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Homologous haplotypes, expression, genetic effects and geographic distribution of the wheat yield gene TaGW2

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TaGWA, cloned in earlier research, strongly influences wheat grain width and TKW. Here, we mainly analyzed haplotypes of TaGWB and their effects on TKW and interaction with haplotypes at TaGWA.


About  kb of the promoter sequences of TaGWB and TaGWD were cloned in 34 bread wheat cultivars. Eleven SNPs were detected in the promoter region of TaGWB, forming 4 haplotypes, but no divergence was detected in the TaGWD promoter or coding region. Three molecular markers including CAPS, dCAPS and ACAS, were developed to distinguish the TaGWB haplotypes. Haplotype association analysis indicated that TaGWB has a stronger influence than TaGWA on TKW, and Hap-6B-1 was a favored haplotype increasing grain width and weight that had undergone strong positive selection in global wheat breeding. However, clear geographic distribution differences for TaGWA haplotypes were found; Hap-6A-A was favored in Chinese, Australian and Russian cultivars, whereas Hap-6A-G was preferred in European, American and CIMMYT cultivars. This difference might be caused by a flowering and maturity time difference between the two haplotypes. Hap-6A-A is the earlier type. Haplotype interaction analysis between TaGWA and TaGWB showed additive effects between the favored haplotypes. Hap-6A-A/Hap-6B-1 was the best combination to increase TKW. Relative expression analysis of the three TaGW2 homoeologous genes in 22 cultivars revealed that TaGWA underwent the highest expression. TaGWD was the least expressed during grain development and TaGWB was intermediate. Diversity of the three genes was negatively correlated with their effect on TKW.


Genetic effects, expression patterns and historic changes of haplotypes at three homoeologous genes of TaGW2 influencing yield were dissected in wheat cultivars. Strong and constant selection to favored haplotypes has been found in global wheat breeding during the past century. This research also provides a valuable case for understanding interaction of genes that control complex traits in polyploid species.


Common wheat is a hexaploid species (AABBDD) with a large genome size ( × 109 bp) and abundant repeat sequences (>80%) [1]. Comparative genomics proved the existence of genomic colinearity among cereal crops [2]. As a model plant of cereals, the rice genomic sequence completed in [3, 4], and several yield-related genes [5, 6], such as GS3, GW2, GW5, GW8, TGW6, Ghd7 and GIF1, have been isolated [7–13], providing opportunities for homology-based cloning of yield-related genes in other cereals. The availability of a draft wheat genome sequence [14–17] will promote genome-based research of this extremely important crop. Cloning yield-related genes, exploring the favored alleles and developing functional markers will be important for yield improvement in that crop. This will be the next major focus of wheat genetics and genomics.

Among yield-related genes, current studies on gene function and allele discovery of GW2 are the most in-depth and extensive in cereal crops. Firstly, Song et al.[8] isolated a major yield QTL from rice, which was mapped on short arm of chromosome 2 and designated as OsGW2. It encoded a RING-type protein with E3 ubiquitin ligase activity that negatively regulated grain width, and loss-of-function mutations enhanced grain weight and yield. In maize, Li et al. () [18] found two homologs of OsGW2, viz. ZmGW2-CHR4 and ZmGW2-CHR5, and a SNP in the promoter region of ZmGW2-CHR4 was significantly associated with kernel width (KW) and hundred kernel weight (HKW) in maize. We cloned TaGW2 from chromosome 6A of wheat, and found SNPs in its promoter region, that were significantly associated with KW and TKW. A CAPS marker was developed based on the A/G polymorphism and association analysis indicated that Hap-6A-A increased TKW by more than  g [19]. Recently, a TaGWA-CAPS marker was used to detect variation in a BC2F4 RIL population, as well as a natural population, further demonstrating that TaGWA was significantly associated with grain weight [20]. Yang et al.[21] identified a single-base insertion in the eighth exon of TaGWA causing premature termination in landrace Lankaodali, which ultimately led to increased grain width and grain weight. However, Bednarek et al.[22] showed that the patterns of TaGW2 regulation of grain development might be more complex after studies on RNA interference (RNAi) of expression of TaGW2 in wheat. In consideration of the characteristics of the wheat genome, further dissection of the regulation and expression patterns of the three TaGW2 homoeologous genes on grain weight could have important biological and breeding implications.

In this study, further research focused on sequencing and diversity studies of the promoter regions of TaGWB and TaGWD, functional marker development, and an expression pattern comparison of the three homoeologous TaGW2 loci. Hence, the major objectives were to (1) reveal sequence diversity and distribution characteristics of the three GW2 homoeologous genes by sequence alignment of their ~ kb promoter regions; (2) develop functional markers for TaGWB and TaGWD to distinguish various haplotypes, and discover favored haplotypes for yield improvement through association analysis; (3) evaluate the distributions of different haplotypes in global wheat major production regions, including North America, Europe, Australia, Russia, Mexico and China, and understand the selection intensity and geographical distribution of TaGW2s in different wheat ecological regions; (4) assess the relationships between the expression levels of the three TaGW2 homoeologues and grain size by real-time PCR analysis, and preliminarily evaluate the genetic effects of TaGW2s based on phenotypic variation (R2) for grain traits; and (5) examine interactions among the three TaGW2 loci on chromosomes 6A, 6B and 6D through haplotype combination analysis. It was expected that the study would identify important genes and functional markers for wheat yield improvement.


Major variations in TaGW2soccur in the promoter regions

In the coding sequence of TaGW2 homoeologous genes, 34 wheat accessions (Additional file 1: Table S3) were used to study the nucleotide polymorphism and no divergence was found. Genome walking was used to clone the sequences of the promoter regions of TaGWB and TaGWD, and ~ kb upstream sequences from the ATG start codons were obtained. The core elements of the promoters were predicted with the TSSP program (, and the TATA box and STS (Start Transcription Site) were identified at  bp and  bp upstream from the ATG codon of TaGWB. For TaGWD, the corresponding locations were located at  bp and  bp, respectively. Generally, more variations in TaGW2s occurred in the promoter regions, but the diversity of TaGWB was higher than that of TaGWA, in which eight SNPs forming two haplotypes were found earlier [19]. No divergence was detected in the TaGWD promoter region (Figure 1). Four haplotypes were formed by 11 SNPs within the  kb upstream sequence of TaGWB; these were designated Hap-6B-1, Hap-6B-2, Hap-6B-3 and Hap-6B-4 (Figure 2).

Haplotypes in promoter region of TaGWBhave strong effects on TKW

TaGWB marker development

In the 11 SNPs detected in the TaGWB promoter region (Figure 2), the nucleotide polymorphism at  bp created a restriction enzyme recognition site for BstNI (CCWGG) (Figure 3A). This was employed to develop a cleaved amplified polymorphism sequence (CAPS) marker to distinguish Hap-6B-1 from the other three haplotypes. No restriction enzyme recognition site was found in Hap-6B-1 (A), whereas it existed in the other three haplotypes (C). In addition, ACAS-PCR primer sets designed for SNP T/C worked well and were co-dominant (Figure 3B). The forward primer for ACAS-PCR was genome-specific, and the reverse was allele-specific with artificial mismatches in the 3′-end. Hap-6B-1 and Hap-6B-2 amplified a fragment of  bp, whereas Hap-6B-3 and Hap-6B-4 amplified a  bp fragment. Thus, the ACAS-PCR primer sets reliably discriminated Hap-6B-2 and the other two haplotypes. Finally, only one SNP difference was found at  bp for discriminating Hap-6B-3 and Hap-6B-4. The dCAPS marker was designed with a specific mismatch in the primer to introduce a restriction enzyme HpyII recognition site (Figure 3C) using an available program dCAPS Finder ( This marker effectively discriminated Hap-6B-3 ( bp) and Hap-6B-4 ( bp). Thus, three markers, TaGW2-6B-CAPS, TaGW2-6B-dCAPS and TaGW2-6B-ACAS, were developed to distinguish these haplotypes.

Tests on a set of Chinese Spring (CS) nullisomic-tetrasomic lines confirmed that the three markers were chromosome 6B-specific (Figure 3D). The TaGWB gene was mapped between the markers Xmag and Xwmc on chromosome 6B in the recombinant inbred line (RIL) population derived from Nanda and Wangshuibai (Additional file 2: Figure S1). Based on the wheat consensus SSR genetic map [23], TaGWB was very close to the 6B centromere.

Strong differences in TKW and heading date exist between TaGWBhaplotypes

All three molecular markers, distinguishing the four TaGWB promoter haplotypes were used for genotyping the entries in the Chinese wheat mini-core collection. Previous studies had demonstrated that these accessions were clustered into two sub-populations comprising landraces and modern cultivars [24, 25] by Structure v software [26]. Therefore, association analysis between haplotypes of TaGWB and grain traits took population structure into account.

There were significant differences in TKW between Hap-6B-1 and Hap-6B-4 in the landraces (P < in , P < in ), and phenotypic differences between them were  g and  g in and , respectively (Table 1). This might be caused by differences in KL between Hap-6B-1 and Hap-6B-4 ( mm in ,  mm in ). Among modern cultivars, significant differences were again detected in TKW between Hap-6B-1 and Hap-6B-4 (P < in and ), and the mean TKW differences of Hap-6B-1 and Hap-6B-4 were  g and  g. These differences were due to large differences in KW and KT (Table 1). KW differences between the two groups were  mm and  mm, the KT differences were  mm and  mm, respectively. The significant negative effect of Hap-6B-4 may be the major reason for its elimination in breeding. Compared with the other three haplotypes, Hap-6B-1 was the favored one that increased grain weight. It was noteworthy that Hap-6B-2 was quite close to Hap-6B-1 in effect on grain weight in modern Chinese cultivars.

In addition to kernel weight, haplotype association analyses of heading and maturity dates were also performed (Additional file 3: Figure S2). There were no significant differences between Hap-6B-1 and Hap-6B-4 among the landraces for the two traits, but among modern cultivars heading and maturity date differences between Hap-6B-1 and Hap-6B-4 in both growing seasons were significant. The heading dates in and differed by 13 and 9 days and the corresponding differences for maturity date were 15 and 6 days, respectively. Similarly, Hap-6B-2 was also 11 and 6 days earlier than Hap-6B-4 in heading in the two seasons. For maturity, Hap-6B-2 was 13 and 4 days earlier than Hap-6B-4 in the two seasons respectively. Therefore, it seemed that Hap-6B-1 and Hap-6B-2 were associated not only with larger grain, but also earlier maturity.

Geographic distribution and frequency changes among haplotypes of TaGWA, and TaGWBin global wheat breeding

Geographic distribution of TaGWB haplotypes in Chinese wheats

Wheat production in China is divided into ten ecological zones based on cultivar ecotypes, growing season, and cultivar response to temperature and photoperiod [25, 27]. The distribution of TaGWB haplotypes was evaluated in both landraces and modern cultivars from each zone (Figure 4). Among landraces, selection pressure on haplotypes in the different zones was not as strong as expected, and the frequency of the favored haplotype Hap-6B-1 was generally low. In the winter wheat zones III, IV, V and IX, the frequency of Hap-6B-3 was highest, whereas in spring wheat zones VI and X, Hap-6B-2 was more frequent, and Hap-6B-1 was relatively frequent only in zone VII. However, in modern cultivars, Hap-6B-1 frequencies were higher across all zones (up to 90%), indicating it had undergone strong positive selection during wheat improvement. In detail, Hap-6B-1 was the most frequent haplotype in zones II, V, VI and VII, whereas Hap-6B-2 was most frequent in IV, VIII, IX and X. Association analysis showed that grain size and component parameters of Hap-6B-2 were significantly higher than those of Hap-6B-4, although they were lower than those of Hap-6B-1 (Table 1). Compared with landraces, Hap-6B-1 and Hap-6B-2 frequencies were higher across the ecological zones, presumably due to selective breeding, hence becoming the most frequent haplotypes. In contrast, the frequencies of Hap-6B-3 and Hap-6B-4 significantly decreased and even disappeared in zones IX, VI and VII (Figure 4).

Further evidence showing that TaGW2-6B underwent strong selection in Chinese wheat breeding is provided in Figure 5. The frequency of Hap-6B-1 showed an increasing trend, especially in the s (frequencies higher than 90%). Thus this haplotype tended towards fixation during modern breeding. In contrast, Hap-6B-4 and Hap-6B-3 disappeared from cultivars released after the s.

Global distributions of haplotypes for TaGWA and TaGWB

Previous study showed that Hap-6A-A was favored in China, whereas Hap-6A-G was favored in Europe [19]. In order to evaluate the distribution of all TaGW2 haplotypes in global wheat cultivars, the frequencies of haplotypes at the TaGWA and TaGWB loci were determined in cultivar collections from North America, Australia, China, CIMMYT, Europe and Russia (Figure 6).

Obvious geographic differences in haplotype frequencies for TaGWA were found among the different groups. Hap-6A-A was more frequent in Australian, Chinese and Russian cultivars, whereas Hap-6A-G predominated in U.S., CIMMYT and European collections (Additional file 4: Figure S3). At TaGWB, the superior haplotype Hap-6B-1 was more frequent in all regions, and Hap-6B-4 was virtually absent in all groups. Selection pressure on Hap-6A-A in North America and Europe was apparently very low, in contrast to China, and Hap-6A-G tended to dominate (Additional file 4: Figure S3A-B). The favored haplotype Hap-6B-1 at TaGWB showed a slow growth trend, while Hap-6B-4 gradually decreased or disappeared in all continents (Additional file 4: Figure S3C-D). Therefore, an obvious consistency of globally favored haplotypes was detected at TaGWB, but not at TaGWA.

TaGW2genes negatively regulate wheat grain weight

The average expression level of TaGWA reached a peak at 15 dpf and was significantly higher than that of either TaGWB or TaGWD in all six sampling stages of seed development (Figure 7A). The average relative expression of TaGWB peaked at 10 dpf, and that of TaGWD was 15 dpf. The average relative expression level of TaGWB was higher than that of TaGWD in all six stages except 15 dpf.

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