Geographical variation in heading traits in wild emmer wheat, Triticum dicoccoides. I. Variation in vernalization response and ecological differentiation

 Geographical variation in vernalization response and narrow-sense earliness was investigated for accessions of wild emmer wheat, Triticum dicoccoides, collected in Israel. Wide variation between and within populations was observed in both characters. The analysis of vernalization response showed that 2 accessions from Tabigha were of a strong spring growth habit, and thus wild emmer wheat was classified into four types, i.e., strongly spring type, moderately spring type, moderately winter type, and strongly winter type, according to their vernalization response. Whereas winter types were frequently found in most populations except that of Tabigha, the distribution of spring types was sporadic and restricted to warmer areas. It was thus suggested that spring type in T. dicoccoides might have evolved from a winter prototype as an adaptation to warmer conditions. Within moderately winter and moderately spring types, quantitative differences in vernalization response, measured as Dof₇₀/Dof₂₀ and Dof₂₀/Dof₀, were observed between populations. Inter- and intra-population variation in vernalization response could be explained to some extent by the difference in growing conditions at each habitat. It was clearly indicated that environmental heterogeneity caused ecogenetic differentiation in wild emmer wheat in Israel. Wild emmer wheat also varied considerably for narrow-sense earliness, ranging from 32.9 days to 69.5 days among accessions. However, it was difficult to explain its geographical variation simply by a linear relationship with environmental factors, and a nonlinear relationship and/or unknown microgeographic heterogeneity may be responsible. Received: 18 March 1996/Accepted: 13 December 1996     

Genetic adaptation for vernalization requirement in Nepalese wheat and barley

A large number of accessions of covered and naked barley from eastern Nepal were grown without vernalization, and it was found that naked barley accessions were predominantly spring varieties while covered barley accessions were predominantly winter varieties. Seven accessions were subjected to a range of vernalization periods. Four naked varieties were spring varieties, although one showed some response to vernalization, but the three covered barleys were winter varieties. Although the majority of naked barleys are spring forms, they are winter sown at high altitudes and this does not conform to the distribution of naked barley described by Takahashi (1955). Wheat accessions which came from villages situated at high altitudes tended to have higher vernalization requirements than those which came from lower altitudes. This was taken to indicate local adaptation and a low movement of seeds (gene-flow) between villages. The relationship between vernalization requirement and altitude was not found in barley. Marked but contrasting regional patterns for vernalization requirement occurred in the wheat and covered barley. It was concluded that gene-flow was greater within regions than between them. This regional isolation together with environmental heterogeneity are major diversity promoting mechanisms.     

Discrete developmental roles for temperate cereal grass VERNALIZATION1/FRUITFULL-like genes in flowering competency and the transition to flowering

Members of the grass subfamily Pooideae are characterized by their adaptation to cool temperate climates. Vernalization is the process whereby flowering is accelerated in response to a prolonged period of cold. Winter cereals are tolerant of low temperatures and flower earlier with vernalization, whereas spring cultivars are intolerant of low temperatures and flower later with vernalization. In the pooid grasses wheat (Triticum monococcum, Triticum aestivum) and barley (Hordeum vulgare), vernalization responsiveness is determined by allelic variation at the VERNALIZATION1 (VRN1) and/or VRN2 loci. To determine whether VRN1, and its paralog FRUITFULL2 (FUL2), are involved in vernalization requirement across Pooideae, we determined expression profiles for multiple cultivars of oat (Avena sativa) and wheat with and without cold treatment. Our results demonstrate significant up-regulation of VRN1 expression in leaves of winter oat and wheat in response to vernalization; no treatment effect was found for spring or facultative growth habit oat and wheat. Similar cold-dependent patterns of leaf expression were found for FUL2 in winter oat, but not winter wheat, suggesting a redundant qualitative role for these genes in the quantitative induction of flowering competency of oat. These and other data support the hypothesis that VRN1 is a common regulator of vernalization responsiveness within the crown pooids. Finally, we found that up-regulation of VRN1 in vegetative meristems of oat was significantly later than in leaves. This suggests distinct and conserved roles for temperate cereal grass VRN1/FUL-like genes, first, in systemic signaling to induce flowering competency, and second, in meristems to activate genes involved in the floral transition.     

Molecular and Structural Characterization of Barley Vernalization Genes

Vernalization, the requirement of a period of low temperature to induce transition from the vegetative to reproductive state, is an evolutionarily and economically important trait in the Triticeae. The genetic basis of vernalization in cultivated barley (Hordeum vulgare subsp. vulgare) can be defined using the two-locus VRN-H1/VRN-H2 model. We analyzed the allelic characteristics of HvBM5A, the candidate gene for VRN-H1, from ten cultivated barley accessions and one wild progenitor accession (subsp. spontaneum), representing the three barley growth habits – winter, facultative, and spring. We present multiple lines of evidence, including sequence, linkage map location, and expression, that support HvBM5A being VRN-H1. While the predicted polypeptides from different growth habits are identical, spring accessions contain a deletion in the first intron of HvBM5A that may be important for regulation. While spring HvBM5A alleles are typified by the intron-localized deletion, in some cases, the promoter may also determine the allele type. The presence/absence of the tightly linked ZCCT-H gene family members on chromosome 4H perfectly correlates with growth habit and we conclude that one of the three ZCCT-H genes is VRN-H2. The VRN-H2 locus is present in winter genotypes and deleted from the facultative and spring genotypes analyzed in this study, suggesting the facultative growth habit (cold tolerant, vernalization unresponsive) is a result of deletion of the VRN-H2 locus and presence of a winter HvBM5A allele. All reported barley vernalization QTLs can be explained by the two-locus VRN-H1/VRN-H2 model based on the presence/absence of VRN-H2 and a winter vs. spring HvBM5A allele. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions

FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) are two genes that, unless plants are vernalized, greatly delay flowering time in Arabidopsis thaliana. Natural loss-of-function mutations in FRI cause the early flowering growth habits of many A. thaliana accessions. To quantify the variation among wild accessions due to FRI, and to identify additional genetic loci in wild accessions that influence flowering time, we surveyed the flowering times of 145 accessions in long-day photoperiods, with and without a 30-day vernalization treatment, and genotyped them for two common natural lesions in FRI. FRI is disrupted in at least 84 of the accessions, accounting for only approximately 40% of the flowering-time variation in long days. During efforts to dissect the causes for variation that are independent of known dysfunctional FRI alleles, we found new loss-of-function alleles in FLC, as well as late-flowering alleles that do not map to FRI or FLC. An FLC nonsense mutation was found in the early flowering Van-0 accession, which has otherwise functional FRI. In contrast, Lz-0 flowers late because of high levels of FLC expression, even though it has a deletion in FRI. Finally, eXtreme array mapping identified genomic regions linked to the vernalization-independent, late-flowering habit of Bur-0, which has an alternatively spliced FLC allele that behaves as a null allele.     

A minimum requirements method to isolate large quantities of highly purified DNA from one drop of poultry blood

Requirement of vernalization is an important factor which plays a crucial role in cereals to transit from vegetative to reproductive phase. There are three types of growth habit in barley: winter, spring and facultative types; in which spring type does not require vernalization but winter and facultative genotypes require full and partial vernalization, respectively. Combination of two loci, Vrn-h1 and Vrn-h2, regulates vernalization in barley genotypes. Specific DNA markers have been identified for growth habit regulator genes in barley. In this study, we examined 24 barley genotypes using specific primers for detecting Vrn-h1 and Vrn-h2 loci. Results showed that among all differently suggested primer combinations, a few markers were precisely correlated with seasonal growth habit in barley. The specific markers of 600, 600 and 200 bps were verified for ZCCT-Ha, ZCCT-Hb and ZCCT-Hc loci, respectively. Our field growth habit test showed that cultivar Bahman as a winter growth habit, where all the others genotypes exhibited spring growth habit. By using specific primers for Vrn-h1, only Bahman cultivar produced 616 bp and 830 bp fragments and spring genotypes showed 574 bp or 616 bp alleles without any amplification for 830 bp fragments. Therefore, presence of 616 bp and 830 bp alleles together in each genotype can be considered as an informative marker for winter growth habit in barley. These informative markers can be used easily in barley breeding programmes for detection of growth habit types in the seedling stage.     

Allelic variation at the linked AP1 and PhyC loci in hexaploid wheat is associated but not perfectly correlated with vernalization response

Vernalization requirement is an important trait in temperate crop plants such as wheat and must be considered when selecting varieties for cultivation under different climatic conditions. To determine the growth habit of wheat varieties, plants need to be grown under different vernalization regimes, a lengthy but necessary process for breeders involved in crossing winter with spring germplasm. If haplotypes can be associated with growth habit, then molecular marker assays that are reliable, cheap, and quick can be developed to assist in the selection of plants with the desired phenotype. We have analyzed 81 accessions that have different vernalization requirements and putative different origins of spring habit for sequence variation at the Apetala1 (AP1) locus, which underlies Vrn-1, and at the linked Phytochrome C (PhyC) locus. Good correspondence was found between the AP1 genotype and the PhyC haplotype for 77 of the 81 accessions. Two varieties displayed a recombination event between the AP1 and PhyC loci, and one variety carried a recombinant PhyC gene. In addition, one variety carried an apparent AP1 winter allele, but displayed the Vrn-A1 spring habit. The PhyC haplotype for this variety also indicated the presence of a Vrn-A1 spring allele. Our data suggest that both the AP1 promoter region and PhyC SNPs can be used as diagnostic markers for vernalization response at the vrn-A1 locus, but that neither are perfect tags.     

Monophyletic origin of naked barley inferred from molecular analyses of a marker closely linked to the naked caryopsis gene (<Emphasis Type="Italic">nud</Emphasis>)

To elucidate the origin of naked barley, molecular variation of the marker sKT7 tightly linked to the nud locus was examined. A total of 259 (53 wild, 106 hulled domesticated, and 100 naked domesticated) barley accessions were studied. Restriction analysis of the sKT7 PCR-amplified product revealed the alleles I, II, III, and IV. All four alleles were found in wild barley, but allele IV was found only in a single accession from southwestern Iran. Hulled domesticated accessions showed alleles I, II, or III, but all naked domesticated accessions had allele IV. The distribution of allele IV in wild barley and its pervasive presence in naked domesticated lines support the conclusion that naked barley has a monophyletic origin, probably in southwestern Iran. The available results suggest two scenarios for the origin of naked barley: either directly from a wild barley with allele IV or from a hulled domesticated line with allele IV that later became extinct. Naked domesticated accessions from different regions of the world have extremely homogeneous DNA sequences at the sKT7 locus, supporting the monophyletic origin of naked barley. For allele IV, four haplotypes (IVb to IVe) were found in 30 naked accessions: IVb was predominant (66.7%) and widely distributed, while the other three haplotypes, differing by only one nucleotide at different positions relative to IVb, showed a localized distribution. The geographical distribution of the haplotypes of sKT7 allele IV suggests migration routes of naked domesticated barley in central and eastern Asia.Communicated by F. Salamini     

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.     

The Characterization and Geographical Distribution of the Genes Responsible for Vernalization Requirement in Chinese Bread Wheat

The frequency and distribution of the major vernalization requirement genes and their effects on growth habits were studied.Of the 551 bread wheat genotypes tested,seven allelic combinations of the three Vrn.1 genes were found to be responsible for the spring habit,three for the facultative habit and one for the winter habit.The three Vrn-1 genes behaved additively with the dominant allele of Vrn-A1 exerting the strongest effect.The allele combinations of the facultative genotypes and the discovery of spring genotypes with "winter" allele of Vrn-1 implied the presence of as yet unidentified alleles/genes for vernalization response.The dominant alleles of the three Vrn-1 genes were found in all ten ecological regions where wheat Is cultivated in China,with Vrn-D1 as the most common allele in nine and Vrn-A1 in one.The combination of vrn-A 1vrnB 1Vrn-D1 was the predominant genotype in seven of the regions.Compared with landraces,improved varieties contain a higher proportion of the spring type.This was attributed by a higher frequency of the dominant Vrn-A1 and Vrn-B1 alleles in the latter.Correlations between Vrn-1 allelic constitutions and heading date,spike length,plant type as well as cold tolerance were established.     

VRN1 genes variability in tetraploid wheat species with a spring growth habit

Background

Vernalization genes VRN1 play a major role in the transition from vegetative to reproductive growth in wheat. In di-, tetra- and hexaploid wheats the presence of a dominant allele of at least one VRN1 gene homologue (Vrn-A1, Vrn-B1, Vrn-G1 or Vrn-D1) determines the spring growth habit. Allelic variation between the Vrn-1 and vrn-1 alleles relies on mutations in the promoter region or the first intron. The origin and variability of the dominant VRN1 alleles, determining the spring growth habit in tetraploid wheat species have been poorly studied.

Results

Here we analyzed the growth habit of 228 tetraploid wheat species accessions and 25 % of them were spring type. We analyzed the promoter and first intron regions of VRN1 genes in 57 spring accessions of tetraploid wheats. The spring growth habit of most studied spring accessions was determined by previously identified dominant alleles of VRN1 genes. Genetic experiments proof the dominant inheritance of Vrn-A1d allele which was widely distributed across the accessions of Triticum dicoccoides. Two novel alleles were discovered and designated as Vrn-A1b.7 and Vrn-B1dic. Vrn-A1b.7 had deletions of 20 bp located 137 bp upstream of the start codon and mutations within the VRN-box when compared to the recessive allele of vrn-A1. So far the Vrn-A1d allele was identified only in spring accessions of the T. dicoccoides and T. turgidum species. Vrn-B1dic was identified in T. dicoccoides IG46225 and had 11 % sequence dissimilarity in comparison to the promoter of vrn-B1. The presence of Vrn-A1b.7 and Vrn-B1dic alleles is a predicted cause of the spring growth habit of studied accessions of tetraploid species. Three spring accessions T. aethiopicum K-19059, T. turanicum K-31693 and T. turgidum cv. Blancal possess recessive alleles of both VRN-A1 and VRN-B1 genes. Further investigations are required to determine the source of spring growth habit of these accessions.

Conclusions

New allelic variants of the VRN-A1 and VRN-B1 genes were identified in spring accessions of tetraploid wheats. The origin and evolution of VRN-A1 alleles in di- and tetraploid wheat species was discussed.

     

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.     

Population-based resequencing reveals that the flowering time adaptation of cultivated barley originated east of the Fertile Crescent

Gene resequencing and association analysis present new opportunities to study the evolution of adaptive traits in crop plants. Here we apply these tools to an extensive set of barley accessions to identify a component of the molecular basis of the flowering time adaptation, a trait critical to plant survival. Using an association-based study to relate variation in flowering time to sequence-based polymorphisms in the Ppd-H1 gene, we identify a causative polymorphism (SNP48) that accounts for the observed variation in barley flowering time. This polymorphism also shows latitude-dependent geographical distribution, consistent with the expected clinal variation in phenotype with the nonresponsive form predominating in the north. Networks, genealogies, and phylogenetic trees drawn for the Ppd-H1 haplotypes reveal population structure both in wild barley and in domesticated barley landraces. The spatial distribution of these population groups indicates that phylogeographical analysis of European landraces can provide information relevant to the Neolithic spread of barley cultivation and also has implications for the origins of domesticated barley, including those with the nonresponsive ppd-H1 phenotype. Haplotypes containing the nonresponsive version of SNP48 are present in wild barley accessions, indicating that the nonresponsive phenotype of European landraces originated in wild barley. The wild accessions whose nonresponsive haplotypes are most closely similar to those of landraces are found in Iran, within a region suggested as an area for domestication of barley east of the Fertile Crescent but which has previously been thought to have contributed relatively little to the diversity of European cultivars.     

Transcriptome analysis of the vernalization response in barley (Hordeum vulgare) seedlings

Temperate cereals, such as wheat (Triticum spp.) and barley (Hordeum vulgare), respond to prolonged cold by becoming more tolerant of freezing (cold acclimation) and by becoming competent to flower (vernalization). These responses occur concomitantly during winter, but vernalization continues to influence development during spring. Previous studies identified VERNALIZATION1 (VRN1) as a master regulator of the vernalization response in cereals. The extent to which other genes contribute to this process is unclear. In this study the Barley1 Affymetrix chip was used to assay gene expression in barley seedlings during short or prolonged cold treatment. Gene expression was also assayed in the leaves of plants after prolonged cold treatment, in order to identify genes that show lasting responses to prolonged cold, which might contribute to vernalization-induced flowering. Many genes showed altered expression in response to short or prolonged cold treatment, but these responses differed markedly. A limited number of genes showed lasting responses to prolonged cold treatment. These include genes known to be regulated by vernalization, such as VRN1 and ODDSOC2, and also contigs encoding a calcium binding protein, 23-KD jasmonate induced proteins, an RNase S-like protein, a PR17d secretory protein and a serine acetyltransferase. Some contigs that were up-regulated by short term cold also showed lasting changes in expression after prolonged cold treatment. These include COLD REGULATED 14B (COR14B) and the barley homologue of WHEAT COLD SPECIFIC 19 (WSC19), which were expressed at elevated levels after prolonged cold. Conversely, two C-REPEAT BINDING FACTOR (CBF) genes showed reduced expression after prolonged cold. Overall, these data show that a limited number of barley genes exhibit lasting changes in expression after prolonged cold treatment, highlighting the central role of VRN1 in the vernalization response in cereals.     

Cloning and characterization of SOC1 homologs in barley (Hordeum vulgare) and their expression during seed development and in response to vernalization

A number of genes are involved in the vernalization pathway, such as VRN1, VRN2 and VRN3/FT1, whose function has been studied in barley and wheat. However, the function of the flowering and vernalization integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) has not been well studied in Triticeae, and particularly in barley. Herein, we cloned and characterized two barley SOC1-like homologs, HvSOC1-like1 and HvSOC1-like2. Primary sequence analysis of the predicted HvSOC1-like1 and HvSOC1-like2 proteins showed that they are members of the type II MADS-box protein family. Phylogenetic analysis placed the predicted proteins with other SOC1 and SOC1-like proteins from different species neighboring those from other cereal plant species. Primary and secondary structures of the predicted proteins are conserved to each other and more distant to the recently identified barley ODDSOC1 proteins. Genomic organization of HvSOC1-like1 is very similar to the Arabidopsis and Brachypodium SOC1 genes and localized in highly syntenic chromosomal regions. Regulatory cis-acting elements detected in the HvSOC1-like1 promoter include the CArG-box, implicated in the regulation of SOC1 expression in Arabidopsis. Both HvSOC1-like1 and HvSOCI-like2 are expressed in vegetative and reproductive tissues and at different stages of seed development. Both are upregulated in a particular seed developmental stage suggesting their possible implication in seed development. Furthermore, HvSOC1-like1 was induced in two winter barley cultivars after vernalization treatment pointing to its probable involvement in the vernalization process. The study of the SOC1 genes reported here opens the way for a better understanding of both the vernalization process and seed development and germination in this important cereal crop.