GWA mapping of cold tolerance traits at the seedling stage and validation in two rice biparental mapping populations

Authors: Eizenga, Georgia; SHIMOYAMA, NAOKI - Marquette University; Jackson, Aaron; Edwards, Jeremy; JESSEL, AVERY - Marquette University; SCHLAPPI, MICHAEL - Marquette University

Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 12/4/2019
Publication Date: 1/6/2021
Citation: Eizenga, G.C., Shimoyama, N.S., Jackson, A.K., Edwards, J., Jessel, A.R., Schlappi, M.R. 2021. GWA mapping of cold tolerance traits at the seedling stage and validation in two rice biparental mapping populations. Proceedings of 38th Rice Technical Working Group Meeting, February 24-27, 2020, Orange Beach, Alabama. p 81-82. Electronic Publication.

Interpretive Summary:

Technical Abstract: Cold stress at the seedling stage causes poor stand establishment, necrosis and injures plants. Improved seedling cold tolerance in rice (Oryza sativa L.) would enable earlier planting in the spring in the southern USA, thus potentially reducing the stress of summer high night time temperatures and allowing a longer season for the ratoon crop. The overall goal is to understand the basic genetic and cellular mechanisms responsible for seedling cold tolerance. The specific objective of this study was to identify seedling cold stress QTL and the underlying putative candidate gene(s) by conducting genome-wide association (GWA) mapping with two association mapping panels and QTL mapping with two biparental populations. Previously, the USDA Rice Minicore Collection (RMC) which includes 202 diverse accessions, was evaluated for five traits designed to mimic cold stress response in the natural environment, two traits were associated with cold stress at germination and three at the seedling stage. GWA mapping was conducted with 157 markers including 148 SSR markers, three InDel markers and six SNP markers. This study uncovered 48 GWA-QTL at 39 chromosome regions distributed across all 12 rice chromosomes. Recently, 173 RMC accessions were genotyped with 3.2 million SNPs, thus the GWA mapping was conducted with this high-density genotyping and the same trait data utilizing the mixed linear model in Tassel 5. To further validate the RMC GWA-QTL, 354 accessions from the Rice Diversity Panel 1 (RDP1) were evaluated for two traits allied with seedling stage cold tolerance, Low Temperature Seedling Survivability (LTSS) and Electrolyte Leakage (EL), to identify GWA-QTL and the underlying candidate genes. For LTSS, the one week of cold stress was performed at four different temperatures (8, 10, 12 and 16° C) and for EL, the one week of cold stress also was performed at four temperatures (4, 10, 12 or 16° C). Using the LTSS and EL data collected across the different temperatures and the 700K SNP genotypes for the RDP1 accessions, GWA mapping was conducted with the association mapping pipeline developed for the RDP1. Perusal of the significant SNPs identified across the different temperatures for LTSS and EL uncovered 40 GWA-QTL, identified as Multiple Chilling Phenotype (MP) QTL. Six of these GWA MP-QTL, qMP3-1, qMP6-2, qMP9-4, qMP10-1, qMP10-4 and qMP11-2, overlapped with previously reported RMC GWA-QTL. Database searches revealed eleven candidate genes near MP3-1, MP6-2, MP9-4 and MP10-4. To further validate these GWA-QTL, two biparental mapping populations were developed from three RMC accessions. Carolino 164, an aus accession, was the cold susceptible male parent, and the temperate japonica accessions, Krasnodarskij 3352 and Wir911 were the cold tolerant female parents of the recombinant inbred line (RIL) and backcross inbred line (BIL) populations, respectively. The parents and progeny were genotyped with 7K SNPs and phenotyped for LTSS and EL at 10° C. QTL mapping of 93 Krasnodarskij 3352/Carolino 164 F7 RILs with QTL IciMapping identified six LTSS QTL (qLTSS3, qLTSS4-1, qLTSS4-2, qLTSS6, qLTSS8, qLTSS9) and qEL6. Only qLTSS4-1 was not co-located with a previously identified GWA-QTL and candidate genes associated with various stress tolerances were found in the qLTSS3, qLTSS4-2 and qLTSS8 regions. A vacuolar H- translocating inorganic pyrophosphatase 1 gene (OVP1), associated with cold stress, was located in the qEL6 region. QTL mapping with 93 BC1F5 (Wir911/Carolino 164)/Carolino 164 BILs genotyped with 7K SNPs and phenotyped for LTSS and EL, identified three QTL below the permuted LOD threshold, qLTSS1, qLTSS8 and qEL8. Only qLTSS8 overlapped with RMC and RDP1 GWA-QTL but no relevant candidate genes were discovered. The next steps are to validate selected candidate genes with transgenic studies, the CRISPR/Cas9 system, and/