Haplotype Analysis of the Pre-harvest Sprouting Resistance Locus Phs-A1 Reveals a Causal Role of TaMKK3-A in Global Germplasm

Abstract

Pre-harvest sprouting (PHS) is an important cause of quality loss in many cereal crops and is particularly prevalent and damaging in wheat. Resistance to PHS is therefore a valuable target trait in many breeding programs. The Phs-A1 locus on wheat chromosome arm 4AL has been consistently shown to account for a significant proportion of natural variation to PHS in diverse mapping populations. However, the deployment of sprouting resistance is confounded by the fact that different candidate genes, including the tandem duplicated Plasma Membrane 19 (PM19) genes and the mitogen-activated protein kinase kinase 3 (TaMKK3-A) gene, have been proposed to underlie Phs-A1. To further define the Phs-A1 locus, we constructed a physical map across this interval in hexaploid and tetraploid wheat. We established close proximity of the proposed candidate genes which are located within a 1.2 Mb interval. Genetic characterization of diverse germplasm used in previous genetic mapping studies suggests that TaMKK3-A, and not PM19, is the major gene underlying the Phs-A1 effect in European, North American, Australian and Asian germplasm. We identified the non-dormant TaMKK3-A allele at low frequencies within the A-genome diploid progenitor Triticum urartu genepool, and show an increase in the allele frequency in modern varieties. In United Kingdom varieties, the frequency of the dormant TaMKK3-A allele was significantly higher in bread-making quality varieties compared to feed and biscuit-making cultivars. Analysis of exome capture data from 58 diverse hexaploid wheat accessions identified fourteen haplotypes across the extended Phs-A1 locus and four haplotypes for TaMKK3-A. Analysis of these haplotypes in a collection of United Kingdom and Australian cultivars revealed distinct major dormant and non-dormant Phs-A1 haplotypes in each country, which were either rare or absent in the opposing germplasm set. The diagnostic markers and haplotype information reported in the study will help inform the choice of germplasm and breeding strategies for the deployment of Phs-A1 resistance into breeding germplasm.

Applying the latest advances in genomics and phenomics for trait discovery in polyploid wheat

“…With these data, it becomes clear that, in the absence of the causal polymorphism per se , it is important to recognize the haplotype as the unit of genetic information that underlies traits, rather than a closely associated bi‐allelic SNP. The case study of the wheat pre‐harvest sprouting resistance gene TaMKK3 provides a telling example to this effect (Barrero et al ., ; Torada et al ., ; Shorinola et al ., ). Thus, when making crosses, breeders are shuffling haplotypes to generate new combinations of these blocks of DNA.…”

Exome sequencing of bulked segregants identified a novel TaMKK3-A allele linked to the wheat ERA8 ABA-hypersensitive germination phenotype

“…KASP markers for SNPs identified through whole genome exome resequencing of Zak and ZakERA8 were designed using PolyMarker with default settings (Table S4; Ramirez-Gonzalez et al 2015b). The KASP assay for the naturally occuring preharvest sprouting susceptible (A660) versus resistant (C660) alleles of TaMKK3-A was performed as described in (Shorinola et al 2017). Genomic DNA was extracted from BC 3 F 2 leaf tissue on the Qiagen BioSprint-96, and DNA concentrations determined using the BioTek Gen5 plate reader.…”

“…The X5.2 population likely segregated for a weaker ERA8 phenotype because it lost more dormancy due to longer storage in the freezer before plating (Table S1). Interestingly, SNP_17 is an ERA8-linked missense mutation in the cloned preharvest sprouting tolerance gene, TaMKK3-A (Nakamura et al 2016;Torada et al 2016;Shorinola et al 2017). Because the TaMKK3-A gene was not present in the assigned chromosomes of the RefSeq v1.0 wheat genome reference assembly, its relative position was estimated based on neighboring genes in the draft WGA v0.4 sequence (IWGSC et al 2014;Shorinola et al 2017).…”

“…Based on the hypothesis that the ERA8 phenotype may have resulted from a mutation in the TaMKK3-A coding region, we characterized the TaMKK3-A alleles of Zak, ZakERA8, and Louise using KASP assays (Shorinola et al 2017). The naturally-occuring PHS tolerant allele of TaMKK3-A/Phs1 in wheat results from a C nucleotide at position +660 associated with a recessive dormant phenotype, whereas the PHS susceptible allele is an A nucleotide at position +660 (Torada et al 2016).…”

“…To date, 18 dormancy and 13 PHS studies have identified a QTL on the long arm of chromosome 4A called the Phs1 locus (Liu et al 2008(Liu et al , 2014Mares and Mrva 2014;Shorinola et al 2016;Martinez et al 2018;Zuo et al 2019). This 4A QTL has been cloned as the wheat Mitogen-Activated protein Kinase Kinase 3 gene (TaMKK3-A) (Nakamura et al 2016;Torada et al 2016;Shorinola et al 2017). Another major PHS tolerance QTL on chromosome 3A was cloned from Japanese and U.S. germplasm, the wheat gene MOTHER OF FLOWERING TIME AND TFL1 (TaMFT1) (Nakamura et al 2011;Liu et al 2013b).…”