簇毛麦6VS特异转录序列P21461及P33259的获得及其分子标记在鉴定小麦-簇毛麦抗白粉病育种材料中的应用

簇毛麦6V#2S和6V#4S染色体臂分别携带抗白粉病基因Pm21和Pm V,在与小麦的杂种后代中,抗病基因与外源染色体臂共分离。开发鉴定2条外源染色体臂间多态性的序列,尤其是遗传信息相对缺乏的6V#4S染色体臂的序列,对于其在遗传与育种上的应用具有重要意义。本研究以携带6V#4S·6DL染色体的小麦易位系Pm97033及感病小麦亲本宛7107接种白粉菌的叶片转录组数据为资源,通过差异基因筛选、共线性分析、簇毛麦基因组扩增及测序验证的方法,鉴定出来自6V#4S的表达序列P21461和P33259,其中基于P21461序列设计的引物P461-5在簇毛麦6V#2S和6V#4S染色体臂的扩增产物具有30 bp的In Del和4 nt的多态性。用该引物转化的标记P461-5a可以鉴定抗白粉病小麦品种和高代品系所含的外源染色体,显示其在簇毛麦抗源鉴别和小麦抗病育种辅助选择中潜在的应用价值。根据P33259开发的标记P259-1可以对含有6V#4S染色体臂的材料进行特异扩增,但对6V#2S·6AL易位染色体没有扩增产物,因此P259-1可作为6V#4S·6DL易位染色体的特异分子标记。q RT-PCR分析结果显示,P21461的表达不受白粉菌诱导,而P33259在接菌后12 h和24 h的转录水平比接菌前提高约2倍,推测其可能参与Pm97033与白粉菌的早期互作。     

InDel分子标记WC656的开发及其在鉴别小麦-簇毛麦5VS纯合易位中的应用

簇毛麦是小麦品种改良重要的遗传资源,其5VS上含有硬度基因Dina/Dinb、抗白粉病基因Pm55和抗条锈病基因Yr5V,创制普通小麦-簇毛麦5VS易位系新种质,对于高效利用5VS上的有益基因进行小麦品种遗传改良具有重要意义。为了便于鉴别普通小麦-簇毛麦5VS纯合易位,本研究以mInDel软件分析普通小麦中国春基因组序列,开发了小麦第5同源群短臂的InDel特异分子标记WC656,对小麦品种以及普通小麦-簇毛麦5VS.5AL和5VS.5DL易位系及其衍生后代进行PCR扩增,根据扩增片段大小来区分5AS、5BS、5DS染色体臂以及5VS易位系。结果表明,在21个普通小麦品种、小麦-簇毛麦5VS回交F₂代中杂合易位单株,均扩增出379 bp（5AS）、471 bp（5BS）、422 bp（5DS）3条带。在T5VS.5AL高代品系、回交F₂代中纯合易位单株扩增出471 bp（5BS）、422 bp（5DS）2条带,379 bp（5AS）条带缺失。T5VS.5DL高代品系、回交F₂代中纯合易位单株扩增出379 bp（5AS）、471 bp（5BS）2条带,422 bp（5DS）条带缺失。WC656鉴别的5VS纯合易位结果与顺序FISH-GISH分析结果一致。T5VS.5AL、T5VS.5DL具有抗白粉病、籽粒硬度指数低等优良特性。因此,利用InDel分子标记WC656可以鉴别普通小麦-簇毛麦T5VS.5AL、T5VS.5DL衍生后代中的纯合易位,PCR扩增实验操作相对简便,可以为分子标记辅助选育携有5VS纯合易位的软质弱筋小麦提供帮助。     

一年生簇毛麦α-醇溶蛋白基因的分离、原核表达与功能鉴定

醇溶蛋白是面筋的主要成分之一,对小麦品质具有重要影响。根据数据库中全长α-醇溶蛋白基因设计了1对通用引物,从5份一年生簇毛麦(Dasypyrum villosum)品系中共得到52条序列,长度在816~873 bp之间(GenBank登录号为KJ004676~KJ004727)。核酸序列分析表明,其中有8条假基因,有1条(KJ004680)缺失终止密码子。推导氨基酸序列显示,KJ004677、KJ004686、KJ004714和KJ004696含有1个额外的Cys,其中,前3条序列由于Tyr→Cys所致,而KJ004696则由于Ser→Cys突变。序列间的差异主要出现在N-端重复区和多聚谷氨酰胺I区,根据N端重复区多肽序列的差异将一年生簇毛麦α-醇溶蛋白分为5种类型。为了分析具有额外Cys的α-醇溶蛋白所具有的品质效应,选取KJ004708(具有典型的6个Cys)和KJ004714(具有1个额外的Cys)分别构建表达载体,IPTG诱导后均得到分子量约30 kD的蛋白,与理论值相符;目的条带经切胶串联质谱鉴定证明,这2个α-醇溶蛋白基因在大肠杆菌中正确表达。对表达的蛋白亚基进行纯化、复性和低温冷冻干燥,经4 g粉质仪分析表明,KJ004708和KJ004714均能改善面团的加工品质,其中具有1个额外Cys的KJ004714亚基对面粉品质的改善更为显著。     

普通小麦–簇毛麦种质对菲利普孢囊线虫的抗性分析

菲利普孢囊线虫(Heterodera filipjevi)是在我国新发现的一种小麦病原线虫, 已严重威胁我国小麦的安全生产。小麦近缘种属具有改良小麦所需的许多目标性状, 是丰富小麦遗传变异、选育抗病品种的重要基因资源。采用室内接种鉴定法, 从20份小麦近缘属种材料中发现簇毛麦(Dasypyrum villosum)高抗H. filipjevi。分别对3套普通小麦–簇毛麦染色体附加系和6VS易位系进行接虫抗性鉴定, 结果6V染色体附加系高抗H. filipjevi;含6VS的易位系则表现感病。据此推测, 簇毛麦6V染色体上可能含有抗H. filipjevi基因, 且可能位于6VL上。     

Production and cytogenetics of intergeneric hybrids between Triticum durum-Dasypyrum villosum amphidiploid and Psathyrostachys huashanica

Summary Intergeneric crosses between Triticum durum-Dasypyrum villosum (2n=42, AABBVV), and Psathyrostachys huashanica (2n=14, N ^h N ^h ) were made, the seed set was 1.67%. Intergeneric hybrid were successfully obtained by means of embryo culture for first time. The average chromosome pairing in the hybrid (ABVN ^h ) was 26.61% univalents, and 0.69 bivalents. The chiasmata per cell was 0.69. The chiasmata was higher than that in Triticum durum dihaploid (AB), and lower than that in T. durum-Dasypyrum villosum trihaploid (ABV). The result indicated that the N ^h genome of Psathyrostachys huashanica has no homology with the V genome of Dasypyrum villosum, and the A and B genomes of Triticum durum. The coenocytism, micronuclei cell and variation in chromosome numbers were also observed. The F₁ hybrid was crossed with Triticum aestivum (AABBDD), and resulted in seed set. The hybrid of T. durum-D. villosum amphidiploid x P. huashanica showed partial fertility. It made the possibility for chromosome manipulation among Triticum aestivum, Dasypyrum villosum and Psathyrostachys huashanica.     

簇毛麦(Dasypyrumvillosum)矮秆突变体的鉴定

为给簇毛麦杂交后代群体中自然突变产生的矮秆突变体在小麦遗传改良中合理有效的利用提供理论依据,对该矮秆突变体的生物学及遗传学特性进行了初步研究。该突变体在苗期与对照植株无明显差异,在成熟期突变体株高30 cm,约为对照株高的1/3。突变株茎的倒一、二节间(最上部的两个茎节)长度分别为对照植株长度的1/9和1/6。细胞学观察表明,突变体的茎节细胞纵向延长受到抑制。此外,该突变株还表现出部分雄性不育,自然结实率低。花粉活力检测发现,突变株可育花粉只有20%,在外施GA3后花粉育性可得到恢复,推测该突变体为赤霉素敏感型矮秆突变体。遗传分析表明,矮秆突变性状受一个隐性基因控制。     

Production and meiotic pairing of intergeneric hybrids of Triticum × Dasypyrum species

Summary Crossing experiments between Dasypyrum villosum (2 x) and 14 taxa of Triticum sensu lato (seven diploids, five tetraploids and two hexoploids) were performed. Adult hybrids were obtained in all but three of the combinations with diploid species. A haploid plant was obtained from the combination T. aestivum x D. villosum. Cytogenetic data on the meiotic pairing in the intergeneric hybrids revealed that, in general, very little pairing occurred between the V genome of Dasypyrum and the different genomes in Triticum. There may be comparatively large differences in pairing behaviour in hybrids including different parental accessions, which shows that the Triticum as well as the Dasypyrum genotypes may influence the pairing. The combination T. aestivum x D. hordeaceum (4 x) was also produced and from the meiotic pairing in the hybrid it is evident that D. hordeaceum is an autoploid.     

Evidence for common genetic mechanisms controlling the tolerance of sudden salt stress in the tribe Triticeae

Previous studies in several Triticeae species have suggested that salt tolerance is a polygenic trait, but that genes on some chromosomes confer better tolerance to salt stress than others. This suggests an intriguing possibility that there may be a similar basis for salt tolerance in the species of the tribe Triticeae. In this study, chromosomal control of the tolerance to sudden salt stress, measured as the mean rate of leaf elongation in solution cultures with a single increment of 200 mM NaCl, was investigated in the genomes of cultivated barley (Hordeum vulgare L.), rye (Secale cereale L.), and Dasypyrum villosum (L.) Can-dargy by using disomic addition lines of individual pairs of chromosomes or chromosome arms of each of the three species in the ‘Chinese Spring’ wheat genetic background. It was observed that the chromosomes of homoeologous groups 3, 4, and 5 in barley, 5 and 7 in rye, and 4 and 6 in D. villosum carry loci with significant positive effects on salt tolerance. Increased doses of chromosomes of group 2, however, reduce or do not increase the tolerance to salt stress. These results are in agreement with a previous study of the tolerance of this salt stress regime in wheat and wheatgrass Lophopyrum elongatum. A ranking analysis of the chromosomal effects within each genome of the five Triticeae species investigated in this and previous studies revealed that the chromosomes of homoeologous groups 3 and 5 consistently confer large positive effects on the tolerance of sudden salt stress, while the chromosomes of homoeologous group 2 in increased dose have no or negative effects on the tolerance. This strongly suggests that species of the tribe Triticeae share some common genetic mechanisms of tolerance of sudden salt stress. The findings in this study give credence to the proposal that wild relatives can be exploited in the development of wheat cultivars with greater tolerance to salt stress.     

Effect of the ph1b mutant on chromosome pairing in hybrids between Dasypyrum villosum and Triticum aestivum

Chromosome pairing was analysed in F₁ hybrids of the wheat cultivar ‘Chinese Spring’ (CS) and its ph1b mutant (CSphlb) with Dasypyrum villosum. On average, 1.61 chromosomes per cell paired in the hybrid CS ×D. villosum, but 14.43 in the hybrid CS ph1b×D. villosum. Genomic fluorescence in situ hybridization (GISH) revealed three types of homoeologous association between wheat (W) and D. villosum (D) chromosomes (W‐D, D‐W‐W and D‐W‐D) in pollen mother cells of the CS ph1b×D. villosum hybrid, and only one type (W‐W), in the CS ×D. villosum hybrid. Both F₁ hybrids were self‐sterile. The seed set of the backcross of CS ×D. villosum with CS was 6.67% and that of CS ph1b×D. villosum with CS or CS ph1b was only 0.45%. The chromosome number of BC₁ plants varied from 48 to 72. Translocations of chromosome segments or entire arms between wheat and D. villosum chromosomes were detected by GISH in the BC₁ plants from the backcross of CS ph1b×D. villosum to CS ph1b.     

簇毛麦新型HMW-GS的序列分析及加工品质效应鉴定

利用设计的HMW-GS特异引物,从簇毛麦TA10220中克隆到6条HMW-GS基因序列(1.0~1.7 kb, GenBank登录号为KF887414~KF887419),比普通小麦HMW-GS基因序列小,其中KF887414~KF887417为能正常表达的基因,KF887418和KF887419是编码区出现终止信号的假基因。综合推导氨基酸的序列分析以及基于编码蛋白全序列、N-端和C-端域的进化树分析,认为KF887414属于y型HMW-GS,KF887415~KF887419的氨基酸序列同时具有x和y型的结构特征。将具有完整编码区的4个基因在宿主菌Escherichia coli Rosetta-gami B(DE3)中经IPTG诱导表达,通过SDS-PAGE分析表达的蛋白质和种子提取的HMW-GS,经Western blot进一步检测表达产物,证明蛋白质表达成功。通过His-Trap HP柱纯化表达蛋白,回收产物经SDS-PAGE分析后,低温冷冻干燥备用。将冷冻干燥的回收产物加入中国春基础面粉中,利用微量掺粉试验通过测定粉质参数来鉴定簇毛麦来源的HMW-GS对面粉品质的影响,结果表明,簇毛麦来源的4个HMW-GS对于面粉加工品质具有显著的正向效应。