Molecular differentiation of null alleles at <Emphasis Type="Italic">ZCCT</Emphasis>-<Emphasis Type="Italic">1</Emphasis> genes on the A,B, and D genomes of hexaploid wheat

A dominant allele of the vernalization gene Vrn-2 is the wild type conferring winter growth habit, whereas a recessive vrn-2 allele confers spring growth habit. The recessive vrn-2 allele is mutated due to the deletion of the complete gene (a null form) or alternation of a key amino acid in the VRN-2 protein (a nonfunctional form) in diploid wheat or tetraploid wheat. VRN-2 is also denoted ZCCT due to the presence of a zinc finger and a CCT domain in its protein. There are two paralogous ZCCT genes at the VRN-2 locus in diploid Triticum monococcum and three paralogous ZCCT genes on each of the A and B genomes in tetraploid wheat, but little is known about the allelic variation in VRN-2 in hexaploid wheat. In the study reported here, we performed a one-shot PCR to simultaneously amplify the promoter regions of the three ZCCT-1 genes from hexaploid wheat, including the 302-bp fragment from ZCCT-A1, the 294-bp fragment from ZCCT-B1, and the 320-bp fragment from ZCCT-D1. Each amplicon could be differentiated by electrophoresis in an acrylamide/bisacrylamide gel. This PCR marker for different lengths of the three ZCCT-1 genes was used to search for null alleles in hexaploid wheat. A null allele was found in each of ZCCT-A1, ZCCT-B1, and ZCCT-D1 among 74 cultivars and genetic stocks of U.S. hexaploid wheat. Among 54 Chinese wheat cultivars, breeding lines, and landraces, we identified three accessions carrying a single null allele at ZCCT-A1, three accessions carrying a null allele at ZCCT-B1, and one accession carrying a double null allele at both ZCCT-A1 and ZCCT-D1. The potential application of these natural ZCCT-1 mutant materials in wheat breeding programs and studies on the genetics of wheat is discussed.     

Allelic variation at the <Emphasis Type="Italic">VRN-1</Emphasis> promoter region in polyploid wheat

Vernalization, the requirement of a long exposure to low temperatures to induce flowering, is an essential adaptation of plants to cold winters. We have shown recently that the vernalization gene VRN-1 from diploid wheat Triticum monococcum is the meristem identity gene APETALA1, and that deletions in its promoter were associated with spring growth habit. In this study, we characterized the allelic variation at the VRN-1 promoter region in polyploid wheat. The Vrn-A1a allele has a duplication including the promoter region. Each copy has similar foldback elements inserted at the same location and is flanked by identical host direct duplications (HDD). This allele was found in more than half of the hexaploid varieties but not among the tetraploid lines analyzed here. The Vrn-A1b allele has two mutations in the HDD region and a 20-bp deletion in the 5 UTR compared with the winter allele. The Vrn-A1b allele was found in both tetraploid and hexaploid accessions but at a relatively low frequency. Among the tetraploid wheat accessions, we found two additional alleles with 32 bp and 54 bp deletions that included the HDD region. We found no size polymorphisms in the promoter region among the winter wheat varieties. The dominant Vrn-A1 allele from two spring varieties from Afghanistan and Egypt (Vrn-A1c allele) and all the dominant Vrn-B1 and Vrn-D1 alleles included in this study showed no differences from their respective recessive alleles in promoter sequences. Based on these results, we concluded that the VRN-1 genes should have additional regulatory sites outside the promoter region studied here.     

Genetic and Molecular Characterization of the VRN2 Loci in Tetraploid Wheat

Winter wheat (Triticum spp.) varieties require long exposures to low temperatures to flower, a process called vernalization. The VRN2 locus includes two completely linked zinc finger-CCT domain genes (ZCCT1 and ZCCT2) that act as flowering repressors down-regulated during vernalization. Deletions or mutations in these two genes result in the elimination of the vernalization requirement in diploid wheat (Triticum monococcum). However, natural allelic variation in these genes has not been described so far in polyploid wheat (tetraploid Triticum turgidum and hexaploid Triticum aestivum). A tetraploid wheat population segregating for both VRN-A2 and VRN-B2 loci facilitated the characterization of different alleles. Comparisons between functional and nonfunctional alleles revealed that both ZCCT1 and ZCCT2 genes are able to confer vernalization requirement and that different ZCCT genes are functional in different genomes. ZCCT1 and ZCCT2 proteins from nonfunctional vrn2 alleles have mutations at arginine amino acids at position 16, 35, or 39 of the CCT domain. These positions are conserved between CCT and HEME ACTIVATOR PROTEIN2 (HAP2) proteins, supporting a model in which the action of CCT domains is mediated by their interactions with HAP2/HAP3/HAP5 complexes. This study also revealed natural variation in gene copy number, including a duplication of the functional ZCCT-B2 gene and deletions or duplications of the complete VRN-B2 locus. Allelic variation at the VRN-B2 locus was associated with a partially dominant effect, which suggests that variation in the number of functional ZCCT genes can be used to expand allelic diversity for heading time in polyploid wheat and, hopefully, improve its adaptation to different environments.     

Recurrent deletions of puroindoline genes at the grain hardness locus in four independent lineages of polyploid wheat

Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the A(m) genome of hexaploid Triticum zhukovskyi (A(m)AG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed.     

A new multiplex PCR test for the determination of Vrn-B1 alleles in bread wheat (Triticum aestivum L.)

Winter wheat requires vernalization, a long exposure to low but non-freezing temperatures, to promote reproductive development. The vernalization requirement in bread wheat (Triticum aestivum L.) is mainly controlled by the Vrn-1 genes that are located on chromosomes 5A, 5B and 5D. Dominant alleles confer spring habit and are epistatic to the recessive winter alleles which means that spring varieties carry at least one dominant allele. To date, two dominant and one recessive Vrn-B1 alleles have been described. Vrn-B1a (formerly designated as Vrn-B1) differs from the winter vrn-B1 allele by a large deletion in intron 1. Vrn-B1b has an additional small deletion and is probably derived from Vrn-B1a. The novel allele described here and designated as Vrn-B1c also has a large deletion within intron 1 but with different breakpoints from Vrn-B1a or b, and sequence duplication, showing that this is an independently derived spring allele. By combining an exon 1 primer with previously published PCR primers it was possible to develop a multiplex PCR that distinguished all four alleles simultaneously. The multiplex PCR was validated by testing 320 winter wheat and 137 spring wheat varieties. This demonstrated that the novel Vrn-B1c allele was present in 25 spring varieties of diverse origin, showing this allele to be widely distributed.     

Cbf14 copy number variation in the A, B, and D genomes of diploid and polyploid wheat

Freezing tolerance and winter hardiness are complex traits. In the Triticeae, two loci on the group 5 chromosome homoeologs are repeatedly identified as having major effects on these traits. Recently, we found that segments of the genomic region at one of these loci, Frost resistance-2 (Fr-2) is copy number variable in barley. Freezing-tolerant winter-hardy genotypes have greater tandem copy numbers of the genomic region encompassing the C-repeat binding factor genes Cbf2A and Cbf4B at Fr-H2 than the less freezing-tolerant nonwinter-hardy genotypes. Here we report that in wheat the Cbf14 gene at Fr-2 is copy number variable. Using DNA blot hybridizations, we estimated copy numbers of Cbf14 across the different genomes of diploid and polyploid wheat. Copy numbers of Cbf14 are lower in the B genome than in the A and D genomes across all ploidy levels. Among hexaploid red wheats, winter genotypes harbor greater Cbf14 copy numbers than spring genotypes. Cbf14 copy numbers also vary across the red winter wheats such that hard wheats harbor greater copy numbers than soft wheats. Analysis of hexaploid wheat chromosome 5 substitution lines indicates that Cbf14 copy numbers in the introgressions are stable in the different backgrounds. Taken together our data suggest that higher copy number states existed in the diploid wild ancestors prior to the polyploidization events and that the loss of Cbf14 copies occurred in the cultivated germplasm.     

Map-based analysis of the tenacious glume gene Tg-B1 of wild emmer and its role in wheat domestication

The domestication of wheat was instrumental in spawning the civilization of humankind, and it occurred through genetic mutations that gave rise to types with non-fragile rachises, soft glumes, and free-threshing seed. Wild emmer (Triticum turgidum ssp. dicoccoides), the tetraploid AB-genome progenitor of domesticated wheat has genes that confer tenacious glumes (Tg) that underwent genetic mutations to give rise to free-threshing wheat. Here, we evaluated disomic substitution lines involving chromosomes 2A and 2B of wild emmer accessions substituted for homologous chromosomes in tetraploid and hexaploid backgrounds. The results suggested that both chromosomes 2A and 2B of wild emmer possess genes that inhibit threshability. A population of recombinant inbred lines derived from the tetraploid durum wheat variety Langdon crossed with a Langdon — T. turgidum ssp. dicoccoides accession PI 481521 chromosome 2B disomic substitution line was used to develop a genetic linkage map of 2B, evaluate the genetics of threshability, and map the gene derived from PI 481521 that inhibited threshability. A 2BS linkage map comprised of 58 markers was developed, and markers delineated the gene to a 2.3 cM interval. Comparative analysis with maps containing the tenacious glume gene Tg-D1 on chromosome arm 2DS from Aegilops tauschii, the D genome progenitor of hexaploid wheat, revealed that the gene inhibiting threshability in wild emmer was homoeologous to Tg-D1 and therefore designated Tg-B1. Comparative analysis with rice and Brachypodium distachyon indicated a high level of divergence and poorly conserved colinearity, particularly near the Tg-B1 locus. These results provide a foundation for further studies involving Tg-B1, which, together with Tg-D1, had profound influences on wheat domestication.     

Further genetic analysis of a major quantitative trait locus controlling root length and related traits in common wheat

Roots are essential for normal growth, development, and reproduction of higher plants. Consequently, improvement of root system architecture and functionality is of fundamental importance in crop improvement. However, the genetic mechanisms controlling root morphology and function are still not well understood, especially in common wheat, which possesses a complex and unsequenced hexaploid genome. Here we report a more detailed genetic analysis of qTaLRO-B1, a major quantitative trait locus (QTL) previously detected to affect root length and related traits in common wheat. A pair of QTL isolines with different qTaLRO-B1 alleles was developed. Line 178B, carrying the longer root allele, was significantly more efficient in taking up phosphate nutrient and biomass accumulation than line 178A, with the shorter root allele. We mapped qTaLRO-B1 to a 0.9-cM interval on common wheat chromosome 2BS with seven sequence-tagged-site (STS) markers developed from the genes conserved between wheat and Brachypodium distachyon. The seven STS markers were collinearly conserved in tetraploid wheat, but they covered a much larger genetic distance (22.8 cM) in the latter species. In conclusion, we have converted qTaLRO-B1 into a major gene that affects common wheat root length in a qualitative manner, and improved understanding of the genetic location of qTaLRO-B1 and the chromosomal segment carrying this important locus. The implications of our data for further study of qTaLRO-B1 are discussed.     

Exploring the diploid wheat ancestral A genome through sequence comparison at the high-molecular-weight glutenin locus region

The polyploid nature of hexaploid wheat (T. aestivum, AABBDD) often represents a great challenge in various aspects of research including genetic mapping, map-based cloning of important genes, and sequencing and accurately assembly of its genome. To explore the utility of ancestral diploid species of polyploid wheat, sequence variation of T. urartu (A^uA^u) was analyzed by comparing its 277-kb large genomic region carrying the important Glu-1 locus with the homologous regions from the A genomes of the diploid T. monococcum (A^mA^m), tetraploid T. turgidum (AABB), and hexaploid T. aestivum (AABBDD). Our results revealed that in addition to a high degree of the gene collinearity, nested retroelement structures were also considerably conserved among the A^u genome and the A genomes in polyploid wheats, suggesting that the majority of the repetitive sequences in the A genomes of polyploid wheats originated from the diploid A^u genome. The difference in the compared region between A^u and A is mainly caused by four differential TE insertion and two deletion events between these genomes. The estimated divergence time of A genomes calculated on nucleotide substitution rate in both shared TEs and collinear genes further supports the closer evolutionary relationship of A to A^u than to A^m. The structure conservation in the repetitive regions promoted us to develop repeat junction markers based on the A^u sequence for mapping the A genome in hexaploid wheat. Eighty percent of these repeat junction markers were successfully mapped to the corresponding region in hexaploid wheat, suggesting that T. urartu could serve as a useful resource for developing molecular markers for genetic and breeding studies in hexaploid wheat.     

Genome organisation and retrotransposon driven molecular evolution of the endosperm Hardness (Ha) locus in Triticum aestivum cv Glenlea

Wheat endosperm texture is controlled primarily by a locus (Ha), which comprises Gsp-1, Pina and Pinb genes encoding the so-called grain softness protein, puroindoline-a and puroindoline-b, respectively. Pina and Pinb were detected only on the D-genome of hexaploid wheat and its diploid progenitors while Gsp-1 was on all three homoeologous loci. Hexaploid cultivar Glenlea has a hard phenotype due to a null Pina genotype (D-genome) but the sequence organization is not reported. This study aimed at understanding the evolution of homoeologous Ha loci. Sequencing of three BAC clones from cv Glenlea was performed and sequence analyses delimited the Ha loci which spanned 3,925, 5,330 and 31,607 bp in the A-, B- and D-genomes, respectively. A solo LTR of Angela retroelement, downstream to Gsp-A1 and a fragment of Sabrina retroelement, downstream of Gsp-B1, were discovered. We propose that the insertion of these elements into the intergenic regions have driven the deletions of genomic segments harbouring Pina and Pinb genes in the A- and B-genomes of hexaploid wheat. Similarly, fragments of Romani and Vagabond retroelements were identified between truncated Pina and Pinb genes, indicating their role in the deletion of Pina in Glenlea, leading to its hard texture. Structural differences of the Ha locus region of the A-genome between two hexaploid wheat varieties namely Glenlea and Renan (CR626929), suggested the presence of more than one tetraploid ancestor in the origin of hexaploid wheat.     

Large deletions within the first intron in <Emphasis Type="Italic">VRN-1</Emphasis> are associated with spring growth habit in barley and wheat

The broad adaptability of wheat and barley is in part attributable to their flexible growth habit, in that spring forms have recurrently evolved from the ancestral winter growth habit. In diploid wheat and barley growth habit is determined by allelic variation at the VRN-1 and/or VRN-2 loci, whereas in the polyploid wheat species it is determined primarily by allelic variation at VRN-1. Dominant Vrn-A1 alleles for spring growth habit are frequently associated with mutations in the promoter region in diploid wheat and in the A genome of common wheat. However, several dominant Vrn-A1, Vrn-B1, Vrn-D1 (common wheat) and Vrn-H1 (barley) alleles show no polymorphisms in the promoter region relative to their respective recessive alleles. In this study, we sequenced the complete VRN-1 gene from these accessions and found that all of them have large deletions within the first intron, which overlap in a 4-kb region. Furthermore, a 2.8-kb segment within the 4-kb region showed high sequence conservation among the different recessive alleles. PCR markers for these deletions showed that similar deletions were present in all the accessions with known Vrn-B1 and Vrn-D1 alleles, and in 51 hexaploid spring wheat accessions previously shown to have no polymorphisms in the VRN-A1 promoter region. Twenty-four tetraploid wheat accessions had a similar deletion in VRN-A1 intron 1. We hypothesize that the 2.8-kb conserved region includes regulatory elements important for the vernalization requirement. Epistatic interactions between VRN-H2 and the VRN-H1 allele with the intron 1 deletion suggest that the deleted region may include a recognition site for the flowering repression mediated by the product of the VRN-H2 gene of barley.     

A single nucleotide polymorphism at the Vrn-D1 promoter region in common wheat is associated with vernalization response

Facultative wheat varieties adapt to a particular environment. But the molecular basis for the facultative growth habit is not clear relative to winter and spring growth habit. Two sets of wheat varieties were chosen for this study. Set 1 comprised ten spring accessions and Set 2 comprised ten facultative accessions. All accessions had been tested by the previously described allele-specific markers and shown having the same allelic composition of vrn-A1 vrn-B1 Vrn-D1 and vrn-B3. Here we examined whether differences in growth habit might be associated with as yet unidentified sequence variation at Vrn-D1 locus. A region including the intron 1 deletion, the entire reading frame from a cDNA template and a part of promoter region of the dominant Vrn-D1 gene in each of the accessions was sequenced, and a single nucleotide polymorphism was found between facultative accessions and spring accessions in the CArG-box at the promoter region. The novel allele in facultative accessions was designated as Vrn-D1b. The investigation of an F₂ population segregating for Vrn-D1b and Vrn-D1a (previously, Vrn-D1) in the greenhouse under long days without vernalization showed that the plants with Vrn-D1b homozygous allele headed 32?days later and had about three more leaves than the plants with Vrn-D1a homozygous allele. As Vrn-D1b has the same deletion in intron 1 as Vrn-D1a, and, in addition, a single nucleotide mutation at promoter region, and is associated with facultative growth habit, we suggest that the promoter mutation may modify the basal activity level of an allele of VRN1 that is already active (due to the loss of segments in intron 1). Our finding further supports that both the promoter and intron 1 regulatory affect vernalization response and work independently.     

Mechanisms,origin and heredity of Glu-1Ay silencing in wheat evolution and domestication

Key message

Allotetraploidization drives Glu-1Ay silencing in polyploid wheat.

Abstract

The high-molecular-weight glutenin subunit gene, Glu-1Ay, is always silenced in common wheat via elusive mechanisms. To investigate its silencing and heredity during wheat polyploidization and domestication, the Glu-1Ay gene was characterized in 1246 accessions containing diploid and polyploid wheat worldwide. Eight expressed Glu-1Ay alleles (in 71.81% accessions) and five silenced alleles with a premature termination codon (PTC) were identified in Triticum urartu; 4 expressed alleles (in 41.21% accessions), 13 alleles with PTCs and 1 allele with a WIS 2-1A retrotransposon were present in wild tetraploid wheat; and only silenced alleles with PTC or WIS 2-1A were in cultivated tetra- and hexaploid wheat. Both the PTC number and position in T. urartu Glu-1Ay alleles (one in the N-terminal region) differed from its progeny wild tetraploid wheat (1–5 PTCs mainly in the repetitive domain). The WIS 2-1A insertion occurred?~?0.13 million years ago in wild tetraploid wheat, much later than the allotetraploidization event. The Glu-1Ay alleles with PTCs or WIS 2-1A that arose in wild tetraploid wheat were fully succeeded to cultivated tetraploid and hexaploid wheat. In addition, the Glu-1Ay gene in wild einkorn inherited to cultivated einkorn. Our data demonstrated that the silencing of Glu-1Ay in tetraploid and hexaploid wheat was attributed to the new PTCs and WIS 2-1A insertion in wild tetraploid wheat, and most silenced alleles were delivered to the cultivated tetraploid and hexaploid wheat, providing a clear evolutionary history of the Glu-1Ay gene in the wheat polyploidization and domestication processes.

     

Genome inheritance in populations derived from hexaploid/tetraploid and tetraploid/hexaploid wheat crosses

Hexaploid/tetraploid and tetraploid/hexaploid wheat hybrids were established using the hexaploid (Triticum aestivum L.) bread wheat LRC2010-150 and the tetraploid durum wheat (T. turgidum spp. durum) WID802. Thirty F₂ progeny from each cross were characterised using Diversity Arrays Technology (DArTseq?) markers to determine whether there are differences between the crosses in the proportion of A, B and D genomic material inherited from each parent. Inheritance of the A and B genome from the tetraploid durum parent varied from 32 to 63% among the 60 lines assessed, and results indicated significant differences between the two F₂ populations in the mean overall proportion of chromosomes A and B inherited from each parent. Significant differences were also observed between the crosses in the proportion of chromosomal segments on 2B, 3A, 3B and 4A inherited from the tetraploid parent. The F₂ populations also showed significant differences in the average retention of D chromosomes per line with the tetraploid/hexaploid cross retaining a mean of 2.83 chromosomes while the reciprocal cross retained a mean of 1.8 chromosomes per line. A strong negative correlation was observed in individual lines from both populations between the proportion of the A and B genome inherited from the tetraploid durum parent and the retention of the D genome. The implication of these results for the design of efficient crossing strategies between hexaploid and tetraploid wheats is discussed.     

Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains

Carotenoid ??-hydroxylases attach hydroxyl groups to the ??-ionone rings (??-rings) of carotenoid substrates, resulting in modified structures and functions of carotenoid molecules. We cloned and characterized two genes (each with three homeologs), HYD1 and HYD2, which encode ??-hydroxylases in wheat. The results from bioinformatic and nested degenerate PCR analyses collectively suggest that HYD1 and HYD2 may represent the entire complement of non-heme di-iron ??-hydroxylases in wheat. The homeologs of wheat HYDs exhibited major ??-ring and minor ??-ring hydroxylation activities in carotenoid-accumulating E. coli strains. Distinct expression patterns were observed for different HYD genes and homeologs in vegetative tissues and developing grains of tetraploid and hexaploid wheat, suggesting their functional divergence and differential regulatory control in tissue-, grain development-, and ploidy-specific manners. An intriguing observation was that the expression of HYD1, particularly HYD-B1, reached highest levels at the last stage of tetraploid and hexaploid grain development, suggesting that carotenoids (at least xanthophylls) were still actively synthesized in mature grains. This result challenges the common perception that carotenoids are simply being turned over during wheat grain development after their initial biosynthesis at the early grain development stages. Overall, this improved understanding of carotenoid biosynthetic gene expression and carotenoid metabolism in wheat grains will contribute to the improvement of the nutritional value of wheat grains for human consumption.     

Exploiting the <Emphasis Type="Italic">ZIP4</Emphasis> homologue within the wheat <Emphasis Type="Italic">Ph1</Emphasis> locus has identified two lines exhibiting homoeologous crossover in wheat-wild relative hybrids

Despite possessing related ancestral genomes, hexaploid wheat behaves as a diploid during meiosis. The wheat Ph1 locus promotes accurate synapsis and crossover of homologous chromosomes. Interspecific hybrids between wheat and wild relatives are exploited by breeders to introgress important traits from wild relatives into wheat, although in hybrids between hexaploid wheat and wild relatives, which possess only homoeologues, crossovers do not take place during meiosis at metaphase I. However, in hybrids between Ph1 deletion mutants and wild relatives, crossovers do take place. A single Ph1 deletion (ph1b) mutant has been exploited for the last 40 years for this activity. We show here that chemically induced mutant lines, selected for a mutation in TaZIP4-B2 within the Ph1 locus, exhibit high levels of homoeologous crossovers when crossed with wild relatives. Tazip4-B2 mutant lines may be more stable over multiple generations, as multivalents causing accumulation of chromosome translocations are less frequent. Exploitation of such Tazip4-B2 mutants, rather than mutants with whole Ph1 locus deletions, may therefore improve introgression of wild relative chromosome segments into wheat.