Shefali Mishra

Single-copy gene based 50 K SNP chip for genetic studies and molecular breeding in rice

Single nucleotide polymorphism (SNP) is the most abundant DNA sequence variation present in plant genomes. Here, we report the design and validation of a unique genic-SNP genotyping chip for genetic and evolutionary studies as well as molecular breeding applications in rice. The chip incorporates 50,051 SNPs from 18,980 different genes spanning 12 rice chromosomes, including 3,710 single-copy (SC) genes conserved between wheat and rice, 14,959 SC genes unique to rice, 194 agronomically important cloned rice genes and 117 multi-copy rice genes. Assays with this chip showed high success rate and reproducibility because of the SC gene based array with no sequence redundancy and cross-hybridisation problems. The usefulness of the chip in genetic diversity and phylogenetic studies of cultivated and wild rice germplasm was demonstrated. Furthermore, its efficacy was validated for analysing background recovery in improved mega rice varieties with submergence tolerance developed through marker-assisted backcross breeding.

Haplotype distribution and association of candidate genes with salt tolerance in Indian wild rice germplasm

Key messageThe association of natural genetic variations of salt-responsive candidate genes belonging to different gene families with salt-tolerance phenotype and their haplotype variation in different geographic regions.AbstractSoil salinity covers a large part of the arable land of the world and is a major factor for yield losses in salt-sensitive crops, such as rice. Different gene families that respond to salinity have been identified in rice, but limited success has been achieved in developing salt-tolerant cultivars. Therefore, 21 salt stress-responsive candidate genes belonging to different gene families were re-sequenced to analyse their genetic variation and association with salt tolerance. The average single nucleotide polymorphism (SNP) density was 16 SNPs per kbp amongst these genes. The identified nucleotide and haplotype diversity showed comparatively higher genetic variation in the transporter family genes. Linkage disequilibrium (LD) analysis showed significant associations of SNPs in BADH2, HsfC1B, MIPS1, MIPS2, MYB2, NHX1, NHX2, NHX3, P5CS1, P5CS2, PIP1, SIK1, SOS1, and SOS2 genes with the salt-tolerant phenotype. A combined analysis of SNPs in the 21 candidate genes and eight other HKT transporter genes produced two separate clusters of tolerant genotypes, carrying unique SNPs in the ion transporter and osmoticum-related genes. Haplotype network analysis showed all the major and few minor alleles distributed over distant geographic regions. Minor haplotypes may be recently evolved alleles which migrated to distant geographic regions and may represent recent expansion of Indian wild rice. The analysis of genetic variation in different gene families identified the relationship between adaptive variations and functional significance of the genes. Introgression of the identified alleles from wild relatives may enhance the salt tolerance and consequently rice production in the salinity-affected areas.

Haplotype diversity and association analysis of SNAC1 gene in wild rice germplasm

Effect of SNAC1 gene variants on drought tolerance was investigated in wild rice accessions. A total of 46 INDELs and 44 SNPs were identified including 17 SNPs in the coding region of the gene across the 164 Indian wild rice accessions and four cultivated rice genotypes. Based on the sequence variation, 25 haplotypes were identified including four specific to drought tolerant accessions. Translation of different allele sequences showed existence of five different SNAC1 protein variants. Evolutionary study on the basis of nucleotide sequence variation in the SNAC1 gene with different neutrality test suggested purifying selection in the wild rice accessions. Association analysis revealed that four SNPs were showing significant association with drought tolerance. The significantly associated SNPs and their associated haplotypes can further be used for the development of drought tolerant rice cultivars.

Morphological and Molecular Data Reveal Three Distinct Populations of Indian Wild Rice Oryza rufipogon Griff. Species Complex

Wild relatives of crops possess adaptive mutations for agronomically important traits, which could play significant role in crop improvement for sustainable agriculture. However, global climate change and human activities pose serious threats to the natural habitats leading to erosion of genetic diversity of wild rice populations. The purpose of this study was to explore and characterize India’s huge untapped wild rice diversity in Oryza rufipogon Griff. species complex from a wide range of ecological niches. We made strategic expeditions around diversity hot spots in 64 districts of nine different agro-climatic zones of the country and collected 418 wild rice accessions. Significant variation was observed among the accessions for 46 morphological descriptors, allowing classification into O. nivara, O. rufipogon, and O. sativa f. spontanea morpho-taxonomic groups. Genome-specific pSINE1 markers confirmed all the accessions having AA genome, which were further classified using ecotype-specific pSINE1 markers into annual, perennial, intermediate, and an unknown type. Principal component analysis revealed continuous variation for the morphological traits in each ecotype group. Genetic diversity analysis based on multi-allelic SSR markers clustered these accessions into three major groups and analysis of molecular variance for nine agro-climatic zones showed that 68% of the genetic variation was inherent amongst individuals while only 11% of the variation separated the zones, though there was significant correlation between genetic and spatial distances of the accessions. Model based population structure analysis using genome wide bi-allelic SNP markers revealed three sub-populations designated ‘Pro-Indica,’ ‘Pro-Aus,’ and ‘Mid-Gangetic,’ which showed poor correspondence with the morpho-taxonomic classification or pSINE1 ecotypes. There was Pan-India distribution of the ‘Pro-Indica’ and ‘Pro-Aus’ sub-populations across agro-climatic zones, indicating a more fundamental grouping based on the ancestry closely related to ‘Indica’ and ‘Aus’ groups of rice cultivars. The Pro-Indica population has substantial presence in the Eastern Himalayan Region and Lower Gangetic Plains, whereas ‘Pro-Aus’ sub-population was predominant in the Upper Gangetic Plains, Western Himalayan Region, Gujarat Plains and Hills, and Western Coastal Plains. In contrast ‘Mid-Gangetic’ population was largely concentrated in the Mid Gangetic Plains. The information presented here will be useful in the utilization of wild rice resources for varietal improvement.