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.     

Haplotype analysis of vernalization loci in European barley germplasm reveals novel <Emphasis Type="Italic">VRN-H1</Emphasis> alleles and a predominant winter <Emphasis Type="Italic">VRN-H1/VRN-H2</Emphasis> multi-locus haplotype

In barley, variation in the requirement for vernalization (an extended period of low temperature before flowering can occur) is determined by the VRN-H1, -H2 and -H3 loci. In European cultivated germplasm, most variation in vernalization requirement is accounted for by alleles at VRN-H1 and VRN-H2 only, but the range of allelic variation is largely unexplored. Here we characterise VRN-H1 and VRN-H2 haplotypes in 429 varieties representing a large portion of the acreage sown to barley in Western Europe over the last 60 years. Analysis of genotype, intron I sequencing data and growth habit tests identified three novel VRN-H1 alleles and determined the most frequent VRN-H1 intron I rearrangements. Combined analysis of VRN-H1 and VRN-H2 alleles resulted in the classification of seventeen VRN-H1/VRN-H2 multi-locus haplotypes, three of which account for 79% of varieties. The molecular markers employed here represent powerful diagnostic tools for prediction of growth habit and assessment of varietal purity. These markers will also allow development of germplasm to test the behaviour of individual alleles with the aim of understanding the relationship between allelic variation and adaptation to specific agri-environments.     

Association of barley photoperiod and vernalization genes with QTLs for flowering time and agronomic traits in a BC2DH population and a set of wild barley introgression lines

The control of flowering time has important impacts on crop yield. The variation in response to day length (photoperiod) and low temperature (vernalization) has been selected in barley to provide adaptation to different environments and farming practices. As a further step towards unraveling the genetic mechanisms underlying flowering time control in barley, we investigated the allelic variation of ten known or putative photoperiod and vernalization pathway genes between two genotypes, the spring barley elite cultivar ‘Scarlett’ (Hordeum vulgare ssp. vulgare) and the wild barley accession ‘ISR42-8’ (Hordeum vulgare ssp. spontaneum). The genes studied are Ppd-H1, VRN-H1, VRN-H2, VRN-H3, HvCO1, HvCO2, HvGI, HvFT2, HvFT3 and HvFT4. ‘Scarlett’ and ‘ISR42-8’ are the parents of the BC₂DH advanced backcross population S42 and a set of wild barley introgression lines (S42ILs). The latter are derived from S42 after backcrossing and marker-assisted selection. The genotypes and phenotypes in S42 and S42ILs were utilized to determine the genetic map location of the candidate genes and to test if these genes may exert quantitative trait locus (QTL) effects on flowering time, yield and yield-related traits in the two populations studied. By sequencing the characteristic regions of the genes and genotyping with diagnostic markers, the contrasting allelic constitutions of four known flowering regulation genes were identified as ppd-H1, Vrn-H1, vrn-H2 and vrn-H3 in ‘Scarlett’ and as Ppd-H1, vrn-H1, Vrn-H2 and a novel allele of VRN-H3 in ‘ISR42-8’. All candidate genes could be placed on a barley simple sequence repeat (SSR) map. Seven candidate genes (Ppd-H1, VRN-H2, VRN-H3, HvGI, HvFT2, HvFT3 and HvFT4) were associated with flowering time QTLs in population S42. Four exotic alleles (Ppd-H1, Vrn-H2, vrn-H3 and HvCO1) possibly exhibited significant effects on flowering time in S42ILs. In both populations, the QTL showing the strongest effect corresponded to Ppd-H1. Here, the exotic allele was associated with a reduction of number of days until flowering by 8.0 and 12.7%, respectively. Our data suggest that Ppd-H1, Vrn-H2 and Vrn-H3 may also exert pleiotropic effects on yield and yield-related traits.     

Validation of the <Emphasis Type="Italic">VRN-H2</Emphasis>/<Emphasis Type="Italic">VRN-H1</Emphasis> epistatic model in barley reveals that intron length variation in <Emphasis Type="Italic">VRN-H1</Emphasis> may account for a continuum of vernalization sensitivity

The epistatic interaction of alleles at the VRN-H1 and VRN-H2 loci determines vernalization sensitivity in barley. To validate the current molecular model for the two-locus epistasis, we crossed homozygous vernalization-insensitive plants harboring a predicted “winter type” allele at either VRN-H1 (Dicktoo) or VRN-H2 (Oregon Wolfe Barley Dominant), or at both VRN-H (Calicuchima-sib) loci and measured the flowering time of unvernalized F₂ progeny under long-day photoperiod. We assessed whether the spring growth habit of Calicuchima-sib is an exception to the two-locus epistatic model or contains novel “spring” alleles at VRN-H1 (HvBM5A) and/or VRN-H2 (ZCCT-H) by determining allele sequence variants at these loci and their effects relative to growth habit. We found that (a) progeny with predicted “winter type” alleles at both VRN-H1 and VRN-H2 alleles exhibited an extremely delayed flowering (i.e. vernalization-sensitive) phenotype in two out of the three F₂ populations, (b) sequence flanking the vernalization critical region of HvBM5A intron 1 likely influences degree of vernalization sensitivity, (c) a winter habit is retained when ZCCT-Ha has been deleted, and (d) the ZCCT-H genes have higher levels of allelic polymorphism than other winterhardiness regulatory genes. Our results validate the model explaining the epistatic interaction of VRN-H2 and VRN-H1 under long-day conditions, demonstrate recovery of vernalization-sensitive progeny from crosses of vernalization-insensitive genotypes, show that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity, and provide molecular markers that are accurate predictors of “winter vs spring type” alleles at the VRN-H loci.     

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.     

Regions associated with repression of the barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) <Emphasis Type="Italic">VERNALIZATION1</Emphasis> gene are not required for cold induction

Activity of the VERNALIZATION1 (VRN1) gene is required for flowering in temperate cereals such as wheat and barley. In varieties that require prolonged exposure to cold to flower (vernalization), VRN1 is expressed at low levels and is induced by vernalization to trigger flowering. In other varieties, deletions or insertions in the first intron of the VRN1 gene are associated with increased VRN1 expression in the absence of cold treatment, reducing or eliminating the requirement for vernalization. To characterize natural variation in VRN1, the first intron of the barley (Hordeum vulgare) VRN1 gene (HvVRN1) was assayed for deletions or insertions in a collection of 1,000 barleys from diverse geographical regions. Ten alleles of HvVRN1 containing deletions or insertions in the first intron were identified, including three alleles that have not been described previously. Different HvVRN1 alleles were associated with differing levels of HvVRN1 expression in non-vernalized plants and with different flowering behaviour. Using overlapping deletions, we delineated regions in the HvVRN1 first intron that are associated with low levels of HvVRN1 expression in non-vernalized plants. Deletion of these intronic regions does not prevent induction of HvVRN1 by cold or the maintenance of increased HvVRN1 expression following cold treatment. We suggest that regions within the first intron of HvVRN1 are required to maintain low levels of HvVRN1 expression prior to winter but act independently of the regulatory mechanisms that mediate induction of HvVRN1 by cold during winter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers 1179825, 1179833, 1179836, 1179858.     

The Vrn-H2 locus is a major determinant of flowering time in a facultative × winter growth habit barley (Hordeum vulgare L.) mapping population

With the aim of dissecting the genetic determinants of flowering time, vernalization response, and photoperiod sensitivity, we mapped the candidate genes for Vrn-H2 and Vrn-H1 in a facultative × winter barley mapping population and determined their relationships with flowering time and vernalization via QTL analysis. The Vrn-H2 candidate ZCCT-H genes were completely missing from the facultative parent and present in the winter barley parent. This gene was the major determinant of flowering time under long photoperiods in controlled environment experiments, irrespective of vernalization, and under spring-sown field experiments. It was the sole determinant of vernalization response, but the effect of the deletion was modulated by photoperiods when the vernalization requirement was fulfilled. There was no effect under short photoperiods. The Vrn-H1 candidate gene (HvBM5A) was mapped based on a microsatellite polymorphism we identified in the promoter of this gene. Otherwise, the HvBM5A alleles for the two parents were identical. Therefore, the significant flowering time QTL effect associated with this locus suggests tight linkage rather than pleiotropy. This QTL effect was smaller in magnitude than those associated with the Vrn-H2 locus and was significant in two-way interactions with Vrn-H2. The Vrn-H1 locus had no effect on vernalization response. Our results support the Vrn-H2/Vrn-H1 repressor/structural gene model for vernalization response in barley and suggest that photoperiod may also affect the Vrn genes or tightly linked loci.     

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.     

The FLOWERING LOCUS T-like gene family in barley (Hordeum vulgare)

The FLOWERING LOCUS T (FT) gene plays a central role in integrating flowering signals in Arabidopsis because its expression is regulated antagonistically by the photoperiod and vernalization pathways. FT belongs to a family of six genes characterized by a phosphatidylethanolamine-binding protein (PEBP) domain. In rice (Oryza sativa), 19 PEBP genes were previously described, 13 of which are FT-like genes. Five FT-like genes were found in barley (Hordeum vulgare). HvFT1, HvFT2, HvFT3, and HvFT4 were highly homologous to OsFTL2 (the Hd3a QTL), OsFTL1, OsFTL10, and OsFTL12, respectively, and this relationship was supported by comparative mapping. No rice equivalent was found for HvFT5. HvFT1 was highly expressed under long-day (inductive) conditions at the time of the morphological switch of the shoot apex from vegetative to reproductive growth. HvFT2 and HvFT4 were expressed later in development. HvFT1 was therefore identified as the main barley FT-like gene involved in the switch to flowering. Mapping of HvFT genes suggests that they provide important sources of flowering-time variation in barley. HvFTI was a candidate for VRN-H3, a dominant mutation giving precocious flowering, while HvFT3 was a candidate for Ppd-H2, a major QTL affecting flowering time in short days.     

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.     

Expression analysis of vernalization and day-length response genes in barley (Hordeum vulgare L.) indicates that VRNH2 is a repressor of PPDH2 (HvFT3) under long days

The response to vernalization and the expression of genes associated with responses to vernalization (VRNH1, VRNH2, and VRNH3) and photoperiod (PPDH1 and PPDH2) were analysed in four barley (Hordeum vulgare L.) lines: 'Alexis' (spring), 'Plaisant' (winter), SBCC058, and SBCC106 (Spanish inbred lines), grown under conditions of vernalization and short days (VSD) or no vernalization and long days (NVLD). The four genotypes differ in VRNH1. Their growth habits and responses to vernalization correlated with the level of expression of VRNH1 and the length of intron 1. 'Alexis' and 'Plaisant' behaved as expected. SBCC058 and SBCC106 showed an intermediate growth habit and flowered relatively late in the absence of vernalization. VRNH1 expression was induced by cold for all genotypes. Under VSD, VRNH1 expression was detected in the SBCC genotypes later than in 'Alexis' but earlier than in 'Plaisant'. VRNH2 was repressed under short days while VRNH1 expression increased in parallel. VRNH3 was detected only in 'Alexis' under NVLD, whereas it was not expressed in plants with the active allele of VRNH2 (SBCC058 and 'Plaisant'). Under VSD, PPDH2 was expressed in 'Alexis', SBCC058, and SBCC106, but it was only expressed weakly in 'Alexis' under NVLD. Further analysis of PPDH2 expression in two barley doubled haploid populations revealed that, under long days, HvFT3 and VRNH2 expression levels were related inversely. The timing of VRNH2 expression under a long photoperiod suggests that this gene might be involved in repression of PPDH2 and, indirectly, in the regulation of flowering time through an interaction with the day-length pathway.     

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.     

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.     

The alternative splicing of <Emphasis Type="Italic">EAM8</Emphasis> contributes to early flowering and short-season adaptation in a landrace barley from the Qinghai-Tibetan Plateau

Key message

The early flowering of Lalu was determined to be due to a novel spontaneous eam8 mutation, which resulted in intron retention and the formation of a putative truncated protein.

Abstract

Barley is a staple crop grown over an extensive area in the Qinghai-Tibetan Plateau. Understanding the genetic mechanism for its success in a high altitude is important for crop improvement in marginal environments. Early flowering is a critical adaptive trait that strongly influences reproductive fitness in a short growing season. Loss-of-function mutations at the circadian clock gene EARLY MATURITY 8 (EAM8) promote rapid flowering. In this study, we identified a novel, spontaneous recessive eam8 mutant with an early flowering phenotype in a Tibetan barley landrace Lalu, which is natively grown at a high altitude of approximately 4000 m asl. The co-segregation analysis in a F₂ population derived from the cross Lalu (early flowering)?×?Diqing 1 (late flowering) confirmed that early flowering of Lalu was determined to be due to an allele at EAM8. The eam8 allele from Lalu carries an A/G alternative splicing mutation at position 3257 in intron 3, designated eam8.l; this alternative splicing event leads to intron retention and a putative truncated protein. Of the 134 sequenced barley accessions, which are primarily native to the Qinghai-Tibet Plateau, three accessions carried this mutation. The eam8.l mutation was likely to have originated in wild barley due to the presence of the Lalu haplotype in H. spontaneum from Tibet. Overall, alternative splicing has contributed to the evolution of the barley circadian clock and in the short-season adaptation of local barley germplasm. The study has also identified a novel donor of early-flowering barley which will be useful for barley improvement.

     

Altitudinal and climatic adaptation is mediated by flowering traits and FRI, FLC, and PHYC genes in Arabidopsis

Extensive natural variation has been described for the timing of flowering initiation in many annual plants, including the model wild species Arabidopsis (Arabidopsis thaliana), which is presumed to be involved in adaptation to different climates. However, the environmental factors that might shape this genetic variation, as well as the molecular bases of climatic adaptation by modifications of flowering time, remain mostly unknown. To approach both goals, we characterized the flowering behavior in relation to vernalization of 182 Arabidopsis wild genotypes collected in a native region spanning a broad climatic range. Phenotype-environment association analyses identified strong altitudinal clines (0-2600 m) in seven out of nine flowering-related traits. Altitudinal clines were dissected in terms of minimum winter temperature and precipitation, indicating that these are the main climatic factors that might act as selective pressures on flowering traits. In addition, we used an association analysis approach with four candidate genes, FRIGIDA (FRI), FLOWERING LOCUS C (FLC), PHYTOCHROME C (PHYC), and CRYPTOCHROME2, to decipher the genetic bases of this variation. Eleven different loss-of-function FRI alleles of low frequency accounted for up to 16% of the variation for most traits. Furthermore, an FLC allelic series of six novel putative loss- and change-of-function alleles, with low to moderate frequency, revealed that a broader FLC functional diversification might contribute to flowering variation. Finally, environment-genotype association analyses showed that the spatial patterns of FRI, FLC, and PHYC polymorphisms are significantly associated with winter temperatures and spring and winter precipitations, respectively. These results support that allelic variation in these genes is involved in climatic adaptation.     

Deciphering the genetics of flowering time by an association study on candidate genes in bread wheat (Triticum aestivum L.)

Earliness is very important for the adaptation of wheat to environmental conditions and the achievement of high grain yield. A detailed knowledge of key genetic components of the life cycle would enable an easier control by the breeders. The objective of the study was to investigate the effect of candidate genes on flowering time. Using a collection of hexaploid wheat composed of 235 lines from diverse geographical origins, we conducted an association study for six candidate genes for flowering time and its components (vernalization sensitivity and earliness per se). The effect on the variation of earliness components of polymorphisms within the copies of each gene was tested in ANOVA models accounting for the underlying genetic structure. The collection was structured in five groups that minimized the residual covariance. Vernalization requirement and lateness tend to increase according to the mean latitude of each group. Heading date for an autumnal sowing was mainly determined by the earliness per se. Except for the Constans (CO) gene orthologous of the barley HvCO3, all gene polymorphisms had a significant impact on earliness components. The three traits used to quantify vernalization requirement were primarily associated with polymorphisms at Vrn-1 and then at Vrn-3 and Luminidependens (LD) genes. We found a good correspondence between spring/winter types and genotypes at the three homeologous copies of Vrn-1. Earliness per se was mainly explained by polymorphisms at Vrn-3 and to a lesser extent at Vrn-1, Hd-1 and Gigantea (GI) genes. Vernalization requirement and earliness as a function of geographical origin, as well as the possible role of the breeding practices in the geographical distribution of the alleles and the hypothetical adaptive value of the candidate genes, are discussed.     

Hybridisation‐based target enrichment of phenology genes to dissect the genetic basis of yield and adaptation in barley

Barley (Hordeum vulgare L.) is a major cereal grain widely used for livestock feed, brewing malts and human food. Grain yield is the most important breeding target for genetic improvement and largely depends on optimal timing of flowering. Little is known about the allelic diversity of genes that underlie flowering time in domesticated barley, the genetic changes that have occurred during breeding, and their impact on yield and adaptation. Here, we report a comprehensive genomic assessment of a worldwide collection of 895 barley accessions based on the targeted resequencing of phenology genes. A versatile target‐capture method was used to detect genome‐wide polymorphisms in a panel of 174 flowering time‐related genes, chosen based on prior knowledge from barley, rice and Arabidopsis thaliana. Association studies identified novel polymorphisms that accounted for observed phenotypic variation in phenology and grain yield, and explained improvements in adaptation as a result of historical breeding of Australian barley cultivars. We found that 50% of genetic variants associated with grain yield, and 67% of the plant height variation was also associated with phenology. The precise identification of favourable alleles provides a genomic basis to improve barley yield traits and to enhance adaptation for specific production areas.     

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.