Validation of the major locus for FT on chromosome 7B
Wheat is an allopolyploid cereal crop with a complex genetic background, and contains some linkage regions exceeding 10 Mb (Zhu et al. 2019; Jiang et al. 2022). The simultaneous detection of QFT.ahau-7B.1, QFT.ahau-7B.2, and QFT.ahau-7B.3 within the 560.51-572.42 Mb region (11.91 Mb physical distance) may be due to the highly-linked LD block. In order to define the actual interval of these three stable loci, we extended this target region to 554.78-575.48 Mb (the upstream and downstream of 560.51-572.42 Mb extend a physical distance of 5.73 Mb and 3.06 Mb, respectively) for LD analysis. Subsequent pairwise LD correlation (r2 > 0.6) of the Wheat 90K SNP array resulted in the identification of 55 SNPs in the 554.78-575.48 Mb interval. Of these, 38 SNPs were mapped to four linkage regions, including QFT.ahau-7B.1 (556.46-561.75 Mb), QTL-free block (564.17-565.05 Mb), QFT.ahau-7B.2 (565.74-566.48 Mb), and QFT.ahau-7B.3 (568.50-572.42 Mb) (Fig. 2a, b, c).
Based on three significant SNPs for the three stable loci (BS00010557_51 for QFT.ahau-7B.1, RAC875_c3229_165 for QFT.ahau-7B.2, and BS00060341_51 for QFT.ahau-7B.3), three CAPS markers (CAPS-BS00010557_51, CAPS-RAC875_c3229_165, and CAPS-BS00060341_51, respectively) were developed to validate their associations with FT in 431 wheat varieties (lines). The U-test results showed that there were significant differences in FT between wheat varieties (lines) with the two allelic variations of CAPS-BS00010557_51, CAPS-RAC875_c3229_165, and CAPS-BS00060341_51. The FT of wheat varieties (lines) carrying the T allele for CAPS-BS00010557_51, C allele for CAPS-RAC875_c3229_165, and C allele for CAPS-BS00060341_51 was significantly greater than of that of the varieties (lines) carrying the G allele for CAPS-BS00010557_51, T allele for CAPS-RAC875_c3229_165, and T allele for CAPS-BS00060341_51 (P < 0.01) (Fig. 2e, Table S7). These preliminary results validate the associations of the three stable loci (QFT.ahau-7B.1, QFT.ahau-7B.2, and QFT.ahau-7B.3) with FT in wheat.
Through association mapping, we found that the representative CAPS marker (CAPS-RAC875_c3229_165) for QFT.ahau-7B.2 was significantly associated with all nine sets of FT phenotypic data (including BLUPs) (P values: 5.58 × 10− 7-5.15 × 10− 14; PVE: 10.89–26.33%) from 245 wheat varieties (lines). Additionally, U-test results showed that CAPS-RAC875_c3229_165 for QFT.ahau-7B.2 was highly significantly correlated with all nine sets of FT phenotypic data (correlation coefficient: 6.259–11.728) from 431 wheat varieties (lines). Based on these results, the major stable locus QFT.ahau-7B.2 was selected for exploration of candidate genes.
Identification of the candidate genes underlying QFT.ahau-7B.2
To explore candidate genes underlying QFT.ahau-7B.2, four polymorphic SNPs (AX109460788, AX109363358, AX109501979, and AX111588585) within the target region were chosen to develop CAPS markers (designated CAPS-AX109460788, CAPS-AX111588585, CAPS-AX109363358, and CAPS-AX109501979, respectively) for regional association (Fig. 3a, Table S8) and LD analysis (Fig. 3b). LD analysis revealed that the four CAPS markers were closely linked with the CAPS marker CAPS-RAC875_c3229_165 for QFT.ahau-7B.2. The regional association mapping results showed that two CAPS markers (CAPS-AX109363358 [566.13 Mb] and CAPS-AX109501979 [566.63 Mb]) were significantly associated with FT, while the other two CAPS markers (CAPS-AX109460788 [565.77 Mb] and CAPS-AX111588585 [566.76 Mb]) exhibited no correlation with FT. The peak of -log10(P) was located at CAPS-RAC875_c3229_165 (566.48 Mb) between CAPS-AX109363358 (566.13 Mb) and CAPS-AX109501979 (566.63 Mb). Therefore, QFT.ahau-7B.2 was further focused to the 566.13-566.63 Mb region, in which four genes were annotated (TraesCS7B02G316700, TraesCS7B02G316800, TraesCS7B02G316900, and TraesCS7B02G317000) (Table S9).
We further cloned the promoter, untranslated region, and coding sequence of the above four candidate genes in three freezing-tolerant varieties (‘Jimai 22’, ‘Tai 10604’, and ‘Annong 9267’) and three freezing-sensitive varieties (‘Yangnuomai 1’, ‘Yangmai 16’, and ‘Yumai 9’). The sequence analysis results indicated that all four genes contained numerous mutations in the coding and promoter regions. To study the expression patterns of these four genes in response to freezing stress, we subjected the freezing-tolerant and freezing-sensitive wheat varieties to low temperature treatment (Fig. 3c). The results indicated that TraesCS7B02G316700, which encodes Resistance to Pseudomonas syringae pv. Maculicola 1 (TaRPM1-7BL), and TraesCS7B02G316800, which encodes Type 1 membrane protein-like, exhibited significant differences in expression after low temperature treatment between freezing-tolerant and freezing-sensitive varieties. In particular, TaRPM1-7BL expression was consistently and significantly induced by low temperature (-6°C) in freezing-sensitive varieties compared to tolerant varieties. The TaRPM1-7BL gene remained minimally expressed at 20°C, 4°C, and − 2°C in both tolerant and sensitive varieties as the temperature was decreased. When the temperature was further decreased to -6°C, TaRPM1-7BL expression was significantly upregulated in freezing-tolerant (by 1.11-fold, on average) and freezing-sensitive varieties (by 49.55-fold, on average), compared to 20°C. In addition, the TaRPM1-7BL gene was significant upregulated (by 5.14-fold, on average) in freezing-sensitive varieties compared to tolerant varieties at -6°C. Subsequently, the expression levels of TaRPM1-7BL decreased significantly in both tolerant and sensitive varieties when the temperature returned to 4°C and 20°C. Based on these observations, TaRPM1-7BL was considered as a potential candidate gene underlying QFT.ahau-7B.2 for FT in wheat.
Sequence analysis of TaRPM1-7BL and CAPS marker development
We next cloned the TaRPM1-7BL promoter (approximately 1500-bp), untranslated region, and coding sequence of the freezing-tolerant varieties (‘Jimai 22’, ‘Tai 10604’, and ‘Annong 9267’) and freezing-sensitive varieties (‘Yangnuomai 1’, ‘Yangmai 16’, and ‘Yumai 9’). The TaRPM1-7BL gene was 4874-bp in length, containing a 132-bp 5'UTR, 2 exons, 4 introns, and one 457-bp 3'UTR. The CDS of TaRPM1-7BL was 2676-bp in length, encoding an 891 amino acid NLR disease resistance protein containing a coiled-coil (CC), nucleotide binding site (NBS), and leucine rich repeat (LRR) domain. Sequence alignment analysis showed that there were 76 sequence variations in the TaRPM1-7BL gene between freezing-tolerant and freezing-sensitive varieties, including 11 in the promoter region, 39 in the coding region (20 missense and 19 synonymous), 24 in the intron, one in the 5’UTR, and one in the 3’UTR. The 20 missense mutations in the coding region consisted of one in the CC (amino acids 7-126), ten in the NBS (amino acids 159–376), five in the LRR (amino acids 502–877), and four in the domainless region (Fig. 4a). The promoter sequence of TaRPM1-7BL was predicted to contain seven cis-acting elements responsive to abscisic acid (ABRE), methyl jasmonate (CGTCA-motif and GA-motif), MYB, light (TCCC-motif, GC-motif, I-box, and Sp1), anoxia (GC-motif and ARE), auxin (TGA-element), circadian control (circadian), and cell cycle regulation (MSA-like) (Fig. 4a). These data suggest that TaRPM1-7BL may be widely involved in abiotic stress response, growth, development, photoperiodicity, and cell cycle regulation.
To investigate natural variations in TaRPM1-7BL among different wheat varieties, we extracted whole-exome capture sequencing data of TaRPM1-7BL from 160 wheat varieties with diverse genetic backgrounds for haplotype analysis. After excluding the heterozygous genotypes (13), the TaRPM1-7BL gene was found to have 21 missense mutations and form three haplotypes, including two main haplotypes and one rare haplotype: Hap1 (99, 67.3%), Hap2 (47, 32.0%), and Hap3 (1, 0.7%) (Table S10). Based on cloning sequence results, the two main haplotypes, Hap1 and Hap2, corresponded to the freezing-tolerant and freezing-sensitive haplotypes, respectively. Notably, we identified a rare haplotype present only in the Hongyouzi landrace which results in a change from tryptophan to arginine at amino acid 364 on the basis of Hap1. Therefore, we suggest that the amino acid sequence of TaRPM1-7BL is relatively conserved.
To further validate the association of the TaRPM1-7BL gene with FT, a representative CAPS marker (CAPS-TaRPM1-7BL) was developed based on the missense mutation (A/G) at position + 2555 (in NBS domain) and then used to genotype 431 wheat varieties (lines) and 318 F2 lines derived from a cross of ‘Annong 9267’ × ‘Yumai 9’ (AY). The U-test results showed that there were significant differences in FT between varieties carrying the two alleles of TaRPM1-7BL (P < 0.01). Varieties containing the allele A exhibited stronger FT compared to those containing the G allele (Fig. 4b, c). These results demonstrate that TaRPM1-7BL is associated with FT in wheat.
Tissue expression patterns of TaRPM1-7BL
To investigate the tissue expression patterns of TaRPM1-7BL in response to freezing stress, transcript levels of TaRPM1-7BL were detected at 20°C and − 6°C in roots, stems, leaves, and developing grains (Fig. 5). Relatively low transcript levels of TaRPM1-7BL were observed in all tissues at 20°C. By contrast, TaRPM1-7BL expression increased 14.97-, 3.16-, 15.35-, and 4.29-fold in roots, stems, leaves, and grains, respectively, at -6°C. These results indicate that TaRPM1-7BL is responsive to freezing stress in all tissues, particularly in leaves, and support the association of TaRPM1-7BL with FT in wheat.
Preliminary functional analysis of TaRPM1-7BL
To validate the role of TaRPM1-7BL in regulating FT in wheat, we constructed TaPDS (BSMVTaPDS) and TaRPM1-7BL-silenced (BSMVTaRPM1−7BL) plants in the freezing-sensitive ‘Yumai 9’ background using barley stripe mosaic virus (BSMV)-based VIGS. ‘Yumai 9’ inoculated with BSMV transcripts without exogenous genes (BSMV0) and FES buffer (WT) were used as controls. Both BSMVTaRPM1−7BL and BSMVTaPDS exhibited stripe mosaic symptoms and photobleaching 14 days after virus inoculation, respectively, suggesting the induction of BSMV-mediated silencing (Fig. 6a). qRT-PCR was performed to identify the relative expression level of TaRPM1-7BL in silenced and control plants at 15 days post-inoculation. The results showed that the TaRPM1-7BL gene was silenced and that transcription decreased by 58–94% in BSMVTaRPM1−7BL compared to BSMV0 and WT (Fig. 6b). We observed drooping and wilting in BSMVTaRPM1−7BL, BSMV0, and WT plants after freezing treatment and found that the frost damage of BSMVTaRPM1−7BL plants was significantly lighter than that of BSMV0 and WT plants (Fig. 6c). These data demonstrate that TaRPM1-7BL is a potential candidate gene underlying QFT.ahau-7B.2 for FT in wheat.