Exome-based new allele-specific PCR markers and transferability for sodicity tolerance in bread wheat (Triticum aestivum L.)

Roopali Bhoite; Rosemary Smith; Urmil Bansal; Mirza Dowla; Harbans Bariana; Darshan Sharma

doi:10.1002/pld3.520

Exome-based new allele-specific PCR markers and transferability for sodicity tolerance in bread wheat (Triticum aestivum L.)

Plant Direct. 2023 Aug 18;7(8):e520. doi: 10.1002/pld3.520. eCollection 2023 Aug.

Authors

Roopali Bhoite^{1

2}, Rosemary Smith¹, Urmil Bansal³, Mirza Dowla¹, Harbans Bariana⁴, Darshan Sharma^{1

5}

Affiliations

¹ Grains Genetic Improvement Department of Primary Industries and Regional Development South Perth Western Australia Australia.
² The UWA Institute of Agriculture The University of Western Australia Perth Western Australia Australia.
³ Plant Breeding Institute, School of Life Sciences, Faculty of Science The University of Sydney Cobbitty New South Wales Australia.
⁴ School of Science Western Sydney University Richmond New South Wales Australia.
⁵ College of Science, Health, Engineering and Education Murdoch University Perth Western Australia Australia.

Abstract

Targeted exome-based genotype by sequencing (t-GBS), a sequencing technology that tags SNPs and haplotypes in gene-rich regions was used in previous genome-wide association studies (GWAS) for sodicity tolerance in bread wheat. Thirty-nine novel SNPs including 18 haplotypes for yield and yield-components were identified. The present study aimed at developing SNP-derived markers by precisely locating new SNPs on ~180 bp allelic sequence of t-GBS, marker validation, and SNP functional characterization based on its exonic location. We identified unknown locations of significant SNPs/haplotypes by aligning allelic sequences on to IWGSC RefSeqv1.0 on respective chromosomes. Eighteen out of the target 39 SNP locations fulfilled the criteria for producing PCR markers, among which only eight produced polymorphic signals. These eight markers associated with yield, plants m^-2, heads m^-2, and harvest index, including a pleiotropic marker for yield, harvest index, and grains/head were validated for its amplification efficiency and phenotypic effects in focused identification germplasm strategy (FIGS) wheat set and a doubled haploid (DH) population (Scepter/IG107116). The phenotypic variation explained by these markers are in the range of 4.1-37.6 in the FIGS population. High throughput PCR-based genotyping using new markers and association with phenotypes in FIGS wheat set and DH population validated the effect of functional SNP on closely associated genes-calcineurin B-like- and dirigent protein, basic helix-loop-helix (BHLH-), plant homeodomain (PHD-) and helix-turn-helix myeloblastosis (HTH myb) type -transcription factor. Further, genome-wide SNP annotation using SnpEff tool confirmed that these SNPs are in gene regulatory regions (upstream, 3'-UTR, and intron) modifying gene expression and protein-coding. This integrated approach of marker design for t-GBS alleles, SNP functional annotation, and high-throughput genotyping of functional SNP offers translation solutions across crops and complex traits in crop improvement programs.

Keywords: IWGSC RefSeqv1.0; SNPs; allele‐specific markers; haplotypes; high‐throughput SNP genotyping; pleiotropic SNP; t‐GBS; variant annotation; wheat.