Molecular mapping of vernalization requirement and fertility restoration genes in carrot

Carrot (Daucus carota L.) is a cool-season vegetable normally classified as a biennial species, requiring vernalization to induce flowering. Nevertheless, some cultivars adapted to warmer climates require less vernalization and can be classified as annual. Most modern carrot cultivars are hybrids which rely upon cytoplasmic male-sterility for commercial production. One major gene controlling floral initiation and several genes restoring male fertility have been reported but none have been mapped. The objective of the present work was to develop the first linkage map of carrot locating the genomic regions that control vernalization response and fertility restoration. Using an F₂ progeny, derived from the intercross between the annual cultivar ‘Criolla INTA’ and a petaloid male sterile biennial carrot evaluated over 2 years, both early flowering habit, which we name Vrn1, and restoration of petaloid cytoplasmic male sterility, which we name Rf1, were found to be dominant traits conditioned by single genes. On a map of 355 markers covering all 9 chromosomes with a total map length of 669 cM and an average marker-to-marker distance of 1.88 cM, Vrn1 mapped to chromosome 2 with flanking markers at 0.70 and 0.46 cM, and Rf1 mapped to chromosome 9 with flanking markers at 4.38 and 1.12 cM. These are the first two reproductive traits mapped in the carrot genome, and their map location and flanking markers provide valuable tools for studying traits important for carrot domestication and reproductive biology, as well as facilitating carrot breeding.     

Allelic variation and effects of 16 candidate genes on disease resistance in western Canadian spring wheat cultivars

Recently, we mapped genomic regions associated with resistance to wheat diseases and insensitivity to Pyrenophora tritici-repentis (Ptr) toxins using 81 historical and modern Canadian western spring wheat cultivars genotyped with genome-wide single nucleotide polymorphic (SNP) markers. Here, we investigate the frequency and effects of allelic variants of 50 markers associated with 16 candidate genes that regulate resistance to leaf rust (Puccinia triticina), yellow or stripe rust (P. striiformis f. sp. tritici), tan spot (P. tritici-repentis), and Ptr ToxA reaction in a subset of 70 of the 81 spring wheat cultivars. We evaluated the 70 cultivars in the field for all diseases except Ptr ToxA, which was evaluated in a greenhouse. Using Spearman rank correlation, stepwise discriminant analysis, and partial least squares regression, we identified between 4 and 11 markers as best predictors of each phenotypic trait. Overall, 23 of the 50 markers were associated with one or more of the phenotypic traits of which analysis of variance showed significant differences between allelic variants of 19 markers. In most analyses, markers for Lr34/Yr18 and Tsn1 loci were identified consistently as the best predictor of disease resistance and Ptr ToxA sensitivity, respectively. The same alleles from two Lr34/Yr18 diagnostic SNP markers (wMAS000003 and wMAS000004) not only decreased stripe rust scores up to 1.6 (on a 1 to 9 scale), but also increased grain yield up to 196 kg ha^?1 without affecting maturity. Results from this study could aid spring wheat breeders in selecting the best parental combinations and/or marker-assisted selection to integrate disease resistance with early maturity and short stature.     

The quantitative response of wheat vernalization to environmental variables indicates that vernalization is not a response to cold temperature

The initiation of flowering is a crucial trait that allows temperate plants to flower in the favourable conditions of spring. The timing of flowering initiation is governed by two main mechanisms: vernalization that defines a plant's requirement for a prolonged exposure to cold temperatures; and photoperiod sensitivity defining the need for long days to initiate floral transition. Genetic variability in both vernalization and photoperiod sensitivity largely explains the adaptability of cultivated crop plants such as bread wheat (Triticum aestivum L.) to a wide range of climatic conditions. The major genes controlling wheat vernalization (VRN1, VRN2, and VRN3) and photoperiod sensitivity (PPD1) have been identified, and knowledge of their interactions at the molecular level is growing. However, the quantitative effects of temperature and photoperiod on these genes remain poorly understood. Here it is shown that the distinction between the temperature effects on organ appearance rate and on vernalization sensu stricto is crucial for understanding the quantitative effects of the environmental signal on wheat flowering. By submitting near isogenic lines of wheat differing in their allelic composition at the VRN1 locus to various temperature and photoperiod treatments, it is shown that, at the whole-plant level, the vernalization process has a positive response to temperature with complex interactions with photoperiod. In addition, the phenotypic variation associated with the presence of different spring homoeoalleles of VRN1 is not induced by a residual vernalization requirement. The results demonstrate that a precise definition of vernalization is necessary to understand and model temperature and photoperiod effects on wheat flowering. It is suggested that this definition should be used as the basis for gene expression studies and assessment of functioning of the wheat flowering gene network, including an explicit account of the quantitative effect of environmental variables.     

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.     

Genotype-dependent proteolytic response of spring wheat to water deficiency.

Changes in proteolytic activities in response to water deficiency have been investigated in ten genotypes of spring wheat (Triticum aestivum L.) differing in response to water deficit stress and ability to acclimate. To determine subcellular localization and the type of proteases, mesophyll protoplasts isolated from wheat leaves were purified. Proteolytic activities were assayed using azocasein in the case of vacuolar proteinases at pH 5.0 and 125I-lysozyme in the case of extravacuolar ATP-dependent proteinases at pH 8.2. ATP-dependent proteolytic activity was found to be confined to the extravacuolar fraction while the azocaseinolytic activity to vacuoles. Dehydration increased vacuolar azocaseinolytic activity at both stages of plant development (shooting and heading), but the increase was significantly lower in more tolerant genotypes. The extravacuolar energy-dependent 125I-lysozyme degradation was low at the shooting stage but it was higher in the genotypes with a greater critical water saturation deficit. At the heading phase in the non-acclimated flag leaves ATP-dependent 125I-lysozyme degradation decreased in a genotype-dependent manner, but was enhanced upon acclimation to the same extent irrespective to the genotype ability to acquire dehydration tolerance during acclimation. The results presented indicate that both pathways of protein degradation are interlinked upon dehydration and are genotype dependent.     

The regulatory role of vernalization in the expression of low-temperature-induced genes in wheat and rye

Low temperature is one of the primary stresses limiting the growth and productivity of wheat (Triticum aestivum L.) and rye (Secale cereale L.). Winter cereals low-temperature-acclimate when exposed to temperatures colder than 10°C. However, they gradually lose their ability to tolerate below-freezing temperatures when they are maintained for long periods of time in the optimum range for low-temperature acclimation. The overwinter decline in low-temperature response has been attributed to an inability of cereals to maintain low-temperature-tolerance genes in an up-regulated state once vernalization saturation has been achieved. In the present study, the low-temperature-induced Wcs120 gene family was used to investigate the relationship between low-temperature gene expression and vernalization response at the molecular level in wheat and rye. The level and duration of gene expression determined the degree of low-temperature tolerance, and the vernalization genes were identified as the key factor responsible for the duration of expression of low-temperature-induced genes. Spring-habit cultivars that did not have a vernalization response were unable to maintain low-temperature-induced genes in an up-regulated condition when exposed to 4°C. Consequently, they were unable to achieve the same levels of low-temperature tolerance as winter-habit cultivars. A close association between the point of vernalization saturation and the start of a decline in the Wcs120 gene-family mRNA level and protein accumulation in plants maintained at 4°C indicated that vernalization genes have a regulatory influence over low-temperature gene expression in winter cereals.     

Molecular cloning and characterization of vernalization-related (ver) genes in winter wheat

The enriched cold-induced cDNA library of 1 × 10⁴ plaque-forming units (pfu. was prepared using cDNAs from cold-induced winter wheat, subtracted with mRNA of the control (non cold-induced). Results of the hybridization in situ of differential screening with probes of the control, the vernalized and devernalized wheat cDNAs showed that verc 17 ( verc : vernalization-relatcd cDNA clone) is specific for the veinalization. The insert of lambda of 10 recombinant was subcloned into the sites between Bam HI and Hind III in a pUC 19 plasmid. Analysis by northern blotting with a probe from verc 17 indicated that the verc 17 has negative signals for the control and the devernatized mRNA, but a positive signal for the mRNA of vernalized wheat at about 1.8 kb. The sequence had 20 restriction sites, covered by 17 enzymes. The ver 17 gene showed some homology with the P. yeolii major merozoite surface-antigen gene.     

Molecular characterization of vernalization loci VRN1 in wild and cultivated wheats

Background  

Variability of the VRN1 promoter region of the unique collection of spring polyploid and wild diploid wheat species together with diploid goatgrasses (donor of B and D genomes of polyploid wheats) were investigated. Accessions of wild diploid (T. boeoticum, T. urartu) and tetraploid (T. araraticum, T. timopheevii) species were studied for the first time.     

Genes responding to vernalization in hexaploid wheat

Genotype-specific gene expression in response to vernalization in common wheat was examined by the differential display method. Two near-isogenic lines of Vrn-A1 ( Vrn-A1 for the spring type and vrn-A1 for the winter type) were treated by vernalization of developing embryos in detached-ear cultures. This treatment was effective to promote vrn-A1 genotypes to head at a time equivalent to that of Vrn-A1. Differential cDNA fragments were isolated by the RT-PCR method from embryos subjected to vernalization treatments for 2- and 4-weeks at DAP10 and DAP20 stages, respectively. Among 110 differential cDNA fragments isolated, 48 were examined for their chromosomal locations and designated as wec ( wheat- embryo cold treatment) genes. Seven wec genes showed genotype-specific expression in response to vernalization. The statistical analysis utilizing two recombinant inbred lines showed that four wec genes were significantly associated with heading factors.     

Chromosomal location of genes for vernalization duration (Vrd) in winter bread wheat

In this study, we carried out monosomic analysis of two isogenic lines of cultivar Mironovskaya 808, monogenically dominant for genes Vrd1 and Vrd2 reducing the cultivar vernalization requirement duration from 50 to respectively 35 and 45 days, as well as analysis of substitution lines Cappelle Desperez/Bezostaya 1. Gene Vrdl is localized on chromosome 4A; Vrd2, on chromosome 5D. The third gene, which is not allelic to the former two, is present in cultivar Cappelle Desperez on either the IA, 6A, or 4B chromosome.     

Effect of wheat stem sawfly damage on yield and quality of selected Canadian spring wheat

The wheat stem sawfly, Cephus cinctus Norton (Hymenoptera: Cephidae), has reached outbreak status at most locations in the southern Canadian prairies. Solid-stemmed wheat, Triticum aestivum L., cultivars, which are less susceptible to damage, remain the primary management option. This article quantifies the effect of wheat stem sawfly damage on grain yield and quality at harvest and determines how cultivar selection affects harvest losses. Solid-stemmed cultivars were compared with hollow-stemmed cultivars and with blends of a 1:1 ratio of each. The hollow-stemmed cultivars with the exception of'McKenzie', which had intermediate levels of stem cutting, were all significantly more susceptible to stem cutting than solid-stemmed cultivars. Cultivar blends had lower damage but were still significantly higher than the solid-stemmed cultivars. The solid-stemmed 'AC Eatonia' and 'AC Abbey' had the lowest levels of stem cutting and ranked second and third overall for yield in 2001 and 2002. McKenzie ranked first, which reflects its yield potential in combination with its partial resistance to stem cutting. Lower cutting in AC Eatonia, AC Abbey, McKenzie, and the blend of AC Abbey/ McKenzie was significantly correlated with lower grain losses. Grain lost at harvest has major economic implications if sawfly pressure is moderate to high and susceptible cultivars predominate.     

Molecular characterization and functional analysis of elite genes in wheat and its related species

The tribe Triticeae includes major cereal crops (bread wheat, durum wheat, triticale, barley and rye), as well as abundant forage and lawn grasses. Wheat and its wild related species possess numerous favourable genes for yield improvement, grain quality enhancement, biotic and abiotic stress resistance, and constitute a giant gene pool for wheat improvement. In recent years, significant progress on molecular characterization and functional analysis of elite genes in wheat and its related species have been achieved. In this paper, we review the cloned functional genes correlated with grain quality, biotic and abiotic stress resistance, photosystem and nutrition utilization in wheat and its related species.     

Effects of vernalization duration control genes (Vrd) on agronomical traits in winter bread wheat

In monogenic dominant for Vrd1 or Vrd2 genes almost isogenic lines and cultivars the shortening of the period before hadding duration, plant height reduction, and decrease of frost resistance particularly in the second half of winter and in spring have been shown. As for the determined traits the greater effect was detected for Vrd1 gene, the less one--for Vrd2. Genes Vrd1 and Vrd2 did not significantly influence the quantitative characteristics of yield components except the 1000 kernel weight. The use of monogenic dominant for Vrd2 genotypes is recommended to develop new dwarf cultivars for South step of Ukraine.     

Positional relationships between photoperiod response QTL and photoreceptor and vernalization genes in barley

Winterhardiness has three primary components: photoperiod (day length) sensitivity, vernalization response, and low temperature tolerance. Photoperiod and vernalization regulate the vegetative to reproductive phase transition, and photoperiod regulates expression of key vernalization genes. Using two barley mapping populations, we mapped six individual photoperiod response QTL and determined their positional relationship to the phytochrome and cryptochrome photoreceptor gene families and the vernalization regulatory genes HvBM5A, ZCCT-H, and HvVRT-2. Of the six photoreceptors mapped in the current study (HvPhyA and HvPhyB to 4HS, HvPhyC to 5HL, HvCry1a and HvCry2 to 6HS, and HvCry1b to 2HL), only HvPhyC coincided with a photoperiod response QTL. We recently mapped the candidate genes for the 5HL VRN-H1 (HvBM5A) and 4HL VRN-H2 (ZCCT-H) loci, and in this study, we mapped HvVRT-2, the barley TaVRT-2 ortholog (a wheat flowering repressor regulated by vernalization and photoperiod) to 7HS. Each of these three vernalization genes is located in chromosome regions determining small photoperiod response QTL effects. HvBM5A and HvPhyC are closely linked on 5HL and therefore are currently both positional candidates for the same photoperiod effect. The coincidence of photoperiod-responsive vernalization genes with photoperiod QTL suggests vernalization genes should also be considered candidates for photoperiod effects.     

Molecular characterization of of alpha-gliadin genes from wild emmer wheat (Triticum dicoccoides).

According to the two distal and conserved regions of known alpha-gliadin genes, gene-specific primers for alpha-gliadin were designed, and the coding regions of four gliadin genes (i.e. GliTd-1, GliTd-2, GliTd-3 and GliTd-4) with the length of about 800 bp were isolated from the genomic DNA of wild emmer wheat (Triticum dicoccoides). No introns were observed. Sequence comparison indicated that these genes should be classified as alpha-gliadins. GliTd-3 (GenBank accession No.DQ140351) and GliTd-4 (DQ140352) were potentially functional, whereas GliTd-1 (DQ140349) and GliTd-2 (DQ140350) were both pseudogenes by the definition of in-frame stop codons and frameshifts. Six conserved cysteine residues were observed. Sequence analysis suggested that the motif units of repetitive domain for the four newly detected genes were different from the known genes, and the QQQP sequence before the position 60 was more toxic to coeliac patients. Codons for proline were strongly biased. Codons (CAG and CAA) for glutamine were clustered into the specific regions, and the high percentage of pseudogenes resulted from the mutation of CAG --> TAG.