A natural variation of an SVP MADS-box transcription factor in Triticum petropavlovskyi leads to its ectopic expression and contributes to elongated glume

Abstract

Triticum petropavlovskyi, commonly known as Xinjiang rice wheat, is a unique hexaploidy wheat grown in the Xinjiang area. T. petropavlovskyi is marked by its elongated glume (Eg) trait, which is controlled by gene P1pet located on chromosome 7A (Wang et al., 2002Wang H.J. Huang X.Q. Röder M.S. Börner A. Genetic mapping of loci determining long glumes in the genus Triticum.Euphytica. 2002; 123: 287-293Crossref Scopus (18) Google Scholar; Watanabe and Imamura, 2002Watanabe N. Imamura I. Genetic control of long glume phenotype in tetraploid wheat derived from Triticum petropavlovskyi Udacz. et Migusch.Euphytica. 2002; 128: 211-217Crossref Scopus (7) Google Scholar). The origin of T. petropavlovskyi has been disputed. It is generally accepted that P1pet was introduced from tetraploid polish wheat (T. polonicum) via a hybridization with hexaploid common wheat (T. aestivum) (Akond et al., 2008Akond A.S.M.G.M. Watanabe N. Furuta Y. Comparative genetic diversity of Triticum aestivum-Triticum polonicum introgression lines with long glume and Triticum petropavlovskyi by AFLP-based assessment.Genet. Resour. Crop Evol. 2008; 55: 133-141Crossref Scopus (10) Google Scholar; Chen et al., 2016Chen Q. Song J. Du W.P. Xu L.Y. Yu G.R. Possible origin of Triticum petropavlovskyi based on cytological analyses of crosses between T. petropavlovskyi and tetraploid, hexaploid, and synthetic hexaploid (SHW-DPW) wheat accessions.Span J. Agric. Res. 2016; 14: e0713https://doi.org/10.5424/sjar/2016144-8476Crossref Scopus (2) Google Scholar). The P1pet has pleiotropic effects in the increase of spike length, grain length, and grain weight, and decrease of fertility, grain number, and awn length (Okamoto et al., 2013Okamoto Yuki Takumi Shigeo Pleiotropic effects of the elongated glume gene P1 on grain and spikelet shape-related traits in tetraploid wheat.Euphytica. 2013; 194: 207-218Crossref Scopus (14) Google Scholar), indicating the potential impact of P1pet on grain yield-related traits. The cloning of P1pet will provide evidence for the prognosis of whether a mutation at P1 in T. petropavlovskyi or T. polonicum gave rise to the Eg trait and facilitate the utilization of this unique species in breeding for yield improvement. In this study, the locus P1pet was fine mapped via a stepwise mapping approach. By linkage analysis using a recombinant inbred line (RIL) population between T. petropavlovskyi AKSu (AKS) and Chinese Spring (CS) (supplemental Figure 1), the P1pet was mapped into a marker interval between Xwmc826 and Xgpw2119 (supplemental Figure 2A, supplemental Table 1), corresponding to the 101.30–130.24 Mb physical region of 7AS. Further mapping using a chromosome segment substitution line (CSSL) population between T. petropavlovskyi Luopu (LP) and Yangmai158 (YM) allocated P1pet into the 127.91–133.22 Mb region of 7AS, flanked by two SNP markers AX-109335244 and AX-108807463 (supplemental Figure 1 and 2B). The two mapping results shared the 127.91–130.24 Mb overlapping region, in which the P1pet could be allocated. To fine map P1pet, a secondary F2 population derived from a cross between YM and a CSSL line LZP87 (morphologically resembling YM except for the Eg trait from LP, supplemental Figure 2C and 2D). Two P1pet flanking markers, SSR0 and CAPS36261, encompassing the P1pet region, were used to genotype the population for recombinant screening. By phenotyping of 22 recombinants, we fine-mapped P1pet into a marker interval SNP41698 and SSR4, corresponding to a 128.8–129.6 Mb genomic region. Twelve genes (G1–G12) were annotated in this region according to RefSeqv1.1 annotation of CS references (Figure 1A). To predict the candidate gene for P1pet, gene expression profiles of LZP87 and YM at five developmental stages were compared using RNA sequencing (RNA-seq) (Figure 1B). At most of the tested stages, seven genes (G3–G9) were barely expressed; four genes (G1, G10, G11, and G12) were expressed, but they had little difference in expression level and no difference in genome sequences between two parents. Thus, these genes were precluded from P1pet candidates. G2 (TraesCS7A02G175200) encodes an SVP-like MADS-box transcription factor TaVRT-A2. It has been characterized as a key regulator in floral transition in wheat (Kane et al., 2005Kane N.A. Danyluk J. Tardif G. Ouellet F. Laliberte J.F. Limin A.E. Fowler D.B. Sarhan F. TaVRT-2, a member of the StMADS-11 clade of flowering repressors, is regulated by vernalization and photoperiod in wheat.Plant Physiol. 2005; 138: 2354-2363Crossref PubMed Scopus (103) Google Scholar; Xie et al., 2019Xie L. Zhang Y. Wang K. Luo X. Xu D. Tian X. Li L. Ye X. Xia X. Li W. et al.TaVrt2, an SVP-like gene, cooperates with TaVrn1 to regulate vernalization-induced flowering in wheat.New Phytol. 2019; https://doi.org/10.1111/nph.16339Crossref Scopus (18) Google Scholar). At all tested stages, TaVRT-A2 had very high expression level in LZP87, while it was barely expressed in YM. qRT–PCR analysis validated the above expression pattern (Figure 1C). The 6113-bp genomic sequences of TaVRT-A2pet (P1pet) cloned from AKS and LP were identical. The corresponding sequence in CS was 6516 bp and designated TaVRT-A2cs (P1cs). Sequence comparison revealed the presence of an insertion-deletion (Indel) polymorphism in intron-1, i.e., a 560-bp sequence in TaVRT-A2cs was substituted by a 157-bp sequence in TaVRT-A2pet (Figure 1D). A marker targeting this Indel, SVPA2-indel (supplemental Table 1), was developed. A total of 893 lines (94 AKS/CS RIL lines, 191 LP/YM CSSL lines, and 608 LZP87/YM F2 lines) were genotyped. Co-segregating of SVPA2-indel's 157-bp allele with the Eg trait further validated that TaVRT-A2pet is the P1pet candidate (Figure 1E). A mutant NAU32 with reduced glume length (GL) was identified by EMS mutagenesis of LP (Figure 1F). TaVRT-A2pet from NAU32 had a mutation of G > A transition at 4926 bp from its start codon. This mutation, located in the guanine (G) residue of the GT canonical splice donor site within intron-6 of TaVRT-A2pet, caused its non-splicing. We did detect the non-splicing of an intron-6 transcript in NAU32, demonstrated by the presence of an 815-bp transcript in NAU32 compared with 681 bp in LP and cDNA sequencing (Figure 1G). This mutation led to a frame-shift and generated a premature termination codon at 15 bp in intron-6. We propose that the mutated TaVRT-A2pet coded a truncated protein, which lost its function in regulating glume elongation. To further verify the function of TaVRT-A2pet, five independent transgenic plants overexpressing TaVRT-A2 were generated. Compared with the receptor variety Fielder (9.1 mm), they all showed increases in GL, ranging from 9.3 mm (OE-10) and 10.5 mm (OE-20), to more than 12.0 mm (OE-17, OE-7, and OE-15 being 12.3, 12.5, and 13.1 mm, respectively) (Figure 1H). The levels of increased GL are seemly positively correlated to TaVRT-A2 expression (Figure 1I). These results verified the function of TaVRT-A2 in regulating Eg. To estimate the origin of P1pet, we investigated the distribution of P1pet among Triticum species. TaVRT-A2 sequences from 21 samples from five Triticum species (including 15 wheat varieties, and T. spelta, T. dicoccoides, T. durum, T. turgidum, and T. urartu) were compared. They all showed a 560-bp sequence with only one to two SNPs and were P1cs haplotype (supplemental Data 1). A total of 278 wheat varieties, 13 accessions of T. polonicum, and two accessions of T. turanicum were genotyped using SVPA2-indel. All wheat varieties were P1cs haplotype (data not show), and all T. polonicum accessions were P1pet haplotype (supplemental Figure 3A). Cloning of TaVRT-A2 (P1pol) from T. polonicum showed that genomic sequences of P1pol and P1pet were 100% identical. This established the suggestion that P1pet may be introduced from T. polonicum (supplemental Figure 3B). Notably, T. turanicum had the Eg trait but was P1cs haplotype (supplemental Figure 3A), implying its different origin from the Eg trait. The mechanism of the TaVRT-A2pet in regulating Eg was preliminarily investigated. RNA-seq revealed TaVRT-A2 was highly and distinctively expressed in spikes of LZP87, while barely expressed in YM at five spike developmental stages. TaVRT-A2 homologous genes, TaVRT-B2, TaVRT-D2, and their paralogous TaSVP1s and TaSVP2s (Schilling et al., 2020Schilling S. Kennedy A. Pan S. Jermiin L.S. Melzer R. Genome-wide analysis of MIKC-type MADS-box genes in wheat: pervasive duplications, functional conservation and putative neofunctionalization.New Phytol. 2020; 225: 511-529Crossref PubMed Scopus (83) Google Scholar), were barely expressed or only expressed at low levels across all the above stages (supplemental Figure 4A). In leaves and stems at glume primordium and lemma primordium stages, an increased and ectopic expression level of TaVRT-A2 in LZP87 relative to YM were also observed (supplemental Figure 4B). Interestingly, despite loss of function of TaVRT-A2 in NAU32, its expression is comparable to that in LP, due probably to the absence of the 560-bp sequence (supplemental Figure 4C). Thus, we assumed that the 560-bp allele of TaVRT-A2 in intron-1 may be associated with low TaVRT-A2 expression. Methylation-specific PCR assay (supplemental Figure 5A, supplemental Data 2) and methylation-sensitive restriction enzyme digestion by endonucleases HpaII and/or MspI (supplemental Figure 5B) were performed to investigate the DNA methylation level of the 560-bp sequence. The results revealed that the 560-bp sequence was hypomethylated, suggesting that DNA methylation is unlikely to be the reason for low TaVRT-A2 expression of the P1cs haplotype. We further searched for conserved motifs within the 560-bp sequence that may act as cis-regulators. The TaVRT-A2 homologs from Triticeae and orthologs from other Poaceae species all contained a relatively conserved P1cs haplotype rather than the P1pet haplotype (supplemental Data 3). Multiple sequence alignment identified two highly conserved motifs within the 560-bp sequence in intron-1 (supplemental Figure 6). It was proposed that the 157-bp sequence substitution consisted of reccurring sequence units from the local rearrangement flanking either side of the substitution (Adamski et al., 2021Adamski N.M. Simmonds J. Brinton J.F. Backhaus A.E. Chen Y. Smedley M. Hayta S. Florio T. Crane P. Scott P. et al.Ectopic expression of Triticum polonicum VRT-A2 underlies elongated glumes and grains in hexaploid wheat in a dosage-dependent manner.Plant Cell. 2021; https://doi.org/10.1093/plcell/koab119Crossref PubMed Scopus (12) Google Scholar), and thus it unlikely contained putative DNA binding motifs for transcription factors. We speculated that the identified conserved motif or motifs within the 560-bp sequence may act as cis-regulatory for TaVRT-A2 expression. The loss of the 560-bp sequence in intron-1 may lead to ectopic expression TaVRT-A2pet and thus Eg morphology. The genetic effects of P1pet on other agronomic traits was assessed. Unlike the bimodal-distribution of GL, four traits (SL, KL, KW, and TGW) showed normal distribution patterns in the AKS/CS RIL population (supplemental Figure 7). Phenotypic correlations indicated that GL was positively and significantly correlated with SL (0.63–0.75) and KL (0.25–0.40), while not KW and TGW (supplemental Figure 8). The genetic effects of P1pet were evaluated by comparing NAU32 with wild-type LP. Apart from the shortened glume, NAU32 exhibits reduced SL, lemma length, KL, and grain weight, while increased grain numbers per spike (supplemental Table 2). These results demonstrated the pleiotropic effects of P1pet, suggesting that optimizing P1pet gene expression may have potential value for improvement of wheat grain yield. In summary, our study validated the function of TaVRT-A2pet in regulating multiple spike- and grain-related traits in wheat. The cloning of P1pet provided further evidence for the presence of frequent gene exchanges among different ploidy Triticum species during evolution and domestication of common wheat. Our study also expanded the understanding of spatial and temporal cis-regulation of SVP genes from different sub-genomes in leading to their functional diversification. The pleiotropic effects of P1pet demonstrated its potential value in breeding for grain yield, either by marker-assisted selection using the developed CSSLs or by optimizing its expression using molecular tools. This research was supported by the funds from the National Key Research and Development Program , China (grant nos. 2016YFD0100302 and 2020YFE0202900 ), the National Natural Science Foundation of China , China (grant no. 91935304 ), the International Cooperation and Exchange of the National Natural Science Foundation of China , China (grant no. 31661143005 ), the Jiangsu Agricultural Technology System , China (JATS) (no. 2020411 ), the SAAS Program for Excellent Research Team , China, and the Key Research and Development Major Project of Ningxia Hui Autonomous Region , China (no. 2019BBF02022-04 ).

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