长穗偃麦草4E染色体上的K／Na判别性状基因研究

在１５０ｍｍｏｌ／ＬＮａＣｌ胁迫下,长穗偃麦草（Ａｇｒｏｐｙｒｏｎｅｌｏｎｇａｔｕｍ,２ｎ＝７０）４Ｅ染色体代换普通小麦４Ｄ染色体后能显著降低胁迫下植株上部的Ｎａ离子含量而维持较高水平的Ｋ离子含量。     

长穗偃麦草基因组中与耐低磷营养胁迫有关的基因的染色体定位

以中国春－长穗偃麦草二体异附加系和二体异代换系为材料，对其耐低磷营养胁迫特性进行鉴定和遗传分析，结果表明（１）长穗偃麦草的４Ｅ一＾ 色体携有耐低营养胁迫的基因，且其效应远远超过背景亲本中国春。     

小麦×长穗偃麦草的受精和早期胚胎发育

用石蜡切片法,对小麦（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ）和长穗偃麦草（Ｅｌｙｔｒｉｇｉａｅｌｏｎｇａｔａ）杂交的受精和早期胚胎发育进行了观察。结果表明,长穗偃麦草花粉在小麦柱头上萌发良好,花粉管可顺利长入花柱和胚囊。观察的１７０个小麦子房中,１７６５％发生了双受精,产生了胚和胚乳;９４１％发生了单卵受精,只产生胚而无胚乳;４７１％发生了单极核受精,只产生胚乳而无胚;总受精率为３１７７％;成胚率为２７０６％。由于胚乳的缺乏或发育异常及败育,最终难以获得有生活力的种子。为小麦与长穗偃麦草远缘杂交提供了细胞胚胎学证据。     

长穗偃麦草Actin基因片段克隆及表达模式分析

根据已报道的其他植物肌动蛋白(Actin,ACT)基因的保守序列设计一对简并性引物,以长穗偃麦草根中总RNA为模板,采用RT-PCR方法克隆出ACT基因,命名为EeACT。结果表明,该基因片段长度598 bp,编码198个氨基酸,与其他植物ACT氨基酸序列同源性较高,达90%以上。低温、盐及干旱处理12 h,EeACT在其地上部与根中的表达水平没有显著差异,说明EeACT表达稳定。     

小麦x长穗偃麦草的受精和早期胚胎发育

用石蜡切片法,对小麦(Triticum aestivum)和长穗偃麦草(Elytrigia elongata)杂交的受精和早期胚胎发育进行了观察。结果表明,长穗偃麦草花粉在小麦柱头上萌发良好,花粉管可顺利长人花柱和胚囊。 观察的170个小麦子房中,17.65％发生了双受精,产生了胚和胚乳;9.41％发生了单卵受精,只产生胚而无胚乳;4.71％。发生了单极核受精,只产生胚乳而无胚;总受精率为31.77％;成胚率为27.06％。由于胚乳的缺乏或发育异常及败育,最终难以获得有生活力的种子。为小麦与长穗偃麦草远缘杂交提供了细胞胚胎学证据。     

硬粒小麦 长穗偃麦草2E和4E附加系及E染色体传递

为探讨长穗偃麦草E染色体在硬粒小麦背景中的传递特点,利用染色体特异分子标记、基因组原位杂交(GISH)、非变性荧光原位杂交(ND FISH)等方法,对小偃麦8801(AABBEE)与硬粒小麦(AABB)杂交后代中选育的株系Du_No.2和Du_No.4进行了分析。结果表明：(1)分子标记检测株系Du_No.2及Du_No.4分别能扩增出长穗偃麦草2E、4E染色体特异条带。(2)GISH和ND FISH分析显示,株系Du_No.2和Du_No.4分别附加了1条2E和4E染色体,表明株系Du_No.2 和Du_No.4分别为硬粒小麦 长穗偃麦草2E和4E单体附加系。(3)2个株系的减数分裂过程观察发现,后期Ⅰ、Ⅱ和末期Ⅱ都有E染色体分离异常现象,且株系Du_No.2和 Du_No.4的异常率分别为22.24%和36.18%。(4)2个株系分别与硬粒小麦进行正反杂交的后代PCR分析表明, 2E和4E染色体经雄配子的传递率分别为4.41%和2.17%,而通过雌配子的传递率都为零,表明2E和4E染色体在硬粒小麦背景中能通过雄配子传递,但不通过雌配子的传递。该研究为创建全套硬粒小麦 长穗偃麦草双体附加系及代换系提供基础。     

长穗偃麦草分子育种基础研究进展

长穗偃麦草(Elytrigiaelongata)为禾本科小麦族偃麦草属植物,是一种丛生的多年生冷季型牧草。长穗偃麦草原产于欧洲南部、小亚细亚和俄罗斯南部,在美国、加拿大和澳大利亚等国大面积种植。其引入我国后,1956年开始用作小麦(Triticumaestivum)远缘杂交的野生亲本,鲜有作为牧草大面积种植的报道。长穗偃麦草具有耐盐碱、耐涝和抗病等特点,可作为耐盐碱牧草用于建设“滨海草带”,利于避免草粮争地/争水,实现碳中和,保障我国粮食安全。全世界已育成推广了10余个长穗偃麦草品种,但我国尚无引种或自主选育品种,不利于“滨海草带”的建立。长穗偃麦草遗传背景复杂,基础研究薄弱,育种技术远远落后于稻麦等粮食作物。该文对长穗偃麦草分子育种基础,如育种历程、加速育种、资源创新、组织培养、基因组序列及分子标记进行综述,以期为长穗偃麦草品种选育和“滨海草带”建设提供参考。     

利用生化及分子标记确定长穗偃麦草（Elytrigia elongatum,EE.2n=14） …

利用限制性片段长度多态性（ＲＦＬＰ）及等电聚焦（ＩＥＦ）技术确定普通小麦中国春－二倍体长穗偃麦草７个异附加系所附加的外源染以体与小麦染色体的部分同源性,共有８个生化标记,１３个ＲＦＬＰ标记在亲本间揭示了多态性,结果表明：长穗偃麦草的１Ｅ,２Ｅ,３Ｅ,４Ｅ,５Ｅ,６Ｅ,７Ｅ７条染色体分别与小麦染色体的１、２、３、４、５、６、７７个部分同源群具有部分同源关系,偃麦草的１Ｅ与７Ｅ、５Ｅ与７Ｅ染色体间可能     

长穗偃麦草酸性磷酸酶与碱性磷酸酶编码基因的染色体定位

以一套中国春-长穗偃麦草二体异附加系与二体异代换系为材料,用等电聚焦(IEF)研究长穗偃麦草基因组中酸性磷酸酶(AcPh)与碱性磷酸酶(APH)编码基因的染色体定位。结果表明,AcPh大多聚焦于pH5～7范围内,其编码基因位于3E染色体,而APH编码基因则位于4E染色体。由于5E染色体的附加,AcPh活性带强度显著减弱。     

THINOPYRUM BESSARABICUM和THINO-PYRUM ELONGATUM的基因组关系研究

对 2个八倍体 C.S- Thinopyrum bessarabicum( AABBDDJJ,2 n=8x=56)和 Goshawk( GHK) - Thinopyrum elongatum( AABBDDEE,2 n=8x=56)的根尖细胞染色体进行 C-分带 ,从中分检出 Th.bessarabicum和 Th.elongatum的各自染色体进行核型分析 ,结果表明 :Th.bessarabicum和 Th.elongatum的大多数染色体都具有端带 ,但 Th.bessarabicum的端带更强 ,很少有中间带 ;而 Th.elongatum的染色体除 E1外其它染色体的端带较弱 ,而且带纹较丰富 ,有较多的中间带。对 C.S- Th.bessarabicum和 GHK- Th.elongatum进行有性杂交 ,其杂交种F1的 PMC染色体在 MI的平均配对构型为 1 5.50 5.0 3 [ 1 4.2 0 ○ 0 .40 0 .2 1 ,其中 Th.bessarabicum和 Th.elongatum染色体平均配对成 2 .5个二价体 ,由 C-分带显示大多数 Th.bessarabicum和 Th.elongatum染色体呈单价体状态。因此推断 ,Th.bessarabicum和 Th.elongatum两物种的染色体应属不同的染色体组     

基于高通量测序的长穗偃麦草功能分子标记发掘和分析

长穗偃麦草是小麦重要的近源物种,含有丰富的抗逆基因,广泛地应用于小麦的遗传改良育种。本研究利用高通量测序,获得长穗偃麦草的转录组测序信息,利用比较基因组学方法研究其与小麦、水稻和玉米等作物的遗传关系,评估它们之间的亲缘关系。同时,将长穗偃麦草的高通量序列比对到小麦基因,利用软件Freebayes和SAMtools/Bcftools发掘功能基因的变异位点,并对这些含有变异位点的功能基因进行注释分析,揭示长穗偃麦草优异性状形成的分子机制,这将为长穗偃麦草优异基因资源的开发和应用奠定重要基础。     

兼抗白粉、条锈病小偃麦渗入系CH7124抗性遗传及细胞学鉴定

CH7124是通过八倍体小偃麦TAI8335与感病小麦杂交、回交育成的兼抗白粉病、条锈病的小偃麦种质系。利用抗性接种鉴定、细胞学和基因组原位杂交(GISH)技术相结合的方法,对CH7124的抗性来源、遗传方式及细胞学特征进行了分析和鉴定。结果表明,CH7124在苗期和成株期对条锈菌系CYR29、CYR31、CYR32、CYR33和白粉菌系E09、E20、E21、E26表现为免疫或近免疫,其抗性来自中间偃麦草,受1对显性核基因控制;CH7124的根尖细胞染色体数目为2n=42,花粉母细胞减数分裂中期I(PMC MI)绝大多数细胞内可观察到21个二价体,平均配对构型为2n=0.30 I+20.79 II+0.04 III;与普通小麦中国春、绵阳11的杂种F1中,有80%以上的花粉母细胞可观察到2n=21Ⅱ的染色体构型,其平均配对构型均为2n=21II。说明CH7124具有与普通小麦相似的染色体结构和规则的配对构型。由于利用以中间偃麦草总DNA为标记探针的原位杂交未观察到可见的外源DNA杂交信号,进一步证明CH7124是一个小麦-中间偃麦草的隐形异源渗入系。     

小麦与高冰草不对称体细胞杂种F_5代部分株系的根尖细胞染色体分析

将普通小麦“济南177”(Triticum oestivum cv．Jinan 177)原生质体(受体)和经紫外线照射的高冰草Agropyron elongatum)原生质体(供体)用PEG(聚乙二醇,polyethylene glycol)法诱导融合,获得外形偏向小麦的不对称体细胞杂种及后代。经过田间繁育,现已到F5代。对来源于同一个体细胞杂种克隆的不同株系(Ⅱ-2,8-l,Ⅱ-I-8)的F5代的根尖细胞染色体进行核型分析,并与其亲本比较,结果表明,杂种株系染色体的形态、数目在遗传上均趋于稳定。各杂种株系的染色体与亲本小麦“济南177”之间有多处显著的不同,杂种株系之间也存在差异,推测高冰草的染色体小片段可能进入了受体小麦的染色体中。     

Molecular cytogenetic characterization of wheat--Thinopyrum elongatum addition, substitution and translocation lines with a novel source of resistance to wheat Fusarium Head Blight

Thinopyrum elongatum(2n = 2x = 14,EE),a wild relative of wheat,has been suggested as a potentially novel source of resistance to several major wheat diseases including Fusarium Head Blight(FHB).In this study,a series of wheat(cv.Chinese Spring,CS) substitution and ditelosomic lines,including Th.elongatum additions,were assessed for TypeⅡresistance to FHB.Results indicated that the lines containing chromosome 7E of Th.elongatum gave a high level of resistance to FHB,wherein the infection did not spread beyond the inoculated floret.Furthermore,it was determined that the novel resistance gene(s) of 7E was located on the short-arm(7ES) based on sharp difference in FHB resistance between the two 7E ditelosomic lines for each arm.On the other hand,Th.elongatum chromosomes 5E and 6E likely contain gene(s) for susceptibility to FHB because the disease spreads rapidly within the inoculated spikes of these lines. Genomic in situ hybridization(GISH) analysis revealed that the alien chromosomes in the addition and substitution lines were intact,and the lines did not contain discernible genomic aberrations.GISH and multicolor-GISH analyses were further performed on three translocation lines that also showed high levels of resistance to FHB.Lines TA3499 and TA3695 were shown to contain one pair of wheat-Th. elongatum translocated chromosomes involving fragments of 7D plus a segment of the 7E,while line TA3493 was found to contain one pair of wheat-Th.elongatum translocated chromosomes involving the D- and A-genome chromosomes of wheat.Thus,this study has established that the short-arm of chromosome 7E of Th.elongatum harbors gene(s) highly resistant to the spreading of FHB,and chromatin of 7E introgressed into wheat chromosomes largely retained the resistance,implicating the feasibility of using these lines as novel material for breeding FHB-resistant wheat cultivars.     

Isolation and Evolution Mode Analysis of NBS–LRR Resistance Gene Analogs from Hexaploid Wheat

Based on conserved regions of a sequence of a previously isolated powdery mildew (Pm) resistance gene, Pm3b, from hexaploid wheat (Triticum aestivum L.), a pair of primers was designed, and 11 resistance gene analogs (RGAs) were obtained using a polymerase chain reaction-based cloning approach. Three RGAs were deemed as pseudogenes, while the remaining eight corresponded to protein-encoding genes. At the nucleotide level, all these RGAs shared a sequence identity of 99% and showed 86% sequence identity with Pm3b. Phylogenetic analysis revealed that the eight protein-encoding genes were paraphyletic with Pm3 alleles. Positively selected sites were identified using the Selecton 2.1 program, and these were located on secondary structures. Based on these findings, the following two inferences could be made on the mode of evolution of the nucleotide-binding site and leucine-rich repeat (LRR) class of resistance genes. First, the majority of evolution events must have occurred primarily in the LRR domain, and this might have contributed to an increase in the proportion of evolution events in other domains. Second, these evolutionary events in the LRR domain must have occurred initially in secondary structures and then in the β-sheet. The crystalline structure models of RGAs were constructed. De-Shun Feng and Yan Li contributed equally to this paper. Sequence data of 2Q2, 2Q3, 2Q4, 2Q7, 2Q9, 2Q11, 2Q12, 12Q11, 15Q1, and 15Q7 from this article have been deposited at GenBank under accession numbers EF157980, EF157981, EF157982, EF157983, EF157984, EF157985, EF157986, EF157987, EF157988, EF157989, and EF157990.     

四倍体小麦背景中长穗偃麦草E染色体传递特征

通过细胞学方法和染色体特异分子标记鉴定六倍体小偃麦(AABBEE)与硬粒小麦(AABB)杂交的自交后代F₂和F₃植株,探讨长穗偃麦草染色体在硬粒小麦背景中世代间的传递特征,并筛选硬粒小麦-长穗偃麦草E染色体附加系。对218个F₂单株染色体数检测表明,2n=28植株占41.7%,2n=29植株占18.3%,其余40.0%植株的染色体数在2n=31~42范围内。分子标记鉴定表明,在F₂代2n=29单体附加植株中,不同的长穗偃麦草染色体传递率之间存在明显差异,1E传递率最高,3E和6E传递率最低。在F₂代2n=30单株中,1E、4E、7E和5E染色体相互组合产生的双单体多,6E参与组合较少,未检测到2E或3E与其他染色体的组合单株。在1E~7E单体附加株自交后代F₃中,E染色体传递率变化范围为9.1%~27.5%,1E传递率最高,6E传递率最低,与F₂的传递率一致。从F₃代中选育出1E~7E单体附加及少数二体附加,所有单体附加均可育。这些附加E染色体材料将对小麦代换系和易位系的创制提供有益的中间材料。     

Physical localization of the 18S-5.8S-26S rDNA and sequence analysis of ITS regions in Thinopyrum ponticum (Poaceae: Triticeae): implications for concerted evolution

Fluorescence in situ hybridization was used in Thinopyrum ponticum, a decaploid species, and its related diploid species, to investigate the distribution of the 18S-5.8S-26S rDNA. The distribution of rDNA was similar in all three diploid species (Th. bessarabicum, Th. elongatum and Pseudoroegneria stipifolia). Two pairs of loci were observed in each somatic cell at metaphase and interphase. One pair was located near the terminal end and the other in the interstitial regions of the short arms of one pair of chromosomes. However, all of the major loci in Th. ponticum were located on the terminal end of the short arms of chromosomes, and one chromosome had only one major locus. The maximum number of major loci detected on metaphase spreads was 20, which was the sum of that of its progenitors. The interstitial loci that exist in the possible diploid genome donor species were probably 'lost' during the evolutionary process of the decaploid species. A number of minor loci were also detected on whole regions of two pairs of homologous chromosomes. These results suggested that the position of rDNA loci in the Triticeae might be changeable rather than fixed. Positional changes of 18S-5.8S-26S rDNA loci between Th. ponticum and its candidate genome donors indicate that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donors. The possible evolutionary significance of the distribution of the rDNA is also discussed. Internal transcribed spacer (ITS) regions of nuclear DNA in Th. ponticum were investigated by PCR amplification and sequencing. The sequence data from five positive clones selected at random, together with restriction site analysis, indicated that the ITS repeated units are nearly homogeneous in this autoallodecapolypoid species. Combined with in situ hybridization results, the data led to the conclusion that the ITS region has experienced interlocus as well as intralocus concerted evolution. Phylogenetic analyses showed that the sequences from Th. ponticum have concerted to the E genome repeat type.     

The introgressed segment carrying rust resistance genes Yr17, Lr37 and Sr38 in wheat can be assayed by a cloned disease resistance gene-like sequence

A cloned gene sequence (Vrga1D), with features of the nucleotide-binding-site leucine-rich repeat class of disease resistance (R) gene sequence super family, was previously shown to belong to a family of five gene members derived from a Triticum ventricosum Ces. (syn. Aegilops ventricosa Tausch) segment in wheat (Triticum aestivum L.). This gene family was introgressed, together with the linked rust resistance genes Yr17, Lr37 and Sr38 from T. ventricosum, to wheat chromosome 2AS. An independently derived T. ventricosum segment carrying a leaf rust resistance gene in a French wheat cultivar, was shown to exhibit a rust resistance response equivalent to Lr37 as well as Yr17 and Sr38. DNA probes from different regions of the Vrga1D clone consistently detected the presence of RFLPs associated with the introgressed segment carrying the resistance genes Yr17, Lr37 and Sr38 present in diverse wheat genotypes from Australia, Canada, France and the UK. Our results showed that the transfer of the T. ventricosum- derived Vrga1 gene members and the rust resistance genes were always accompanied by the loss of a corresponding set of Vrga1-related gene members in recipient wheat cultivars presumed to be of homoeoallelic origin. A PCR assay, based on sequences from the 3"-untranslated region of a Vrga1 gene member isolated from the T. ventricosum donor line of the introgressed segment, was developed. The PCR assay detected the presence of the introgressed rust resistance genes across the diverse wheat backgrounds and should be useful in marker- assisted selection in wheat breeding. Received: 24 December 1999 / Accepted: 13 June 2000