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

Abstract

Using bulked segregant analysis of exome sequence, we fine-mapped the ABA-hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A. Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat 'Zak.' ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660^S allele as Zak, whereas Louise carries the PHS-tolerant MKK3-A-C660^R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.

Fig. 1

The BSA-exome-seq method. a Genetics. The BC₃F_2:3 population was developed by crossing Zak WT (+/+) and BC₂F₃ ZakERA8 (ERA8/ERA8). BC₃F_2:3 genomic DNA was used for exome-seq. The phenotype was BC₃F_2:3 grain germination on ABA. b Selection of bulked lines. Compared to the parental ERA8 (purple arrow) and WT (green arrow) germination on ABA, 17 ERA8-like and 15 WT-like lines were chosen for bulked segregant analysis. c The number of 125 bp paired-end Illumina HiSeq 2000 reads generated for each parent, WT bulk (WT_b), and ERA8 bulk (ERA8_b) are shown. d Alignment of parental reads to the Chinese Spring IWGSC reference sequence (v 1.0), where varietal differences from Chinese Spring were shared by the Zak WT and ERA8 parents, and mutagen-induced (C/G to T/A) were ERA8-specific. e Unlinked SNPs showed random variation in WT_b versus ERA8_b, whereas ERA8-linked SNPs did not. An example of an exon alignment and bulk frequency ratios is shown. f KASP markers were designed to score SNPs. SNP_17 is shown (color figure online)

Fig. 2

Bulk sequence frequencies aligned to the wheat genome. a The outer track is the physical size (million bp per tick mark) of each chromosome based on the RefSeq v1.0 wheat reference genome sequence. b Bulk frequencies (BF) of the ERA8_b (purple) and WT_b (green) were aligned to the reference genome and were calculated as a fraction of the alternate allele read depth over the total number of reads. c The BF differences (BFD) were calculated from the differences between the BF of ERA8_b and the BF of WT_b (color figure online)

Fig. 3

Fine mapping ERA8 on chromosome 4A. QTL analysis was performed using mutagen-induced SNPs and germination index on ABA in the: a Zak/ZakERA8 BC₃F₃ from crosses X5.1 and X5.2, b Louise/ZakERA8 F_6:7, and c the Otis/ZakERA8 F_2:3 populations. Population sizes were 424, 207, and 108 individuals, respectively. The Louise/ZakERA8 population was phenotyped over three environments (E1, E2, and E3). Marker distances (cM) of the 4A region of interest were calculated using JoinMap. SNP positions are marked above the graph

Fig. 4

The ERA8 interval. a Initial BSA based on exome-seq of 32 BC₃F_2:3 identified the ERA8 region (gray) on chromosome 4A. The centromeric region is in red. b QTL analysis relative to EMS-induced SNPs in 424 BC₃F_2:3 mapped a 4.6 cM region containing the ERA8-linked interval (purple bar) spanning c the 4 unique genes containing SNP_6 through SNP_29. The number of predicted genes between SNPs is in brackets. The TaMKK3-A position is estimated based on previous mapping studies. d Lines containing recombination events in the SNP_6 to SNP_29 region were placed into groups I to VII based on whether they carried the ERA8 (purple) or Zak WT (white) SNP allele. e The ABA germination phenotype of groups I-VII is shown as a box and whisker plot where dots show the percent germination of individual BC₃F_2:3-4 lines. The population parents ERA8 and Zak, and BC₃F_2:3 line where all four SNPs had the ERA8 genotype (ERA8-g) or Zak genotype (Zak-g) are shown as controls. ERA8-like (purple) and Zak-like (green) groups were determined based on Student’s t test (p ≤ 0.05) compared to the parents. n = the number of lines per group (color figure online)

Fig. 5

ABA sensitivity of Louise (blue), ERA8 (purple), Zak WT (green), and the Louise/ZakERA8 RIL(gray) population. The parents (Louise, ERA8, and Zak WT) percent germination after 5 days of imbibition over an after-ripening time course in environment 1 were assayed in: a whole seeds on 2 µM ABA and b cut seeds on 0, 2, 5, and 10 µM ABA. c The RIL population was assayed across three environments with the percent germination after 2, 3, and 1 day of imbibition on 5 µM, 2 µM, and 2 µM of ABA shown, respectively. Environments: E1) F₆ seed from the greenhouse 2013; E2) F₇ seed from the field 2014; and E3) F₇ seed from the field 2015 (color figure online)

Fig. 6

Alleles of the TaMKK3-A gene. Increased dormancy and PHS tolerance may result either from the recessive Phs1 allele at + 660 bp or from the semi-dominant EMS-induced ERA8 allele at + 1093 bp of MKK3-A. Nucleotide and amino acid differences are shown for the S (susceptible) and R (resistant) alleles. The genotypes of ERA8, Zak, Otis, and Louise are shown at both positions