to 20-60% loss in rice production. Effective management of YSB infestation is challenged by the non-availability of adequate sources of resistance and poor understanding of resistance mechanisms, thus necessitating studies for generating resources to breed YSB resistant rice and to understand rice-YSB interaction. In this study, by using bulk-segregant analysis in combination with next-generation sequencing, Quantitative Trait Loci (QTL) intervals in five rice chromosomes were mapped that could be associated with YSB resistance at the vegetative phase in a resistant rice line named SM92. Further, multiple SNP markers that showed significant association with YSB resistance in rice chromosomes 1, 5, 10, and 12 were developed. RNA-sequencing of the susceptible and resistant lines revealed several genes present in the candidate QTL intervals to be differentially regulated upon YSB infestation. Comparative transcriptome analysis revealed a putative candidate gene that was predicted to encode an alpha-amylase inhibitor. Analysis of the transcriptome and metabolite profiles further revealed a possible link between phenylpropanoid metabolism and YSB resistance. Taken together, our study provides deeper insights into rice-YSB interaction and enhances the understanding of YSB resistance mechanism. Importantly, a promising breeding line and markers for YSB resistance have been developed that can potentially aid in marker-assisted breeding of YSB resistance among elite rice cultivars.

Impairment in a member of AP2/ERF and F-box family protein enhances complete panicle exsertion in rice.

Complete panicle exsertion (CPE) is an economically important quantitative trait that contributes to grain yield in rice. We deployed an integrated approach for understanding the molecular mechanism of CPE using a stable ethyl methanesulfonate mutant line, CPE-109 of the Samba Mahsuri (SM) variety of rice (Oryza sativa), which exhibits CPE. Two consistent genomic regions were identified for CPE through quantitative trait locus (QTL) mapping [qCPE-4 (28.24-31.22 Mb) and qCPE-12 (2.30-3.18 Mb)] and QTL-sequencing [chr 4 (31.21-33.69 Mb) and chr 12 (0.12-3.15 Mb)]. Two non-synonymous single nucleotide polymorphisms, namely KASP 12-12 (T→C; chr12:1269983) in Os12g0126300, encoding an AP2/ERF transcription factor, and KASP 12-16 (G→A; chr12:1515198) in Os12g0131400, encoding an F-box domain-containing protein, explained 81.05% and 59.61% of the phenotypic variance, respectively, and exhibited strong co-segregation with CPE in F2 mapping populations, advanced generation lines, and CPE-exhibiting SM mutants through KASP assays. Down-regulation of these genes in CPE-109 compared with SM (wild type) was observed in transcriptome sequencing of flag leaves, which was validated through qRT-PCR. We propose that the abrogation of Os12g0126300 and Os12g0131400 in CPE-109 combinatorially influences down-regulation of ethylene biosynthetic genes, Os01g0192900 (ACC synthase) and Os05g0497300 (ethylene-responsive factor-2), and up-regulation of a gibberellic acid synthetic gene, Os06g0569900 (ent-kaurene synthase) and the two cytokinin biosynthetic genes Os07g0486700 (cytokinin-O-glucosyltransferase 2) and Os10g0479500 (similar to carboxy-lyase), which results in complete panicle exsertion.

Mutation resource of Samba Mahsuri revealed the presence of high extent of variations among key traits for rice improvement.

To create novel variants for morphological, physiological, and biotic stress tolerance traits, induced mutations were created using Ethyl Methane Sulphonate (EMS) in the background of Samba Mahsuri (BPT 5204), a popular and mega rice variety of India. A population derived from 10, 500 M1 plants and their descendants were phenotyped for a wide range of traits leading to the identification of 124 mutants having variations in key agro-morphological traits, and 106 mutants exhibiting variation for physiological traits. Higher yield is the ultimate goal of crop improvement and we identified 574 mutants having higher yield compared to wild type by having better yield attributing traits. Further, a total of 50 mutants showed better panicle exertion phenotypes as compared to Samba Mahsuri leading to enhancement of yield. Upon rigorous screening for three major biotic stresses, 8 mutants showed enhanced tolerance for yellow stem borer (YSB), and 13 different mutants each showed enhanced tolerance for sheath blight (ShB) and bacterial leaf blight (BLB), respectively. In addition, screening at multiple locations that have diverse field isolates identified 3, 3, and 5 lines for tolerance to ShB, YSB and BLB, respectively. On the whole, 1231 desired mutant lines identified at M2 were forwarded to an advanced generation (M5). PCR based allele mining indicated that the BLB tolerant mutants have a different allele than the reported alleles for well-known genes affecting bacterial blight resistance. Whole genome re-sequencing revealed substantial variation in comparison to Samba Mahsuri. The lines showing enhanced tolerance to important biotic stresses (YSB, ShB and BLB) as well as several economically important traits are unique genetic resources which can be utilized for the identification of novel genes/alleles for different traits. The lines which have better agronomic features can be used as pre-breeding lines. The entire mutant population is maintained as a national resource for genetic improvement of the rice crop.

Conversion of partial restorer Swarna into restorer by transferring fertility restorer Rf gene(s) through marker assisted back cross breeding (MABB) in rice.

The major constraints in hybrid rice breeding are availability of limited number of parental lines with specific desirable traits and lower frequency of restorers among elite breeding lines. The popular, high-yielding mega-rice variety Swarna, has been identified to be a partial restorer (as it has only one of major fertility restorer genes, Rf4) and hence cannot be utilized directly in the hybrid rice breeding. To convert the partial restorer to complete restorer, a cross was made between Swarna and a stable restorer KMR3R possessing Rf3 and Rf4 genes and developed BC1F5 and BC2F4 populations by marker-assisted back cross breeding (MABB). The SSR marker DRRM-RF3-10 linked to Rf3 gene located on chromosome 1, clearly distinguished restorers from partial restorers. All the improved lines of Swarna possessing Rf3 and Rf4 genes showed complete fertility restoration in test crosses with higher grain yield heterosis. Few rice hybrids developed by using converted restorers were evaluated in multi location testing under the All India Co-ordinated Rice Improvement Project (AICRIP). The results indicated that new rice hybrids expressed higher heterosis with matching grain quality attributes like Swarna. This study provides significantly novel and relevant restorers to enhance and economize future hybrid rice breeding programs.

A novel mechanism of gall midge resistance in the rice variety Kavya revealed by microarray analysis.

The Asian rice gall midge [Orseolia oryzae (Wood-Mason)] is an important rice pest causing an annual average yield loss of about US $80 million in India. Rice varieties possess several discrete resistance (R) genes conferring resistance against the pest in two distinct ways, i.e., with (HR+ type) or without (HR- type) the expression of hypersensitive reaction (HR). The aim of the present work is to understand the molecular basis of compatible and incompatible (HR- type) rice gall midge interactions between the rice variety Kavya and the two gall midge biotypes: the virulent GMB4M and the avirulent GMB1 using transcriptional microarray gene expression analysis. A large number of differentially expressed genes (602genes in incompatible interaction and 1,330 genes in compatible interaction with at least twofold changes, p value <0.05) was obtained from the microarray analysis that could be grouped into six clusters based on their induction during both or either of the interactions. MapMan software was used for functional characterization of these genes into 13 categories (BINs). Real-time polymerase chain reaction validation of 26 genes selected through the analysis revealed four genes viz. NADPH oxidase, AtrbohF, cinnamoyl-CoA reductase, and von Willebrand factor type A domain containing protein coding genes to be significantly upregulated during the incompatible interaction. But most of the signature genes related to HR+ type resistance like salicylic acid pathway-related genes and disease resistance protein coding genes were downregulated. On the other hand, during the compatible interaction, genes related to primary metabolism and nutrient transport were upregulated and genes for defense and signaling were downregulated. We propose a hypothesis that HR- type of resistance in the rice variety Kavya against gall midge could be due to the constitutive expression of an R gene and a case of extreme resistance which is devoid of cell death. Compatible interaction, however, modulated a large number of differentially expressed transcripts to reprogram cell organization, cell remodeling, and relocation of nutrients through transport to support insect growth.