Exploiting the diversity of <Emphasis Type="Italic">Viviparous-1</Emphasis> gene associated with pre-harvest sprouting tolerance in European wheat varieties

Pre-harvest sprouting (PHS) reduces the quality of wheat (Triticum aestivum L.) and the economic value of the grain. The objective of this study was to evaluate the diversity of the Viviparous-1B (Vp-1B) gene associated with PHS tolerance in a collection of 490 widely grown winter wheat varieties from central and northern Europe. Four alleles of Vp-1B were found in the wheat varieties tested, three of which (Vp-1Ba, Vp-1Bb and Vp-1Bc) had previously been identified in Chinese wheat varieties. The fourth was a new allele which had a 25-bp of deletion in the third intron region compared with the nucleotide sequence of Vp-1Ba, and was designated as Vp-1Bd. The frequencies of different alleles in this set of European wheat germplasm were: Vp-1Ba (54%) > Vp-1Bc (21%) > Vp-1Bd (20%) > Vp-1Ba + c (4%) > Vp-1Bb (1%), with Vp-1Bb being present only in two French varieties, ‘Altria’ and ‘Recital’. In addition, the frequencies of the alleles differed in varieties from different European countries. For example, Vp-1Ba had the highest frequency (76%) in varieties included in the UK National List (NL), but was least frequent in the Recommended List (RL) of Sweden (19%). Similarly, Vp-1Bc was present with the highest frequency (58%) in wheat varieties from Sweden, and the lowest in UK NL varieties (8%) while Vp-1Bd had the highest frequency of 32% in German varieties, and the lowest in Sweden varieties with only 8%. The Vp-1Ba allele was present in over half of the UK wheat varieties tested but the frequency was lower in RL varieties than in NL ones. Furthermore, heterogeneities were found between Vp-1Ba and Vp-1Bc in the varieties from Sweden, Netherlands, Germany and UK.     

Rich allelic variations of Viviparous-1A and their associations with seed dormancy/pre-harvest sprouting of common wheat

The allelic variations of Vp-1B have been confirmed to have close association with seed dormancy (SD) and pre-harvest sprouting (PHS) of Chinese wheat in previous research, but little was known regarding whether the alleles of two other orthologs of Vp1 on 3AL (Vp-1A) and 3DL (Vp-1D) are also present and related to these traits. In view of this, 11 primer pairs flanking the whole sequences of these two orthologs were designed to investigate their allelic variations. The results identified six alleles of Vp-1A using the primer pair A17-19 among 81 wheat cultivars and advanced lines, which were designated as Vp-1Aa, Vp-1Ab, Vp-1Ac, Vp-1Ad, Vp-1Ae, and Vp-1Af. Except for Vp-1Ac, the other five alleles were proven novel, but no allelic variation was found in Vp-1D. On sequence analysis of alleles of Vp-1A, five deletions were observed, all occurring in the same region holding many TTC repeats. Of the six alleles detected in this study, four (Vp-1Aa, Vp-1Ac, Vp-1Ae, and Vp-1Af) were generally distributed in varieties exhibiting higher average germination index (GI, range 0.46–0.56) and spike sprouting (SS, range 39.6–49.4%); however, the alleles Vp-1Ab and Vp-1Ad were distributed in genotypes carrying higher SD (GI 0.19–0.26) and stronger PHS resistance (SS 12.3–17.2%). On Spearman correlation analysis, the allele Vp-1Ab had significantly negative correlation with GI (−0.479) and SS (−0.542) at the 0.01 level, and the three alleles Vp-1Aa, Vp-1Ac, and Vp-1Ae had significantly positive correlation with GI [0.311 (0.05 level), 0.401 (0.01 level), and 0.294 (0.05 level)] and SS [0.283 (0.05 level), 0.309 (0.05 level), and 0.266 (0.05 level)]. The other alleles, including Vp-1Ad and Vp-1Af, also exhibited correlation, albeit not significant, with these two traits. This negative correlation showed that Vp-1Ab helped to improve SD and PHS tolerance, but Vp-1Aa, Vp-1Ac, and Vp-1Ae appeared to exert the opposite effect. To further confirm the association between alleles of Vp-1A and the two traits, a recombinant inbred line (RIL) population with 157 lines was genotyped using the primer pair A17-19, developed from the cross between Wanxianbaimaizi (Vp-1Ab) and Jing411 (Vp-1Ac). General linear model analysis indicated that variation in Vp-1A had a significant (P < 0.001) association with the two traits, explaining 23.4% of the variation in GI and 16.7% of the variation in SS in the population across three crop seasons.     

The use of <Emphasis Type="Italic">Vp1</Emphasis> in real time RT-PCR to select for pre-harvest sprouting tolerance in triticale

In spite of the availability of laboratory and field tests there is still a major problem to select pre-harvest sprouting (PHS) tolerant triticale varieties in a reliable, field-independent way. One approach to minimize the influence of environmental conditions and physio-morphological traits on PHS detection is using molecular genetic tools. The ‘viviparous’ Vp1 gene has been repeatedly described to play an important role in dormancy in wheat. A quantitative RT-PCR assay based on the expression of the Vp1 gene has been developed. Specific primers were designed for detecting Vp1 in both wheat and triticale. The expression levels of Vp1 were normalized using reference genes and relatively quantified with the comparative C_t-method. However, the first results indicate that the achieved Vp1 expression levels at 50 days post anthesis are not useful to select for PHS tolerance, both in wheat and triticale. This negative outcome so far is possibly due to the existence of several splicing events or to the late assaying moment in the kernel development, when Vp1 expression is found to be low.     

分子标记Tamyb10D和TaDFR-B的有效性验证及对小麦抗穗发芽基因型的筛选

为了比较与小麦穗发芽抗性相关分子标记的有效性以及在白粒小麦品种中筛选出抗穗发芽的基因型材料,选择已报道的2个与穗发芽抗性相关的标记Tamyb10D和Ta DFR-B,对2套白粒小麦试材(78,103份)的穗发芽抗性进行综合筛选。结果表明:标记Tamyb10D可有效地用于白粒小麦穗发芽抗性筛选,而标记Ta DFR-B不适用于白粒小麦品种(系)材料的穗发芽抗性筛选的鉴定。通过分子标记Tamyb10D的筛选结果和发芽指数的评价,结合以前用分子标记Vp1B3的检测结果,在103份试材中筛选出5份具有高抗穗发芽基因型材料(GI10%),分别是:阆中白麦子、万县白麦子、陪陵须须白麦、川362和小白玉花,其单倍型分别是:Tamyb10D/Vp-1Bb、Tamyb10D/Vp-1Bb、Tamyb10D/Vp-1Bb、Tamyb10D/Vp-1Bb、Tamyb10D/Vp-1Bc;在另外一套(78份)试材中筛选出10份具高抗有穗发芽基因型材料(GI10%),分别为西农6028、克群、百农3217、开封124、郑州742、西昌5762、小偃5号、克壮、许跃6号和武农99,其单倍型均为Tamyb10D/Vp-1Bc。     

Grain dormancy in fixed lines of white-grained wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) grown under controlled environmental conditions

Pre-harvest sprouting (PHS) in wheat (Triticum aestivum L.) can be a significant problem, causing deleterious effects on grain quality. However, the adverse impacts of PHS can be reduced by introgressing genes controlling grain dormancy into white-grained bread wheat. Screening for grain dormancy typically involves germination testing of harvest-ripe grain grown in a glasshouse or field. However, the more uniform environmental conditions provided by temperature controlled glasshouses (i.e. controlled environmental conditions—CEC) may provide significant benefits for the assessment of grain dormancy. In this study, the dormancy phenotype of grain grown under CEC incorporating an extended photoperiod, was compared with 2 years of data from field grown material. Four dormant double haploid lines (derived from SW95-50213 and AUS1408) and two locally adapted non-dormant cultivars EGA Gregory and EGA Wills were compared in three replicated experiments grown under CEC (22 ± 3°C and 24 h photoperiod). The germination response of harvest-ripe grain was examined to assess the expression of grain dormancy. Two measures of germination, the predicted time to 50% germination (G ₅₀) and a weighted germination index, both clearly differentiated dormant and non-dormant lines grown under CEC. In addition, levels of grain dormancy were similar to field-grown plants. These results demonstrated that CEC with an extended photoperiod can be used for rapid and reliable characterisation of grain dormancy in fixed lines of bread wheat.     

Characterization of DFR allelic variations and their associations with pre-harvest sprouting resistance in a set of red-grained Chinese wheat germplasm

Pre-harvest sprouting (PHS) of wheat greatly reduces the quality and economic value of grain, and PHS resistance is one of the most important traits in wheat breeding. Red-grained wheat varieties are generally more resistant to PHS than white-grained ones; however, some are still susceptible. The red pigment of red-grained wheat is synthesized through the flavonoid biosynthetic pathway, in which the dihydroflavonol-4-reductase gene (DFR) is one of the genes involved in anthocyanin synthesis. In this study, a set of 120 red-grained Chinese wheat cultivars and lines with distinct PHS resistance were used to characterize TaDFR genotype variations and their association with PHS resistance. Whereas no variation or functional variation of TaDFR genes was detected on chromosomes 3A and 3D, a novel TaDFR allele, designated TaDFR-Bb, was explored on chromosome 3B. Compared with TaDFR-Ba, an 8 bp insertion (CTCTAGGA) was identified in the promoter region of TaDFR-B in most of the PHS resistant red-grained wheat varieties and advanced lines. Based on this, a CAPS marker was designed and validated with a set of Chinese red-grained wheat cultivars and lines with distinct PHS resistance. In most cases, TaDFR-Bb was associated with higher PHS resistance. An association study indicated that wheat varieties with the 8 bp insertion (average seed germination index 23.6 %) were significantly more resistant (P < 0.01) to PHS than those without the insertion (average seed germination index 69.5 %). Further study on gene expression demonstrated that the insertion led to increased TaDFR-B expression in cultivars with PHS resistance. Transient expression of TaDFR-B in coleoptiles of wheat cv. Chinese Spring revealed that increasing TaDFR gene expression did not induce the synthesis of anthocyanins.     

Conferring resistance to pre-harvest sprouting in durum wheat by a QTL identified in <Emphasis Type="Italic">Triticum spelta</Emphasis>

Pre-harvest sprouting (PHS) causes significant yield loss and degrade the end-use quality of wheat, especially in regions with prolonged wet weather during the harvesting season. Unfortunately, the gene pool of Triticum durum (tetraploid durum wheat) has narrow genetic base for PHS resistance. Therefore, finding out new genetic resources from other wheat species to develop PHS resistance in durum wheat is of importance. A major PHS resistance QTL, Qphs.sicau-3B.1, was mapped on chromosome 3BL in a recombinant inbred line population derived from ‘CSCR6’ (Triticum spelta), a PHS resistant hexaploid wheat and ‘Lang’, a PHS susceptible Australian hexaploid wheat cultivar. This QTL, Qphs.sicau-3B.1, is positioned between DArT marker wPt-3107 and wPt-6785. Two SCAR markers (Ph3B.1 and Ph3B.2) were developed to track this major QTL and were used to assay a BC₂F₈ tetraploid population derived from a cross between the durum wheat ‘Bellaroi’ (PHS susceptible) and ‘CSCR6’ (PHS resistant). Phenotypic assay and marker-assisted selection revealed five stable tetraploid lines were highly PHS resistant. This study has successfully established that PHS-resistance QTL from hexaploid wheat could be efficiently introgressed into tetraploid durum wheat. This tetraploid wheat germplasm could be useful in developing PHS resistant durum cultivars with higher yield and good end-use quality.     

Increased grain dormancy in white-grained wheat by introgression of preharvest sprouting tolerance QTLs

White-grained wheat cultivars have long been recognized to be less resistant to preharvest sprouting (PHS) than the red-grained ones. Previously two QTLs for grain dormancy, QPhs.ocs-3A.1 (QPhs-3AS) and QPhs.ocs-4A.1 (QPhs-4AL) were identified in a highly dormant Japanese red wheat, Zenkoujikomugi (Zen). Aiming at improvement of PHS tolerance in white-grained wheat, the introgression effect of these two QTLs in a white-grained population consisting of 40 recombinant inbred lines (RILs) developed from a cross between Zen and white-grained Spica was examined here. Random 20 RILs with red grains were also developed from the same cross and used as a control population. The RILs were grown in the field and in the glasshouse to evaluate the grain dormancy by germination test. Several SSR markers closely linked to the QPhs-3AS and QPhs-4AL were used to estimate the alleles at the QTLs. Dormancy variation in the RILs was significantly associated with the differences for grain color and the alleles at QPhs-3AS over several years. Although allelic variation was detected in a SSR marker closely linked to QPhs-4AL there was no difference in germination data between the Zen-allele and the Spica-allele groups. As expected, the red-grained RILs with the Zen allele at QPhs-3AS were the most dormant. Some white-grained RILs with the Zen allele at QPhs-3AS showed higher dormancy compared to the red-grained RILs with the alternative allele. These results demonstrated that introgression of the QPhs-3AS gene could contribute to the increased grain dormancy in white-grained wheat.     

A molecular approach to combat spatio-temporal variation in insecticidal gene (<Emphasis Type="Italic">Cry1Ac</Emphasis>) expression in cotton

Spatio-temporal expression of an insecticidal gene (Cry1Ac) in pre existing transgenic lines of transgenic cotton was studied. Seasonal decline in expression of Cry1Ac differed significantly among different cotton lines tested in the field conditions. The leaves of the Bt cotton plants were found to have the highest levels of toxin expression followed by squares, bolls, anthers and petals. Expression of the gene decreased consistently with the age of plants. Toxin expression in fruiting parts was not enough to confer full resistance against bollworms. The reduction in efficacy of transgenic cotton plants late in the season was attributed to reduction in promoter activity. For this purpose, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (rbcS) promoter was isolated from Gossypium arboreum that was further cloned upstream of an insecticidal gene (Cry1Ac) in expression vector pCAMBIA 1301. A local cotton cultivar NIAB-846 was transformed with Cry1Ac driven by rbcS promoter. The same cotton cultivar was also transformed with Cry1Ac gene driven by 35SCaMV promoter to compare the expression pattern of insecticidal gene under two different promoters. The results showed that rbcS is an efficient promoter to drive the expression of Cry1Ac gene consistent throughout the life of cotton plant as compared to 35S promoter. The use of tissue specific promoter is also useful for addressing the biosafety issues as the promoter activity is limited to green parts of plants, hence no gene expression in roots, cotton seed and other cotton products and by products.     

Gene expression analysis of four WIR1-like genes in floret tissues of European winter wheat after challenge with <Emphasis Type="Italic">G. zeae</Emphasis>

Fusarium head blight (FHB) is a highly destructive disease of wheat and other cereals which causes serious mycotoxin contaminations of grain. A number of molecular mapping studies led to the detection of QTL with small to moderate effects on FHB resistance in European winter wheat. Genes involved in the defence reaction of these genotypes remain largely unknown. WIR1 (wheat induced resistance 1) genes have been shown to be upregulated in cereals during attack of various fungal pathogens; however, their role in resistance is ambiguous. In this study, the expression of three WIR1 genes and a gene with high sequence similarity to WIR1 was investigated in European winter wheat genotypes after inoculation with Giberella zeae. Floret tissues of four winter wheat genotypes (Dream, Lynx, G16-92, Hussar) were challenged with G. zeae conidia or water (control) and sampled six times during 0–96 h after inoculation. Quantitative real-time PCR showed that all four genes were highly upregulated in the resistant genotypes compared to the susceptible ones. WIR1b and a gene with sequence similarity to WIR1 genes mapped to chromosome 5DS in the G16-92/Hussar mapping population. Two genes annotated as WIR1a mapped in the interval of a FHB resistance QTL on chromosome 7BS in the Dream/Lynx mapping population. These could be considered possible candidate genes for quantitative FHB resistance.     

Mapping QTLs for pre-harvest sprouting traits in the spring wheat cross ‘RL4452/AC Domain’

Pre-harvest sprouting (PHS) in spring wheat (Triticum aestivum L.) is a major downgrading factors for grain producers and can significantly reduce end-use quality. PHS resistance is a complex trait influenced by genotype, environment and plant morphological factors. A population of 185 doubled haploid (DH) lines from the spring wheat cross ‘RL4452/AC Domain’ were used as the mapping population to detect quantitative trait loci (QTLs) associated with three PHS traits, germination index (GI), sprouting index (SI) and falling number (FN). Six major QTLs linked with PHS traits were mapped on bread wheat chromosomes 3A, 3D, 4A (2 loci), 4B and 7D. ‘AC Domain’ alleles contributed to PHS resistance on 3A, 4A (locus-2) and 4B, and ‘RL4452’ alleles contributed resistance on 3D, 4A (locus-1) and 7D. QTLs detected on chromosome 4B controlling FN (QFN.crc-4B), GI (QGI.crc-4B) and SI (QSI.crc-4B) were coincident, and explained the largest amount of phenotypic variation in FN (22%), GI (67%) and SI (26%), respectively.     

Mapping genes controlling anthocyanin pigmentation on the glume and pericarp in tetraploid wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> L.)

A novel gene, designated Pg (purple glume), controlling anthocyanin pigmentation of the glume was identified and mapped in an F₂ population from the durum wheat (Triticum durum) cross TRI 15744/TRI 2719. This gene was close to one of the two complementary dominant genes, controlling anthocyanin pigmentation of the pericarp (gene Pp3) in the centromere region of chromosome 2A; the other Pp gene (Pp1) was mapped on the short arm of chromosome 7B, near gene Pc controlling anthocyanin pigmentation of the culm and co-segregating with Pls (purple leaf sheath) and Plb (purple leaf blade). On the basis of the mapping results, the Pp3, Pc, Pls and Plb genes of T. durum were regarded as allelic to the T. aestivum Pp3, Pc-B1, Pls-B1 and Plb-B1 loci. The likely allelism of Pp1 in T. durum and T. aestivum remains in dispute, the present durum Pp gene mapped to the short arm of chromosome 7B, whereas in common wheat it was reportedly located on the long arm.     

Genetic analysis of apical lethality in <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

Investigations were carried out to determine the nature and number of genes governing apical lethality (apical death) in a number of intervarietal crosses of wheat. Genetic analysis of data in segregating generations of the cross WR95/HW2041 and its reciprocal cross revealed that WR95 carries a recessive gene that leads to the death of certain individuals when combined with another recessive gene derived from HW2041. The phenomenon, which is denoted here as “apical lethality”, is controlled by two complementary recessive genes coming together from two different parents in certain F₂ individuals. The gene symbols apd ₁ in WR95 and apd ₂ in HW2041 are proposed for these genes of apical lethality, respectively. Uniculms observed in the F₂ generation are heterozygous (apd ₁ apd ₁ Apd ₂ apd ₂) and, therefore, the uniculmness trait does not breed true. Of the wheat genotypes tested, the gene apd ₂ was found to be present in CL983, CL1019, Lok-1, HW2041, HD2329, HW2011, WH147, HW2042, HW2047, WR196, WR544, WR798 and WR936, while the remaining genotypes, including some of the exotics such as Atila, carried both Apd ₁ and Apd ₂ in the homozygous condition.     

转反义TRX s基因对小麦萌发过程中储藏蛋白降解的影响

以转反义TRX s基因豫麦18和对照（豫麦18）为试材，测定了小麦种子萌发过程中胚乳内thiocalsin、苹果酸脱氢酶、谷丙转氨酶的活性以及游离氨基酸含量的变化。结果表明，反义TRX s基因的导入能够增加对thiocalsin和苹果酸脱氢酶的抑制，降低其活性，使储存蛋白更难于被降解，谷丙转氨酶增高的速度减慢，种子氨基酸代谢减弱。说明蛋白质代谢缓慢是转反义TRX s基因小麦抗穗发芽的一个主要原因。     

Identification of molecular markers linked to adult plant leaf rust resistance gene <Emphasis Type="Italic">Lr48</Emphasis> in wheat and detection of <Emphasis Type="Italic">Lr48</Emphasis> in the Thatcher near-isogenic line with gene <Emphasis Type="Italic">Lr25</Emphasis>

The recessive adult plant resistance (APR) gene Lr48 in wheat was tagged with flanking random amplified polymorphic DNA (RAPD) markers. Markers S336₇₇₅ in coupling and S3₄₅₀ in repulsion with Lr48 were identified in wheat line CSP44. Tests of these markers on available Thatcher near-isogenic lines (NILs) detected the likely presence of Lr48 in TcLr25. A test of allelism of APR involving the cross TcLr25 × CSP44 indicated that Lr48 was present in both lines. A separate experiment on inheritance of resistance in an F₂ population of TcLr25 × Agra Local confirmed the presence of a dominant seedling resistance gene (Lr25) and a recessive APR gene (Lr48) in TcLr25. This study demonstrated the value of molecular markers in identifying the presence of masked genes in genetic stocks where direct phenotyping failed to detect their presence.     

Analysis of hybrid lethality in F<Subscript>1</Subscript> wheat-rye hybrid embryos

The effect of the Embryo lethality mutant (Eml) of rye was studied in crosses between hexaploid wheat and corresponding inbred rye line (L2). Histological analysis of hybrid embryos revealed morphological differences 16 days after pollination. Eml was found to arrest the formation of shoot meristem but had no influence on root meristem formation. The effect of Eml cannot be overcome by in vitro embryo rescue via direct regeneration on Kruse medium. The possibility of complementary interactions between wheat and rye genes and of changes in gene expression through increased variation in dosage-regulated gene expression during hybrid formation is discussed.     

Vp1 expression profiles during kernel development in six genotypes of wheat, triticale and rye

During the last few decades, the physiological and genetic background of dormancy, and correlated pre-harvest sprouting (PHS) have been intensively investigated. Special attention has often been paid to genetic factors that may explain and predict PHS susceptible behaviour. A major candidate is the Vp1 gene which is involved in embryo development and maturation as well as in dormancy establishment. In this study, Vp1 gene expression during kernel development was studied in wheat, triticale and rye as a potential biomarker for selecting PHS tolerant varieties in cereal breeding programs. Plants of known PHS tolerant and PHS susceptible varieties were grown under controlled conditions from flowering until harvest ripeness. During that period, kernels were regularly harvested for RNA extraction and cDNA synthesis. Calibrated and normalized relative Vp1 expression levels were obtained in an RT-qPCR assay. During kernel development, Vp1 expression levels generally showed a typical peak during the soft dough stage, after which they decreased and remained low until harvest maturity. Differences in Vp1 expression levels could be observed between the PHS susceptible and PHS tolerant varieties of wheat, with the PHS tolerant variety showing higher levels of relative Vp1 expression compared to the PHS susceptible variety. In triticale, however, this difference was only seen once and could not be confirmed in further experiments. It seems that the Vp1 gene in triticale behaves in a similar way as in rye, in which no specific trends could be observed.     

Allelic variation for the rust resistance gene <Emphasis Type="Italic">Lr34</Emphasis>/<Emphasis Type="Italic">Yr18</Emphasis> in Canadian wheat cultivars

The wheat (Triticum aestivum L.) gene Lr34/Yr18 conditions resistance to leaf rust, stripe rust, and stem rust, along with other diseases such as powdery mildew. This makes it one of the most important genes in wheat. In Canada, Lr34 has provided effective leaf rust resistance since it was first incorporated into the cultivar Glenlea, registered in 1972. Recently, molecular markers were discovered that are either closely linked to this locus, or contained within the gene. Canadian wheat cultivars released from 1900 to 2007, breeding lines and related parental lines, were tested for sequence based markers caSNP12, caIND11, caIND10, caSNP4, microsatellite markers wms1220, cam11, csLVMS1, swm10, csLV34, and insertion site based polymorphism marker caISBP1. Thirty different molecular marker haplotypes were found among the 375 lines tested; 5 haplotypes had the resistance allele for Lr34, and 25 haplotypes had a susceptibility allele at this locus. The numbers of lines in each haplotype group varied from 1 to 140. The largest group was represented by the leaf rust susceptible cultivar “Thatcher” and many lines derived from “Thatcher”. The 5 haplotypes that had the resistance allele for Lr34 were identical for the markers tested within the coding region of the gene but differed in the linked markers wms1220, caISBP1, cam11, and csLV34. The presence of the resistance or susceptibility allele at the Lr34 locus was tracked through the ancestries of the Canadian wheat classes, revealing that the resistance allele was present in many cultivars released since the 1970s, but not generally in the older cultivars.