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First published online November 12, 2008; 10.1104/pp.108.129353 Plant Physiology 149:245-257 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Genetic and Molecular Characterization of the VRN2 Loci in Tetraploid Wheat1,[W],[OA]Department of Plant Sciences, University of California, Davis, California 95616–8515 (A.D., C.L., J.D.); Instituto de Recursos Biológicos, Instituto Nacional de Tecnología Agropecuaria, Buenos Aires, Argentina (G.T.); and Department of Plant and Soil Sciences, Oklahoma State University, Stillwater, Oklahoma 74078 (L.Y.)
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.
Wheat (Triticum aestivum) is one of the major crop species and occupies a wide range of environments from 65°N to 45°S (Lantican et al., 2005  ). This wide adaptability is favored by diverse growth habits, which include winter and spring forms. Winter wheats are sown in autumn and require long exposures to cold temperatures (vernalization) to accelerate flowering. The vernalization requirement prevents flower development during winter, protecting sensitive floral organs from freezing temperatures. Spring wheats are planted in the spring or in the fall (in regions with mild winters) and do not have a vernalization requirement.
The three major genes responsible for natural variation in vernalization requirement in wheat (and also in barley [Hordeum vulgare]) are VRN1, VRN2, and VRN3. VRN1 is a homolog of the Arabidopsis (Arabidopsis thaliana) meristem identity gene APETALA1, which determines the transition between the production of leaves and flowers at the shoot apical meristem (Danyluk et al., 2003
VRN3 is a homolog of the Arabidopsis photoperiod gene FLOWERING LOCUS T, and in both species this gene up-regulates VRN1 transcription under long days (Yan et al., 2006
In diploid wheat, the VRN2 locus includes two tandemly duplicated genes designated ZCCT1 and ZCCT2 (Yan et al., 2004b
In wheat and barley accessions with winter growth habit, ZCCT transcripts show a progressive decrease during vernalization (under long days) that is not observed in control plants kept at nonvernalizing temperatures (Yan et al., 2004b
All diploid wheat and barley accessions with winter growth habit studied so far have at least one functional ZCCT gene, whereas those with spring growth habit associated with recessive vrn2 alleles have deletions encompassing all ZCCT genes or carry mutations in conserved amino acids of the CCT domains (Yan et al., 2004b
RNA interference of ZCCT1 in hexaploid winter wheat variety Jagger (T. aestivum; 2n = 42; genomes AABBDD) reduces ZCCT1 transcript levels and accelerates flowering, suggesting that the VRN2 locus also plays a significant role in the regulation of flowering in hexaploid wheat (Yan et al., 2004b Allelic variation for VRN2 has not been described so far for tetraploid or hexaploid wheat, likely due to the fact that in polyploid wheat simultaneous loss-of-function mutations at all VRN2 homoeoloci are required to confer spring growth habit. In addition, allelic variation for VRN2 would be detected only when alleles for winter growth habit are present at all VRN1 loci, as alleles for spring growth habit are dominant and epistatic on VRN2.
ZCCT1 and ZCCT2 Sequences from Diploid and Tetraploid Wheat Species
The genomic region encompassing the VRN2 locus was previously sequenced from diploid wheat T. monococcum (Yan et al., 2004b
Additional ZCCT genes were sequenced from winter accessions of diploid Triticum and Aegilops species with genomes similar to the A, B, and D genomes of hexaploid wheat. These species included Triticum urartu, the donor of the A genome (Dvorak et al., 1988 ); Aegilops speltoides (S genome), the closest extant diploid species to the B genome of tetraploid and hexaploid wheat (Dvorak and Zhang, 1990); and Aegilops tauschii, the donor of the D genome of hexaploid wheat (Kihara, 1944).
The sequences for ZCCT-D1 (FJ173818) and ZCCT-D2 (FJ173822) were obtained from Ae. tauschii BAC clones 2H24, 14E16, and 78I09 (Akhunov et al., 2005
The hybridization of Southern blots including DraI-digested DNAs from wild and cultivated tetraploid and hexaploid Triticum accessions (see "Materials and Methods") revealed contrasting patterns of allelic diversity for different ZCCT genes. The shortest overlapping DraI restriction fragments (767 bp from ZCCT-A1 and 771 bp from ZCCT-B1) showed limited variation among accessions. Limited variation was also found for the 1,420-bp fragment corresponding to the ZCCT-A2 gene (Fig. 2 ). On the contrary, the restriction fragments within the region corresponding to the ZCCT-B2a and ZCCT-B2b genes were variable in size (approximately 3–5 kb), generating multiple haplotypes (Fig. 2, lanes 1–5). Some cultivated durum lines showed two fragments in this region and others only one (Fig. 2).
The largest RFLP fragment corresponds to a third and more divergent ZCCT copy (ZCCT-A3). The ZCCT-A3 putative coding region is only 81% to 82% identical to the other two ZCCT genes and has a shorter first exon that does not include the predicted zinc finger characteristic of other ZCCT proteins. So far, ZCCT-A3 has been found only in the A genome, 16.2 kb upstream of ZCCT-A2 (Dubcovsky and Dvorak, 2007 ). It is not yet known if ZCCT-A3 is translated into a functional protein; therefore, it was not included in the allelic diversity study. In addition to the variation in restriction fragment size, the ZCCT-B1 and ZCCT-B2 genes showed polymorphisms in copy number. Six T. turgidum subsp. dicoccoides accessions from Rosh Pinna, Israel, showed unusually strong hybridization signals at the restriction fragments corresponding to ZCCT-B1 and ZCCT-B2 (one accession is shown in Fig. 2, lane 8). The VRN-A2 fragments from the same accessions showed no increase in hybridization intensity, confirming equal loading of DNA on the Southern blots. Based on this result, we concluded that the copy number of the ZCCT-B1 and ZCCT-B2 genes was amplified in the Rosh Pinna accessions. One spring accession of T. turgidum subsp. dicoccon (PI470739), collected in the mountains of Kars, Turkey (1,590 m above sea level), showed a deletion of the restriction fragments corresponding to the ZCCT-B2 gene(s) and reduced hybridization intensity of the 767/771-bp fragment corresponding to ZCCT-A1 and ZCCT-B1 overlapping fragments (Fig. 2, lanes 10 and 11). The deletion of both ZCCT-B1 and ZCCT-B2 genes in PI470739 was confirmed by PCR using primers specific for ZCCT-B1 (VRN2/B1/F3-R6) and ZCCT-B2 (VRN2/B2/F2-R5) genes (Supplemental Table S1). These primers failed to amplify any ZCCT fragment from PI470739.
The effect of the VRN2 loci on flowering time in tetraploid wheat was studied using a plant segregating simultaneously for the vrn-B2 deletion from PI470739 and the nonfunctional vrn-Am2 from T. monococcum accession DV92. The development of this plant is described in "Materials and Methods" and in Figure 3 .
A total of 42 plants with winter growth habit were selected from the progeny of a BC3F1 line heterozygous for different vernalization genes (Fig. 3G). The winter growth habit indicates that these plants are homozygous for the recessive vrn-A1 and vrn-B1 alleles. Using molecular markers for VRN-A2 (Fig. 3C) and VRN-B2 (Fig. 3H), we selected plant 521 (BC3F2-521 hereafter), which was heterozygous for both VRN-A2 and VRN-B2 and homozygous for the recessive vrn-A1 and vrn-B1 alleles (Fig. 3). The progeny of this plant were used to test the effect of the different VRN2 alleles on flowering time.
Several plants from the progeny of BC3F2-521 were analyzed for chromosome number and all showed 28 chromosomes, indicating that the vrn-Am2 gene was incorporated either as a complete chromosome substitution line or as a translocation line. To differentiate between these two possibilities, this line was analyzed with two molecular markers for the short and long arms of homoeologous group 5. The marker for the PINA gene (Bonafede et al., 2007
The nonvernalized progeny of line BC3F2-521 segregated into two nonoverlapping groups for flowering time. The first group included 13 early-flowering plants that headed in less than 60 d (average, 53.0 ± 0.4 d) and were classified as spring, whereas the second group included 41 late-flowering plants that took more than 90 d for heading (average, 139.1 ± 4.6 d) and were classified as winter (Fig. 4A
). The group with spring growth habit was less variable than the group with winter growth habit, which showed two peaks, likely associated with the presence of homozygous and heterozygous lines (Fig. 4A). The observed ratio between spring and winter plants differed significantly from a 1:15 ratio segregation (two dominant genes;
The same plants were genotyped with VRN-A2 and VRN-B2 markers to determine which locus was responsible for the observed segregation in heading time (Fig. 5 ). No significant differences were detected among the VRN-A2 genotypic classes (Fig. 5A), indicating that both T. turgidum (PI470739) and T. monococcum DV92 have recessive vrn-A2 alleles. This indicates that none of the ZCCT genes present at the VRN-A2 locus is able to confer a vernalization requirement.
Genotyping with the codominant SNF-B2 marker tightly linked to the VRN-B2 locus showed that all 13 spring plants were homozygous for the recessive vrn-B2 allele from PI470739 (Fig. 5B), a result that was confirmed using PCR primers specific for the ZCCT-B1 and ZCCT-B2 genes (Supplemental Table S1). These results indicate that variation at the VRN-B2 locus was responsible for the segregation in flowering time observed in the progeny from BC3F2-521. The effect of the functional VRN-B2 allele on heading time was partially dominant. Plants homozygous for the functional VRN-B2 allele (Langdon/Durelle) flowered 159 ± 5.3 d after sowing, whereas those heterozygous for VRN-B2 flowered significantly (P < 0.001) earlier (120 ± 4.3 d after sowing). Heading time of the heterozygous plants was only 14 d later than the midpoint between the two homozygous classes (106 d). Using these numbers, the degree of dominance was calculated to be 0.26 (14 d/53 d, with 0 = completely additive and 1 = completely dominant). This value indicates a relatively small dominant effect. The differences in heading time between the VRN-B2 allelic classes disappeared when the plants were vernalized. Vernalized plants carrying the functional VRN-B2 allele headed almost at the same time (73.7 ± 1.4 d) as the vernalized plants homozygous for the vrn-B2 deletion (74.1 ± 1.2 d; P = 0.26). Differences between the VRN-A2 allelic classes were also not significant (P = 0.13). A two-way factorial ANOVA including vernalization and VRN-B2 alleles as factors showed a highly significant interaction between VRN-B2 alleles and vernalization (P < 0.0001), confirming that the effect of this locus on heading time was the result of differences in vernalization requirement.
The ZCCT1 and ZCCT2 genes present in the functional VRN-B2 and nonfunctional VRN-A2 loci from BC3F2-521 were sequenced to determine if the differences in functionality were associated with specific mutations in the conserved CCT domain. For comparison, a consensus CCT sequence was generated from different classes of CO-like proteins found in plants (Griffiths et al., 2003
ZCCT-A1 The predicted ZCCT-A1 protein corresponding to the nonfunctional VRN-A2 locus from BC3F2-521 has a mutation from R to C at position 39 of the CCT domain (designated R39C hereafter; Fig. 6). This position of the CCT domain is well conserved among CCT domains from other CO-like proteins and HAP2 proteins (Fig. 6). The R39C mutation was detected in all 37 cultivated T. turgidum subsp. durum accessions analyzed in this study but was polymorphic in cultivated T. turgidum subsp. dicoccon (present in 11 of 22 accessions) and wild T. turgidum subsp. dicoccoides (present in 10 of 19 accessions; Table II ). One accession from Asia Minor (PI355454) showed an additional R35Q mutation. The 11 accessions of T. turgidum subsp. dicoccon that lack the R39C mutation all have the R35W mutation. The R35W mutation was also found in eight of the nine accessions of T. turgidum subsp. dicoccoides that do not have the R39C mutation. T. turgidum subsp. dicoccoides accession 10-85 collected at Ammiad in Israel was the only one with no mutations in the CCT domain from ZCCT-A1 (Table II).
In addition to the R39C mutation, the ZCCT-A1 protein has a deletion of seven amino acids relative to the ZCCT-Am1 protein from T. monococcum. These seven amino acids are located immediately downstream of the putative zinc finger domain from amino acids 49 to 55 (numbers are relative to the initial Met in ZCCT-Am1). A screening using primers VRN2/22F+R (Supplemental Table S1) showed that the same deletion was present in all 78 tetraploid wheats tested in this study (Table II) and in 103 of 107 T. urartu accessions. The ZCCT-A1 genes from 15 T. urartu accessions were fully sequenced, and none of them have mutations in the CCT domain.
ZCCT-A2 All 48 accessions of cultivated tetraploid wheat sequenced for this gene have the R16C mutation in the CCT domain. This mutation was also found in the 15 accessions of T. urartu and four accessions of T. monococcum (ZCCT-Am2) but was not detected in the predicted ZCCT2 proteins from Ae. tauschii or Ae. speltoides (Table I; Fig. 6). Three of the four T. monococcum accessions (including DV92) have an R39C mutation in addition to the R16C mutation in ZCCT-Am2.
ZCCT-B1
ZCCT-B2 A screening for the 1-bp indel characteristic of the ZCCT-B2 gene duplication using PCR primers VRN2/B2/F2+R5 (Supplemental Table S1) failed to detect the duplication in T. turgidum subsp. dicoccoides. Although the detection of the two ZCCT-B2 forms with this PCR marker is sufficient to confirm the presence of the duplication, the detection of a single sequence needs to be interpreted with caution because it can indicate either the absence of the duplication or the absence of the 1-bp indel polymorphism. The fact that all of the T. turgidum subsp. dicoccoides accessions included in the RFLP screen show a single ZCCT-B2 fragment (with the exception of the Rosh Pinna accessions) provides additional indirect evidence for the absence of the duplication in most wild accessions. In T. turgidum subsp. dicoccon, the presence of the ZCCT-B2 duplication was confirmed in the four accessions that carry the R39C mutation at the ZCCT-B1 gene (PI319868, PI319869, PI355454, and PI352347; Table II). The presence of the ZCCT-B2 duplication was also confirmed among most of the modern T. turgidum subsp. durum varieties (36 of 37), with Messapia as the only exception (Table II). Many of the cultivated durum varieties showed two fragments in the RFLP screening (Fig. 2).
Quantification of transcript levels of ZCCT1 and ZCCT2 in tetraploid wheat leaves collected from 3-, 4-, and 5-week-old plants showed that the average transcript levels of ZCCT2 were significantly higher than those of ZCCT1 for all three time points (Fig. 7 ). Since quantitative reverse transcription-PCR primers (Supplemental Table S1) were designed to differentiate ZCCT1 from ZCCT2 but not A from B genome copies of the same gene, the transcript levels presented in Figure 7 include both A and B homoeologues for each gene.
The results presented here indicate that the differences among ZCCT proteins coded by genes corresponding to functional and nonfunctional VRN2 alleles are concentrated in the CCT domain. This 43-amino acid domain is well conserved in CO and CO-like proteins (defined as being more similar to CO than to other Arabidopsis proteins like TOC1) from mosses, gymnosperms, and angiosperms, indicating an ancient origin (Griffiths et al., 2003 ). The CCT domains are involved in the nuclear localization of CO and CO-like proteins but also have additional roles. In Arabidopsis, the co-7 CCT mutation does not alter the nuclear localization of the CO protein but delays flowering significantly (Robson et al., 2001). It is possible that some mutations in the CCT domain may limit its ability to interact with other proteins (Kurup et al., 2000).
It was shown recently that the CCT domains from Arabidopsis CO and COL15 can interact with several AtHAP3 and AtHAP5 proteins in yeast, and this interaction was confirmed in plant cells and in vitro (Ben-Naim et al., 2006
CCT domains and HAP2 proteins have similar amino acids at 18 positions, which are also well conserved within each group of proteins from mosses to vascular plants (Wenkel et al., 2006 Interestingly, the three CCT mutations identified here in ZCCT proteins coded by genes located in nonfunctional VRN2 loci are located at positions 16, 35, and 39, which are conserved both between and within the CCT domains and HAP2 proteins (Fig. 6). These three positions of the CCT domain are conserved in many plants, including mosses. Position 16 is either K or R (both positively charged amino acids) in Arabidopsis CO, CO-like (except those of class II), and all HAP2 proteins, whereas Arg residues at positions 35 and 39 are invariant among the same proteins (Fig. 6). All three mutations (R16C, R35W, and R39C) are associated with high negative BLOSUM62 scores (–3), which are indicative of changes involving amino acids with very different biochemical properties.
Taken together, the high negative BLOSUM62 scores and the conserved CCT/HAP2 positions where these mutations occurred suggest that these three mutations have a high probability of disrupting or altering the function of the mutant ZCCT proteins. The importance of CCT position 35 has been confirmed independently in Arabidopsis CO, as an induced ethylmethane sulfonate mutation at this position (co-7) produces a severe effect on flowering time (Robson et al., 2001
Assuming that the mutations at CCT positions 16, 35, and 39 can disrupt the function of the ZCCT proteins, the following model can explain the complex results presented here. We propose that both ZCCT1 and ZCCT2 have the ability to delay flowering and confer a vernalization requirement. We will refer to this model hereafter as the "two-ZCCT" hypothesis to facilitate the discussion. The first corollary of this hypothesis is that the presence of a functional copy of at least one of these two genes would be sufficient to confer a vernalization requirement. The second corollary of this hypothesis is that mutations in both genes are required to completely disrupt the function of a particular VRN2 locus. The following arguments are presented to support this hypothesis.
Similarity of ZCCT1 and ZCCT2 CCT Domains
Nonfunctional vrn2 Alleles
The available information from T. monococcum also supports the two-ZCCT hypothesis. Cultivated T. monococcum accession DV92 has a recessive vrn-Am2 allele that is associated with the R35W mutation in ZCCT-Am1 and both R16C and R39C mutations in ZCCT-Am2 (Yan et al., 2004b
Based on the limited information available at the time of cloning VRN-Am2, Yan et al. (2004b)
Functional VRN2 Alleles The same is true for the winter accessions of T. urartu. The 15 accessions of T. urartu sequenced so far all have the R16C mutation in the CCT domain of ZCCT-A2 and no mutations in ZCCT-A1 (Table I). This suggests that the winter growth habit in T. urartu is also conferred by ZCCT-A1. The molecular characterization of the functional VRN-B2 allele provided the strongest support to the two-ZCCT hypothesis. The ZCCT-B1 protein found in the parental lines of BC3F2-521 (Langdon/Durelle) has an R39C mutation identical to the one found in the ZCCT-A1 protein from the nonfunctional VRN-A2 allele (Table I). The low BLOSUM62 score (–3) and the fact that this mutation alters a conserved position across HAP2 proteins and CCT domains (Fig. 6) suggest that this ZCCT-B1 protein is nonfunctional. In contrast, the ZCCT-B2 protein has no mutations in the conserved amino acids of the CCT domain. The Q mutation found in ZCCT-B2 is associated with a positive BLOSUM62 score (+2), indicative of similar biochemical properties. In addition, CCT position 2 is variable among the CCT domains of ZCCT2 and CO-like proteins and is not conserved with the HAP2 proteins (Fig. 6). These observations suggest that this mutation may not have a negative impact on the structure or function of ZCCT-B2 and that this protein rather than ZCCT-B1 is the one conferring the strong vernalization requirement observed in the late-flowering lines from the BC3F2-521 progeny.
Functional VRN-S2 (Ae. speltoides) and VRN-D2 (Ae. tauschii) Alleles The lack of mutations in ZCCT1 and ZCCT2 in the functional VRN2 alleles from these two diploid species is consistent with the two-ZCCT hypothesis, but it does not provide new information about the relative importance of these genes for the establishment of the vernalization requirement. The absence of mutations in the CCT domain of the ZCCT-D1 and ZCCT-D2 genes in diploid Ae. tauschii (Table I) suggests that the D genome has the potential to contribute two functional ZCCT copies to common wheat. In summary, the hypothesis that both ZCCT1 and ZCCT2 genes can confer vernalization requirement explains well the different results on VRN2 allelic variation described in this and previous studies.
The R16C mutation in the ZCCT-A2 protein seems to be fixed in the A genome of tetraploid wheat, since it is present in all A and Am diploid species sequences determined so far. However, the R39C mutation in the ZCCT-A1 protein is still polymorphic among the wild and cultivated T. turgidum subsp. dicoccoides. Approximately half of the accessions of these two subspecies have the R39C mutation, whereas the others do not. The R39C mutation was present in all 37 T. turgidum subsp. durum varieties analyzed in this study (Table II), suggesting that this mutation was fixed during the domestication of the modern free-threshing tetraploid wheats.
Eight of the nine T. turgidum subsp. dicoccoides accessions and all of the T. turgidum subsp. dicoccon accessions that lack the R39C mutation in ZCCT-A1 carry a R35W mutation identical to the one detected in T. monococcum accession DV92 (Table II). Since there is strong evidence indicating that mutations at CCT position 35 result in nonfunctional proteins (Robson et al., 2001 The R39C mutation in the ZCCT-B1 gene was also polymorphic among the T. turgidum subsp. dicoccon and T. turgidum subsp. dicoccoides accessions but was fixed in all of the T. turgidum subsp. durum varieties analyzed here (Table II). On the contrary, none of the ZCCT-B2 proteins from these 37 accessions of cultivated durum wheat has mutations in the CCT domain. This result suggests that winter growth habit in cultivated tetraploid wheat is conferred mainly by the ZCCT-B2 gene(s) and that in some T. turgidum subsp. dicoccon and T. turgidum subsp. dicoccoides accessions both the ZCCT-B1 and ZCCT-B2 genes can delay flowering under long days.
The analysis of the progeny of BC3F2-521 showed that the effect of the functional VRN-B2 locus on heading time was partially dominant (degree of dominance = 0.26), which agrees with previous results reported in barley (Szücs et al., 2007
The duplication of the functional ZCCT-B2 locus provides a simple explanation for the higher transcript levels of ZCCT2 relative to ZCCT1 in tetraploid wheat (Fig. 7, A and B copies combined). The opposite result was observed before in T. monococcum, where ZCCT-Am1 transcripts were more abundant than those of ZCCT-Am2 (Yan et al., 2004b In addition to the internal duplication of the ZCCT-B2 gene in cultivated wheat, other deletion and duplication events affected the complete VRN-B2 locus. The deletion of all ZCCT genes from the B genome found in T. turgidum subsp. dicoccon accession PI470739 was instrumental in demonstrating the dosage effect of functional ZCCT genes in polyploid wheat. The RFLP screening also revealed the existence of a duplication of the complete VRN-B2 locus affecting both ZCCT-B1 and ZCCT-B2 genes (T. turgidum subsp. dicoccoides from Rosh Pinna). The copy number of ZCCT-B1 and ZCCT-B2 in these accessions is currently unknown, but the intensity of the hybridization signal suggests the presence of several copies (Fig. 2). We have initiated the crosses required to study the effect of this duplication on flowering time.
Accessions with a spring growth habit determined only by deletions or mutations in the VRN2 locus are frequent in cultivated barley (Dubcovsky et al., 2005 ; Szücs et al., 2007) and diploid wheat (Yan et al., 2004b). These VRN2 mutations are also found in combination with dominant VRN1 alleles. These results suggest that vrn2 mutations alone or in combination with some dominant VRN1 alleles might confer different responses to environmental cues from those conferred by those VRN1 mutations alone. The discovery that durum wheat varieties have nonfunctional vrn-A2 alleles and the development of a codominant marker tightly linked to the vrn-B2 deletion (PI470739) will facilitate the development of spring durum wheat varieties with no functional VRN2 loci. These nonfunctional VRN2 alleles can then be used alone or in combination with different dominant VRN1 alleles to develop spring durum wheat varieties with new allelic diversity in heading time. Allelic variation for VRN2 can be widened also in the opposite direction by adding more copies of functional ZCCT genes to cultivated durum wheat. This is expected to increase vernalization requirement and/or delay flowering, although its final effect will depend on other vernalization genes present in the genetic background. The ZCCT-A1 allele with no mutations in the CCT domain (T. turgidum subsp. dicoccoides accession 10-85) can be used to replace the nonfunctional ZCCT-A1 gene in cultivated durum wheat. In addition, the duplicated VRN-B2 allele present in the T. turgidum subsp. dicoccoides accessions from Rosh Pinna may be deployed in cultivated durum wheat.
Allelic variation in the ZCCT closest homolog in rice (Ghd7) has shown significant contributions of this locus to both the productivity and adaptability of cultivated rice on a global scale (Xue et al., 2008
Plant Materials and Growing Conditions
Triticum monococcum accession DV92 was the source of the nonfunctional vrn-Am2 allele (Yan et al., 2004b Seeds were imbibed for 24 h at 4°C to promote synchronized germination. Seedlings were transferred to pots and watered with nutrition solution. Unvernalized plants were grown in a greenhouse at room temperature (20°C–25°C) and long-day photoperiod (8 h of dark/16 h of light). For the vernalization experiments, plants were first grown for 3 weeks at the same conditions described above, transferred to a cold room at 4°C and a long-day photoperiod for 4 weeks, and then transferred back to the greenhouse to score heading date. Heading date was recorded at complete spike emergence. Methods used for sequencing BAC clone 738D05 (VRN-B2 locus), hybridization, PCR, and quantitative reverse transcription PCR, together with the markers for VRN-Am2, VRN-B2, VRN-A1, and PINA loci, are described in the Supplemental Data. Primers for all of the experiments are described in Supplemental Table S1.
The following crosses and selections were performed to introduce the nonfunctional vrn-Am2 allele (R35W) from T. monococcum accession DV92 and the null vrn-B2 allele from T. turgidum subsp. dicoccon PI470739 into tetraploid wheat. T. monococcum accession DV92 was crossed with cultivated tetraploid wheat Langdon (Fig. 3A), which carries dominant VRN-A1 and recessive vrn-B1 alleles (Fu et al., 2005 Plant BC1#2 was crossed with the tetraploid winter wheat Durelle to incorporate the recessive vrn-Am2 allele into a winter background (Fig. 3D). The BC2F1 plant from this cross was self-pollinated, and a population of 80 BC2F2 plants was generated and grown in a greenhouse without vernalization (Fig. 3E). This population showed a 3:1 (62:18) segregation between winter and spring growth habit, as expected for a population segregating only for VRN-A1. Winter BC2F2 lines (homozygous for recessive vrn-A1 and vrn-B1 alleles) were screened with the VRN-Am2 CAPS marker, and three lines homozygous for the recessive vrn-Am2 allele were selected (Fig. 3F).
The selected BC2F2 lines were crossed with T. turgidum subsp. dicoccon accession PI470739 (Fig. 3F), which is homozygous for a deletion encompassing both ZCCT-B1 and ZCCT-B2 genes (recessive vrn-B2 allele). Three BC3F1 plants were self-pollinated, and the resulting BC3F2 seeds were grown in a greenhouse without vernalization to select winter BC3F2 plants (Fig. 3G). The winter lines (homozygous vrn-A1 and vrn-B1) were then screened with the VRN-Am2 CAPS marker and with a codominant marker for SNF-B2 (Fig. 3H), a gene tightly linked to VRN2 (Yan et al., 2004b Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers FJ173816 to FJ173824 and FJ427399.
The following materials are available in the online version of this article.
We thank Dr. J. Dvorak and M.C. Luo for supplying seeds for several wheat accessions and the Southern blots used in Figure 2, the National Small Grains Collection for supplying germplasm, and X. Zhang, C. Miguita, and M. Lau for excellent technical assistance. Received September 4, 2008; accepted November 4, 2008; published November 12, 2008.
1 This work was supported by the National Research Initiative of the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service (grant no. 2007–35301–17737) and by a Vaadia-Binational Agricultural Research and Development Fund Postdoctoral Fellowship Award (grant no. FI–386–06 to A.D.).  The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) are: Assaf Distelfeld (adistel{at}ucdavis.edu) and Jorge Dubcovsky (jdubcovsky{at}ucdavis.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.129353 * Corresponding author; e-mail jdubcovsky{at}ucdavis.edu.
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ABSTRACT
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2 = 29.3, P < 0.0001) but not from a 1:3 ratio (one dominant gene; 
0.01, *** P 
