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.     

Genetics of flowering time in bread wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis>: complementary interaction between vernalization-insensitive and photoperiod-insensitive mutations imparts very early flowering habit to spring wheat

Time to flowering in the winter growth habit bread wheat is dependent on vernalization (exposure to cold conditions) and exposure to long days (photoperiod). Dominant Vrn-1 (Vrn-A1, Vrn-B1 and Vrn-D1) alleles are associated with vernalization independent spring growth habit. The semidominant Ppd-D1a mutation confers photoperiod-insensitivity or rapid flowering in wheat under short day and long day conditions. The objective of this study was to reveal the nature of interaction between Vrn-1 and Ppd-D1a mutations (active alleles of the respective genes vrn-1 and Ppd-D1b). Twelve Indian spring wheat cultivars and the spring wheat landrace Chinese Spring were characterized for their flowering times by seeding them every month for five years under natural field conditions in New Delhi. Near isogenic Vrn-1 Ppd-D1 and Vrn-1 Ppd-D1a lines constructed in two genetic backgrounds were also phenotyped for flowering time by seeding in two different seasons. The wheat lines of Vrn-A1a Vrn-B1 Vrn-D1 Ppd-D1a, Vrn-A1a Vrn-B1 Ppd-D1a and Vrn-A1a Vrn-D1 Ppd-D1a (or Vrn-1 Ppd-D1a) genotypes flowered several weeks earlier than that of Vrn-A1a Vrn-B1 Vrn-D1 Ppd-D1b, Vrn-A1b Ppd-D1b and Vrn-D1 Ppd-D1b (or Vrn-1 Ppd-D1b) genotypes. The flowering time phenotypes of the isogenic vernalization-insensitive lines confirmed that Ppd-D1a hastened flowering by several weeks. It was concluded that complementary interaction between Vrn-1 and Ppd-D1a active alleles imparted super/very-early flowering habit to spring wheats. The early and late flowering wheat varieties showed differences in flowering time between short day and long day conditions. The flowering time in Vrn-1 Ppd-D1a genotypes was hastened by higher temperatures under long day conditions. The ambient air temperature and photoperiod parameters for flowering in spring wheat were estimated at 25°C and 12 h, respectively.     

Cold hardiness of wheat near-isogenic lines differing in vernalization alleles

Four major genes in wheat (Triticum aestivum L.), with the dominant alleles designated Vrn-A1, Vrn-B1, Vrn-D1, and Vrn4, are known to have large effects on the vernalization response, but the effects on cold hardiness are ambiguous. Near-isogenic experimental lines (NILs) in a Triple Dirk (TD) genetic background with different vernalization alleles were evaluated for cold hardiness. Although TD is homozygous dominant for Vrn-A1 (formerly Vrn1) and Vrn-B1 (formerly Vrn2), four of the lines are each homozygous dominant for a different vernalization gene, and one line is homozygous recessive for all four vernalization genes. Following establishment, the plants were initially acclimated for 6 weeks in a growth chamber and then stressed in a low temperature freezer from which they were removed over a range of temperatures as the chamber temperature was lowered 1.3°C h^–1. Temperatures resulting in no regrowth from 50% of the plants (LT₅₀) were determined by estimating the inflection point of the sigmoidal response curve by nonlinear regression. The LT₅₀ values were –6.7°C for cv. TD, –6.6°C for the Vrn-A1 and Vrn4 lines, –8.1°C for the Vrn-D1 (formerly Vrn3) line, –9.4°C for the Vrn-B1 line, and –11.7°C for the homozygous recessive winter line. The LT₅₀ of the true winter line was significantly lower than those of all the other lines. Significant differences were also observed between some, but not all, of the lines possessing dominant vernalization alleles. The presence of dominant vernalization alleles at one of the four loci studied significantly reduced cold hardiness following acclimation.     

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.

     

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.     

Molecular characterization of a novel vernalization allele <Emphasis Type="Italic">Vrn-B1d</Emphasis> and its effect on heading time in Chinese wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) landrace Hongchunmai

Flowering time of wheat cultivars contributes greatly to the adaptability to environmental conditions and it is largely controlled by vernalization genes. In this study, 262 Chinese mini-core wheat cultivars were used to identify the allelic variation at VRN-B1 locus. A novel dominant allele Vrn-B1d was found in Chinese spring wheat landrace cultivar Hongchunmai. This allele contained several genetic divergence within the first intron comparing to the recessive allele vrn-B1, including one large 6850-bp deletion (670–7519 bp), one small 187-bp deletion (7851–8037 bp), one unique SNP (T to C, 7845 bp), and one 4-bp mutation (TTTT to ACAA, 7847–7850 bp). Meanwhile, it was also different from the three known dominant alleles at VRN-B1 locus. Two pairs of primers were designed to identify the novel allele Vrn-B1d and other four known alleles of VRN-B1. A multiplex PCR was established to discriminate all five alleles simultaneously. The greenhouse experiment with high temperature (non-vernalizing condition) and long light showed that F₂ plants containing Vrn-B1d allele headed significantly earlier than those with recessive vrn-B1 allele, suggesting that Vrn-B1d is a dominant allele conferring the spring growth habit. This study provides a useful germplasm and molecular markers for wheat breeding.     

黄淮南片冬麦区主导品种春化基因及冬春性分析

以1950～2007年黄淮南片冬麦区的127个主导小麦品种为材料,利用第5同源群的春化基因分子标记对其进行了春化基因检测,并分析了小麦品种的春化基因与其冬春性的对应关系及黄淮南片冬麦区8次品种更换中春化基因与品种冬春性的演变规律.结果表明,参试品种中没有品种携带显性Vrn-A1基因,7个品种含有Vrn-B1基因(5.5%),2个品种含有Vrn-B1+Vrn-D1基因(1.6%),56个品种含有Vrn-D1基因(44.1%).春化基因类型与品种冬春特性基本相符,春化基因控制着小麦品种的冬春特性.主导品种含春化显性基因频率的变化趋势与冬春性变化规律存在较大差异,与传统方法相比,仅用春化基因来确定品种冬春性存在一定的不完善之处.采用春化基因分子标记与传统的冬春性鉴定方法相结合来认识品种冬春性、预测品种的抗寒性对黄淮南片冬麦区的小麦品种利用更具有指导意义.     

Epistatic interaction between vernalization genes Vrn-Am1 and Vrn-Am2 in diploid wheat

Genes Vrn-A(m)1 and Vrn-A(m)2 control the vernalization requirement in diploid wheat (Triticum monococcum). The epistatic interaction between these two genes on flowering date was studied here using a factorial analysis of variance. One hundred and two F2 plants were classified according to their genotypes for molecular markers tightly linked to Vrn-A(m)1 and Vrn-A(m)2. Mean comparisons showed that the VrnA(m)2 allele for winter growth habit was dominant to the vrn-A(m)2 allele for spring growth habit and that the Vrn-A(m)1 allele for spring growth habit was dominant to the vrn-A(m)1 allele for winter growth habit. A significant interaction was found between these two genes, suggesting that they work in the same developmental pathway. Plants homozygous for the recessive vrn-A(m)2 allele for spring growth habit flowered earlier than plants from the Vrn-A(m)2 class independently of the alleles present at Vrn-A(m)1. However, differences in heading date between plants with the Vrn-A(m)1 allele and those with the vrn-A(m)1 allele were significant only when the dominant Vrn-A(m)2 allele was present. A genetic model for the action of these two vernalization genes is proposed in which the role of Vrn-A(m)1 is to counteract the Vrn-A(m)2-mediated delay of flowering.     

Gliadin alleles in Canadian western red spring wheat cultivars: use of two different procedures of acid polyacrylamide gel electrophoresis for gliadin separation.

Gliadin allele compositions of 21 Canadian spring common wheat cultivars, most of which belong to the Canada western red spring (CWRS) class, were studied and great similarity in their genotypes was confirmed. It was found that alleles frequent in the set of Canadian wheats (such as Gli-B1d, Gli-D1j, Gli-A2m, and Gli-D2h) are very rare or absent in common wheat cultivars from other regions and countries studied earlier, indicating that germplasm of CWRS cultivars is rather unique. It may be suggested that alleles frequent in Canadian cultivars relate to important technological characteristics of these wheats and may possibly serve as marker genes during selection for quality traits. Similarity of gliadin electrophoregrams obtained by two different acid polyacryl-amide gel electrophoretic procedures for the same genotype was established, and the component composition of allelic variants of blocks of gliadin components found in the set of Canadian cultivars and in standard cultivars Chinese Spring and Bezostaya 1 are described.     

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.     

<Emphasis Type="Italic">VRN1</Emphasis> 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.

     

Application of RAPD for making DNA markers to Vrn genes

The possibility of using the PCR with arbitrary primers to mark Vrn genes, Vrn3 in particular, was investigated. RAPD-analysis of DNA of near isogenic lines on Vrn1, Vrn2 and Vrn3 loci in genefone of four varieties of winter wheat were carried out. Monogenic dominant lines by Vrn3 locus possessed 657 b.p. polymorphic DNA fragment. The possibility of using this polymorphic DNA amplicon as a molecular marker was confirmed by results of RAPD-analysis of DNA of some commercial wheat varieties of different ecological-geographic origin possessing dominant Vrn3 alleles, as well as by RAPD-analysis of F2 population from cross of monogenic Vrn3 dominant spring line with its recurrent parent--winter wheat variety Mironovskaya 808. The results of RAPD are comparable with hybridological analysis data concerning type of development of plants from F2 population.     

Segregation analysis of heading traits in hexaploid wheat utilizing recombinant inbred lines

The purpose of this study was to analyze the genetic segregation of heading traits in wheat using recombinant inbred lines (RILs) of hexaploid wheat, derived from Triticum aestivum cv. Chinese Spring and T. spelta var. duhameliamum. The population was examined under controlled environmental conditions as well as in the field. This strategy differentiated the effect of three genetic factors (vernalization requirement, photoperiod sensitivity and narrow-sense earliness) and identified their interactions. Correlation analysis showed that photoperiod sensitivity and narrow-sense earliness are critical for heading time in the field. Single-marker analysis using 322 molecular markers segregating among RIL detected a total of 38 linked markers for each genetic factor and heading in the field. In interval analysis, two Vrn genes (Vrn-B1 and Vrn-D1) and Ppd-B1 were mapped on chromosomes 5B, 5D and 2B, respectively. It was noticed that Vrn-B1 on 5B from the spelt wheat conferred a strong-spring habit equivalent to the homologous Vrn-A1. Quantitative trait locus analysis also showed that Ppd-B1 was not detected under the short-day condition without vernalization treatment, and that there were two types of genes for photoperiod sensitivity, dependent on and independent of vernalization treatment.     

Genetic control of the spring habit in old local cultivars and landraces of common wheat from Siberia

The inheritance of the spring habit was studied in 63 old local cultivars and landraces of common wheat from Eastern and Western Siberia and the Tyva Republic. Minimal polymorphism was observed for the dominant Vrn genes, controlling the spring habit in landraces of these regions. The control was digenic and involved the Vrnl and Vrn2 dominant genes in the majority (95%) of cultivars and was monogenic in three cultivars. None of the cultivars had the Vrn3 dominant gene, characteristic of the neighboring regions of China and Central Asia. Among 137 old local cultivars and landraces of Siberia, only one (cultivar Sibirskaya (k-23347) from Irkutsk oblast, was comparable in the response to the natural short day (photoperiod) to Chinese cultivars. Comparison of the results and the data reported for commercial cultivars revealed that the genotype frequencies of the dominant Vrn genes in Siberian landraces and commercial cultivars of common wheat remained essentially unchanged at least for the past 100 years. At the same time, Siberian landraces significantly differed in Vrn dominant gene frequencies from cultivars of the adjacent regions. It was assumed that the control of the spring habit by the two Vrn dominant genes is optimal for the climatic conditions of Siberia.     

The relationship between vernalization requirement and frost tolerance in substitution lines of wheat

Two sets of wheat (Triticum aestivum L.) substitution lines for the homoeologous group 5 chromosomes, 5A, 5B and 5D, carrying vernalization genes (Vrn-A1, Vrn-B1, Vrn-D1) were used to study the relationship between vernalization requirement and winter survival, with respect to the induction and maintenance of frost tolerance. Substitution lines carrying dominant Vrn loci substituted from the spring cultivars Zlatka (5A), Chinese Spring (5D) and the alternative cultivar eská Pesívka (5B) into three different winter wheat backgrounds, Vala, Koútka and Zdar, showed lower winter survival by 20, 36, and 41 % for substitutions of 5B, 5A and 5D, respectively, compared to the original winter cultivars. Reciprocal substitution lines between two winter cultivars Mironovskaya 808 and Bezostaya 1 carrying different recessive alleles, vrn-A1, vrn-B1, vrn-D1, did not exhibit a modified induction of frost tolerance, but the duration of good frost tolerance, as well as the ability to survive the whole winter, was changed. In accordance with the model suggesting that genes for vernalization act as a master switch regulating the duration of frost tolerance, substitutions of homoeologous group 5 chromosomes induced, at first, frost tolerance at a level equal to the parental cultivar, and then, relative to the different extent of saturation of vernalization requirement, they gradually lost both frost tolerance and their ability to re-induce significant frost tolerance with a drop in temperature following warm periods in the winter.     

Use of RAPD- and STS-analysis for marking genes of a homeologic group from chromosome 5 of common wheat

The search for STS (sequence-tagged site) and RAPD (random amplified polymorphic DNA) markers tightly linked to some genes of homeologous group 5 chromosomes of common wheat Triticum aestivum L., more specifically, awns inhibitor genes (B1), vernalization response gene (Vrn1), and homeologous chromosome pairing gene (Ph1), was conducted. To estimate the linkage of the gene with the marker, wheat lines marked with recessive alleles b1 and vrn1 were used. RELP (restriction fragment length polymorphism) and SSR (simple sequence repeat) analyses of isogenic wheat lines were conducted to characterize the chromosomal region transferred to the isogenic line from the donor parent. In RAPD analysis of isogenic wheat lines marked with recessive alleles b1 and vrn1, 95 arbitrary primers were used. To develop STS markers, analysis of the primary structure of RELP markers Xpsr426 and Xcdo504, tightly linked to the Vrn1 gene, and the Xpsr1201 marker, located at the Ph1 locus, was carried out. Two markers that are tightly linked to the Vrn1 gene (5AL)--RAPD marker Xr405 and STS marker Xsts426--were obtained in this work. In addition, there is every reason to believe that Xsts426 can be used as a PCR marker of genes Vrn2 (5BL) and Vrn3 (5DL), while Xsts1201, of the gene Ph1 (5BL).     

Effect of the 5R(5A) alien chromosome substitution on the growth habit and winter hardiness of wheat

The growth habit, ear emergence time, and frost tolerance of wheat/rye substitution lines have been studied in cultivars Rang and Mironovskaya Krupnozernaya whose chromosome 5A is substituted with chromosome 5R of Onkhoyskaya rye. Hybrid analysis has demonstrated that the spring habit of the recipient cultivars Rang and Mironovskaya Krupnozernaya is controlled by dominant gene Vrn-A1 located in chromosome 5A. Onokhoyskaya rye has a dominant gene for the spring habit (Sp1) located in chromosome 5R. It has been found that the resultant 5R(5A) alien-substitution lines have a winter type of development and ears do not emerge during summer in plants sown in spring. The change in growth habit has been shown to be related to the absence of the rye Spl gene expression in the substitution lines. The winter hardiness of winter 5R(5A) alien-substitution lines has been studied under the environmental conditions of Novosibirsk. Testing the lines in the first winter demonstrated that their winter survival is 20-27%. The possible presence of the frost resistance gene homeoallelic to the known genes Fr1 and Fr2 of the common wheat located on chromosomes 5A and 5D, respectively, is discussed.