中间偃麦草的GISH分析

以染色体组为E^eE^e的二倍体长穗偃麦草（Thinopyrum elongatum,2n=2x=14)、染色体组为E^bE^b的二倍体比萨偃麦草（Th.bessarabicum,2n=2x=14）、染色体组为StStStSt的四倍体拟鹅冠草（Pseudoroegneiria strigosa,2n=4x=28)的总基因组DNA为探针,对中间偃麦草（Th.intermedium)进行GISH分析。结果表明,中间偃麦草是由2个亲缘关系较近的染色体组、1个亲缘关系较远的染色体组构成;中间偃麦草所含的亲缘关系较近的染色体组分别与二倍长穗偃麦草染色体组E^e、比萨偃麦草染色体组E^b、以及1个亲缘关系较远的染色体组与拟鹅冠草染色体组St基本相似,但不完全一样,因此,中间偃麦草的染色体组用E^etE^etE^btStSt表示。     

小麦抗赤霉病利器——他山之石

赤霉病是我国乃至世界小麦(Triticumaestivum)产区的重要病害,给农业生产和人畜健康造成重大威胁。分离鉴定优质抗病基因、培育抗病品种,是控制我国麦区赤霉病的重要手段。最近,山东农业大学孔令让团队完成了二倍体长穗偃麦草(Thinopyrumelongatum)基因组的组装,并在此基础上通过精细定位和图位克隆分离得到来自长穗偃麦草的抗赤霉病基因Fhb7。他们发现Fhb7编码1个谷胱甘肽转移酶,对禾谷镰孢菌(Fusarium graminearum)分泌的包括呕吐毒素等在内的多种毒素具有解毒作用,是1个广谱持久抗病基因。他们还发现Fhb7很可能最初源于内生真菌,经过基因水平转移进入到偃麦草基因组中。此外,Fhb7不影响其它农艺性状,且其抗性不受小麦遗传背景影响。这一系列工作揭示了作物抗病演化中的全新机制,对小麦抗赤霉病育种以及更好地利用长穗偃麦草的丰富基因资源都具有重要意义。     

小麦抗赤霉病利器——他山之石

赤霉病是我国乃至世界小麦(Triticum aestivum)产区的重要病害, 给农业生产和人畜健康造成重大威胁。分离鉴定优质抗病基因、培育抗病品种, 是控制我国麦区赤霉病的重要手段。最近, 山东农业大学孔令让团队完成了二倍体长穗偃麦草(Thinopyrum elongatum)基因组的组装, 并在此基础上通过精细定位和图位克隆分离得到来自长穗偃麦草的抗赤霉病基因Fhb7。他们发现Fhb7编码1个谷胱甘肽转移酶, 对禾谷镰孢菌(Fusarium graminearum)分泌的包括呕吐毒素等在内的多种毒素具有解毒作用, 是1个广谱持久抗病基因。他们还发现Fhb7很可能最初源于内生真菌, 经过基因水平转移进入到偃麦草基因组中。此外, Fhb7不影响其它农艺性状, 且其抗性不受小麦遗传背景影响。这一系列工作揭示了作物抗病演化中的全新机制, 对小麦抗赤霉病育种以及更好地利用长穗偃麦草的丰富基因资源都具有重要意义。     

普通小麦与长穗偃麦草杂种及其衍生材料的细胞遗传学特性

对十倍体长穗偃麦草(Thinopyrum ponticum)与普通小麦杂交F1及其与普通小麦回交BC1F1的形态学和细胞学特性进行了分析。结果表明,长穗偃麦草与普通小麦‘兰考矮早八’衍生F1(‘兰考小偃麦’)的根尖细胞染色体数为56条;花粉母细胞减数分裂中期Ⅰ染色体构型平均值为19.81Ⅰ+15.78Ⅱ+0.75Ⅲ+0.59Ⅳ;基因组荧光原位杂交(GISH)显示,兰考小偃麦中含有35条完整的长穗偃麦草和21条小麦染色体。‘兰考小偃麦’/‘科育818’和‘兰考小偃麦’/‘Cp02-3-5-5’杂交F1的根尖细胞染色体数及其所遗传的长穗偃麦草染色体数分别为50~52和16~22条,且存在染色体易位;花粉母细胞减数分裂中期Ⅰ平均染色体构型为14.54Ⅰ+17.40Ⅱ+0.55Ⅲ+0.14Ⅳ,平均49.4%的细胞出现多价体(三价体或四价体)。这些材料为创造小麦-长穗偃麦草新种质奠定了基础。     

长穗偃麦草中E和E1基因组高分子量麦谷蛋白基因启动子部分序列的进化分析

通过PCR克隆的方法,获得了分别来自二倍体长穗偃麦草的E基因组和四倍体长穗偃麦草的E_1基因组的4个高分子量麦谷蛋白亚基(HMW-GS)基因启动子的部分序列。序列分析表明,它们之间的同源性较高,两个x型亚基启动子序列之间只有1个碱基的差异,而两个y型亚基启动子序列完全相同,x和y型亚基启动子序列之间的长度和部分碱基位点都有差异。推测四倍体长穗偃麦草中的E_1基因组可能起源于二倍体的E基因组。与来自小麦族的A、B、D和G基因组部分亚基基因的启动子序列比较表明,小麦族的这一区域在进化上是相当保守的,不同基因组来源的序列同源性都在90％以上。经过对这些序列的聚类分析,表明长穗偃麦草的y型HMW-GS基因与其他亚基基因的进化关系较远,而x型亚基基因与一个来自小麦1B染色体的亚基基因关系最近。     

普通小麦-中间偃麦草TAI-27中附加染色体的显微切割及特异性探针的筛选

试验以长穗偃麦草基因组DNA为探针 ,与普通小麦 中间偃麦草TAI 2 7进行染色体原位杂交 ,表明有 4条与长穗偃麦草同源的染色体 ;以P .stipifolia (St)基因组DNA为探针 ,有 4条与St同源的染色体 .这说明TAI 2 7中有 4条St染色体 .TAI 2 7是异代换 附加系 .对TAI 2 7中附加的中间偃麦草染色体进行显微切割 ,并建立其微克隆库 ,从中筛选获得了中间偃麦草的特异性探针 ,同源性分析表明该序列为一新序列 .这为进一步筛选抗病、抗逆和优质基因打下基础 .     

一个超矮秆核质杂种小麦的初步研究

在用普通小麦对长穗偃麦草(Etytrigia elongata=Agropyron elongatum,2n=70)的核代换回交过程中,在BC_9F_1发现了一个超矮秆核质杂种小麦,株高35厘米左右,定名为小偃矮。细胞学观察和与普通小麦正反杂交的遗传分析,证明:(1) 小偃矮是一个异源胞质单体附加系(21″W+1′Ag);(2) 长穗偃麦草细胞质和附加的外来染色体没有携带矮秆基因;(3) 矮秆遗传主要受细胞核内一对显性矮化基因控制。     

蓝粒小麦易位系的荧光原位杂交鉴定

普通小麦（Triticum aestivum L.)和长穗偃麦草（Agropyron elongatum (Host)Beauv=Elytriga elongatum(Host)Nevski=Thinopyrum ponticum (Host)Barkworth and Dewey,2n=10x=70)杂交后选育出的蓝粒小麦异代换系（蓝５８）,２n=42其中９９０６中被易位蓝粒片段的相对长度约占易位小麦染色体短臂的１／３,而９９０２中被易位蓝粒片段的相对长度约占易位小麦染色体长臂的１／２,并将９９０２的蓝粒易位片段定位在小麦Ｄ组染色体上;（２）９９１５易位附加和９９０４易位－易位附加,其体细胞染色体数均为４４,其中９９１５的体细胞染色体只有一对发生了易位,另外队了两条长穗偃麦草染色体;而９９０４有两对染色体发生了易位,并易位系中控制蓝粒性状的长穗偃麦草染色体片段的定位和蓝粒小麦易位系的应用进行了讨论。     

St基因组中的CRW同源序列在偃麦草中的FISH分析

为了确定十倍体长穗偃麦草(Thinopyrum ponticum, Liu & Wang)和六倍体中间偃麦草(Th. intermedium, [Host] Barkworth & Dewey )的基因组组成, 根据野生一粒小麦(Triticum boeoticum)着丝粒自主型反转录转座子(CRW)序列设计特异引物, 以二倍体拟鹅观草(Pseudoroegneria spicata, Á Löve )基因组 DNA为模板进行PCR扩增, 筛选到一条St基因组着丝粒区相对特异反转录转座子的部分序列pStC1, 长度为1.755 kb (GenBank登录号: FJ952565), 其中有800 bp与小麦着丝粒反转录转座子(CRW)的LTR区高度同源, 另有小部分片段与其外壳蛋白编码基因(gag)部分同源, 并且包含一段富含AGCAAC碱基的重复序列。以pStC1为探针, 对十倍体长穗偃麦草的FISH检测结果显示其基因组组成为两个St组3个E组(St₁St₂E^eE^bE^x); pStC1与中间偃麦草杂交时, 不仅St基因组上有强烈的荧光信号, 而且E基因组一些染色体的近着丝粒区域也有杂交信号, 说明偃麦草属异源多倍体物种在其形成及进化过程中St与E基因组之间在着丝粒及近着丝粒相关区域可能存在协同进化。     

抗条锈病小偃麦双体异附加系山农87074-519的鉴定

综合利用抗性接种鉴定、细胞学分析、SSR分子标记和基因组原位杂交(GISH)技术相结合的方法,对从长穗偃麦草与小麦复合杂交后代中选育的抗条锈病种质系山农87074-519进行了鉴定。结果表明,山农87074-519的根尖细胞染色体数目2n=44,花粉母细胞减数分裂中期I(PMCMI)绝大多数细胞内可观察到22个二价体,平均染色体构型2n=44=21.82Ⅱ 0.36Ⅰ,它与普通小麦中国春杂种F1的多数花粉母细胞内染色体构型为2n=21Ⅱ 1Ⅰ,因此它是1个附加了1对长穗偃麦草染色体的双体异附加系;以假鹅冠草St基因组总DNA作探针进行原位杂交发现山农87074-519的44条染色体中有2条出现黄绿色杂交信号,且杂交信号遍布整条染色体,证明其附加的长穗偃麦草染色体为St基组;利用SSR分子标记技术,在170对SSR引物中筛选出特异引物BARC165,它能稳定地在山农87074-519中扩增出长穗偃麦草特异标记BARC165268;将长穗偃麦草中BARC165的特异扩增片段克隆测序后制备成探针进行原位杂交,可在山农87074-519的间期染色体和有丝分裂中期染色体检测到杂交信号。山农87074-519综合农艺性状较好,对条锈病免疫,其抗性基因为显性,且位于附加的长穗偃麦草St基组染色体上,暂将其表示为YrSt。该种质系在小麦的遗传改良中具有重要利用价值。     

Identification of the parental chromosomes of the wheat-alien amphiploid Agrotana by genomic in situ hybridization.

Labelled total genomic DNA from four alien species, Thinopyrum ponticum (Host) Beauv. (2n = 70, genomes J1J1J1J2J2), Th. bessarabicum (Savul. &Rayss) Love (2n = 14, genome J), Th. elongatum (Host) Beauv. (2n = 14, genome E), and Haynaldia villosa (L.) Schur. (2n = 14, genome V), were used as probes in combination with blocking wheat DNA for in situ hybridization of the chromosomes of Agrotana, a wheat-alien hybrid (2n = 56) of unknown origin. The results showed that genomic DNA probes from Th. ponticum and Th. bessarabicum both clearly revealed 16 alien and 40 wheat chromosomes in Agrotana, indicating that the J genome present in these two species has a high degree of homology with the alien chromosomes in Agrotana. Biotinylated genomic DNA probe from Th. elongatum identified 10 chromosomes from Agrotana, while some regions of six other chromosomes yielded a weak or no signal. The probe from H. villosa produced no differential labelling of the chromosomes of Agrotana. The genomic formula of Agrotana was designated as AABBDDJJ. We suggest that the alien parent donor species of Agrotana is Th. ponticum rather than Th. bessarabicum. Genomic relationships of the three Thinopyrum species are discussed in relation to the distribution of GISH signals in the chromosomes of Agrotana.     

Identification of Blue-grained Wheat Translocation Lines Using Fluorescence in Situ Hybridization

The blue-grained wheat substitution line (blue 58) originated from wild hybridization between Triticum aestivum L. and Agropyron elongatum (Host) Beauv= Elytrigia elongatum (Host) Nevski= Thinopyrum ponticum (Host) Barkworth and Dewey (2n=10x=70) was irradiated and four translocation lines were screened by fluorescence in situ hybridization from the offsprings. The results obtained include the following: (1) both the two translocation lines, 9906 and 9902, have 42 chromosomes. The length of the translocated blue-grained segment was approximately one-third of the short-arm and one-half of the long-arm of the translocated wheat chromosome in 9906 and 9902, respectively, and the blue-grained translocated segment in 9902 was located on D genome; (2) both 9915 and 9904 have 44 chromosomes. One pair of chromosomes was translocated and two chromosomes from Th. ponticum were added in 9903, while two pairs of chromosomes were translocated in 9904 by blue-grained wheat segment. The location and application of blue-grained wheat translocation lines were discussed.     

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.     

Utilization of blue-grained character in wheat breeding derived from Thinopyrum poticum

The blue grain trait in common wheat （Triticum aestivum L., 2n = 6x = 42, AABBDD）, which is caused by blue pigments in the aleurone layer, was originally derived from the tall wheatgrass （Thinopyrum ponticum Liu ＆ Wang = Agropyron elongatum, 2n = 10x = 70, StStStStEeEeEbEbEXEx） during chromosome engineering research. Over the last few decades, there have been continued interests in the genetic mechanism of this blue coloration and the practical utilization of the blue aleurone character as a phenotypic marker. This article reviews the research history and the recent progress of the studies on blue-grained wheat, with emphases on genetic and biochemical analysis and practical applications of blue-grained wheat.     

Characterization of derivatives from wheat-Thinopyrum wide crosses

Partial amphiploids are lines that contain 42 (38-42) wheat and 14 (14-18) alien chromosomes. They are derived by backcrossing wheat onto hybrids between wheat and either Thinopyrum intermedium (6x) or Th. ponticum (10x). GISH analysis has shown that, with possibly one exception, the alien genomes (chromosome sets) in partial amphiploids are found to be hybrids i.e. composed of chromosomes from more than one alien genome. The individual partial amphiploids are meiotically stable and nearly perfectly fertile, but hybrids between different lines were characterized by varying numbers of unpaired chromosomes and consequently variable degrees of sterility. Translocated chromosomes involving different Thinopyrum genomes or Thinopyrum and wheat genomes were found in partial amphiploids and consequently in the addition lines derived from them. Partial amphiploids have proven to be an excellent tertiary gene pool for wheat improvement, containing resistance to biotic stresses not present in wheat itself. Resistance to Barley Yellow Dwarf Virus (BYDV) and Wheat Streak Mosaic Virus (WSMV) have been found in partial amphiploids and addition lines derived from both Th. intermedium and Th. ponticum. Excellent resistance to Fusarium head blight has been found on a Th. intermedium chromosome that had substituted for chromosome 2D in wheat. Genes for resistance to leaf rust and stem rust have already been incorporated into wheat and tagged with molecular markers.     

蓝粒小麦花青素生物合成途径中的二氢黄酮醇4-还原酶基因的分子克隆

蓝色色素在蓝粒小麦种子糊粉层中的生物合成途径的分子生物学机制至今仍不清楚。应用RT—PCR和RACE方法从蓝粒小麦正在发育的种子中克隆到一个编码二氢黄酮醇4-还原酶的基因(DFR)。推测其为花青素生物合成途径中的一个关键基因,且与蓝粒小麦中蓝色色素形成密切相关;其开放阅读框编码一个包含354个氨基酸残基的多肽,与一些从其他植物中已克隆到的DFR有很高的同源性：大麦(94％)、水稻(83％)、玉米(84％)。从长穗偃麦草(2n=70)、蓝粒小麦、浅蓝粒小麦自交产生的白粒后代小麦以及中国春的基因组中分别分离到一个全长DFR序列。经聚类分析表明DFR cDNA核甘酸序列与从中国春基因组中克隆的DFR具有100％的同源性,且与长穗偃麦草、蓝粒小麦、白粒小麦基因组中分离的DFR均有很高的同源性。4个DFR基因组DNA均含有3个内含子,且它们之间的差异主要在内含子区,表明该基因在进化上很保守。经Southern杂交分析,DFR小麦中至少有3-5个拷贝,不同小麦材料间未见明显差异,但与长穗偃麦草有明显差异,属于一个DFR超基因家族。Northern分析表明该DFR在蓝粒和白粒种子的不同发育时期的表达存在明显差异,都在开花后大约18d表达最强,在同一时期的蓝白种子中,DFR蓝粒种子中的表达量高于白粒。DFR转录本在小麦和长穗偃麦草的幼叶中积累多,但在芽鞘中的表达显著低于幼叶中;在小麦的根和长穗偃麦草的发育种子中均未检测到DFR的表达。推测蓝粒小麦中可能存在调控DFR在蓝粒小麦中表达的调控基因,类似于玉米花青素合成途径中的调节基因。     

Physical mapping of the blue-grained gene(s) from Thinopyrum ponticum by GISH and FISH in a set of translocation lines with different seed colors in wheat.

The original blue-grained wheat, Blue 58, was a substitution line derived from hybridization between common wheat (Triticum aestivum L., 2n=6x=42, ABD) and tall wheatgrass (Thinopyrum ponticum Liu & Wang=Agropyron elongatum, 2n=10x=70, StStEeEbEx), in which one pair of 4D chromosomes was replaced by a pair of alien 4Ag chromosomes (unknown group 4 chromosome from A. ponticum). Blue aleurone might be a useful cytological marker in chromosome engineering and wheat breeding. Cytogenetic analysis showed that blue aleurone was controlled by chromosome 4Ag. GISH analysis proved that the 4Ag was a recombination chromosome; its centromeric and pericentromeric regions were from an E-genome chromosome, but the distal regions of its two arms were from an St-genome chromosome. On its short arm, there was a major pAs1 hybridization band, which was very close to the centromere. GISH and FISH analysis in a set of translocation lines with different seed colors revealed that the gene(s) controlling the blue pigment was located on the long arm of 4Ag. It was physically mapped to the 0.71-0.80 regions (distance measured from the centromere of 4Ag). The blue color is a consequence of dosage of this small chromosome region derived from the St genome. We speculate that the blue-grained gene(s) could activate the anthocyanin biosynthetic pathway of wheat.     

Molecular characterization of a wheat--Thinopyrum ponticum partial amphiploid and its derivatives for resistance to leaf rust.

Leaf rust (caused by Puccinia triticina Eriks.) occurs annually in most wheat-growing areas of the world. Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang has provided several leaf rust resistance genes to protect wheat from this fungal disease. Three chromosome substitution lines, Ji806, Ji807, and Ji859, and two chromosome addition lines, Ji791 and Ji924, with a winter growing habit were developed from crosses between wheat (Triticum aestivum L. em Thell.) and the wheat - Th. ponticum partial amphiploid line 693. These lines were resistant to leaf rust isolates from China. Sequence-tagged site (STS) analysis with the J09-STS marker, which is linked to the gene Lr24, revealed that the partial amphiploid line 693 and all of the substitution and addition lines carried gene Lr24. Genomic in situ hybridization (GISH) analysis was carried out on chromosome preparations using total genomic DNA from Pseudoroegneria strigosa (M. Bieb) A. L?ve (St genome, 2n = 14) as a probe in the presence of total genomic DNA from T. aestivum 'Chinese Spring' wheat (ABD genomes, 2n = 42). The GISH analysis demonstrated that these lines had a pair of chromosomes displaying the typical pattern of a Js genome chromosome. This indicates that the chromosome that carries gene Lr24 belonged to the Js genome of Th. ponticum. In addition to 40 wheat chromosomes, eight Js and eight J genome chromosomes were also differentiated by GISH in the partial amphiploid line 693. Since most sources of Lr24 have a red grain color, the white-colored seeds in all of these substitution and addition lines, together with high protein content in some of the lines, make them very useful as a donor source for winter wheat breeding programs.     

Thinopyrum ponticum and Th. intermedium:the promising source of resistance to fungal and viral diseases of wheat

Thinopyrum ponticum and Th. intermedium provide superior resistance against various diseases in wheat （Ttricum aestivum）. Because of their readily crossing with wheat, many genes for disease resistance have been introduced from the wheatgrasses into wheat. Genes for resistance to leaf rust, stem rust, powdery mildew, Barley yellow dwarf virus, Wheat streak mosaic virus, and its vector, the wheat curl mite, have been transferred into wheat by producing chromosome translocations. These genes offer an opportunity to improve resistance of wheat to the diseases; some of them have been extensively used in protecting wheat from damage of the diseases. Moreover, new resistance to diseases is continuously detected in the progenies of wheat-Thinopyrum derivatives. The present article summaries characterization and application of the genes for fungal and viral disease-resistance derived from Th. ponticum and Th. intermedium.