基于寡核苷酸探针套painting的小麦“中国春”非整倍体高清核型及应用

基于寡核苷酸探针套painting的染色体鉴定技术简单、经济和高效,可以促进小麦品种及亲缘物种染色体识别和变异体鉴定,提高染色体工程效率。我们前期开发了寡核苷酸探针套,包含p As1-1、p As1-3、AFA-4、(GAA)10和p Sc119.2-1共5个探针。本研究通过一次荧光原位杂交(FISH),对源于17个非整倍体的18份材料分析发现,其中14个染色体组成正确,可以清晰识别相应的缺体、四体和端体。还构建了基于寡核苷酸探针套涂染的、能准确识别3个基因组和7个部分同源群染色体的高清核型,发现4个非整倍体发生变异,其中从N5BT5D中鉴定出一个可能的小片段相互易位系T6AS·6AL-6DL和T6DS·6DL-6AL。进一步对7个地方品种、10个栽培品种(系)和1个人工合成小麦分析,发现15条染色体存在多态性,涉及6条B组(除4B)、5条A组(除1A和3A)和4条D组(1D、2D、4D和7D)染色体,可以清晰识别我国小麦生产上广泛应用的3种易位类型(T1RS·1BL、T6VS·6AL及相互易位T1RS·7DL和T7DS·1BL),省去了基因组原位杂交(GISH)程序。另外,对5个亲缘物种分析发现,该探针套可以识别栽培一粒小麦、硬粒小麦Langdon、荆州黑麦、长穗偃麦草(2n=2x=14)全部和中间偃麦草30条染色体,并构建了这5个物种的核型。本研究结果证实该寡核苷酸探针套可以有效用于小麦及亲缘物种染色体鉴定,高清晰的中国春非整倍体核型为小麦染色体工程提供了参考标准。     

基于改进的多色荧光原位杂交技术的染色体分析

为了提高多色荧光原位杂交(M-FISH)技术的鉴定效率,简化M-FISH操作程序,探索和建立新的M-FISH实验及观察体系,本研究以‘荆辉1号’（普通小麦辉县红-荆州黑麦双二倍体）、‘南农9918’（普通小麦-簇毛麦整臂易位系T6VS.6AL）、‘扬麦22’（普通小麦-簇毛麦整臂易位系T6VS.6DL）以及‘ST5V#4S-1’（普通小麦-簇毛麦小片段易位系）等为试验材料,以2个寡核苷酸探针[(GAA)₁₀、pAs1-1]和物种基因组DNA探针作混合探针进行M-FISH分析。结果表明在一次原位杂交中同时使用3种探针,既可以识别出小麦背景中所有的外源染色体或染色体片段,又可以根据(GAA)₁₀和pAs1-1在染色体上的杂交信号分布精确鉴定出小麦和外源染色体（片段）的具体身份。这种改进的M-FISH技术可有效用于小麦及其近缘植物染色体的鉴定,并有助于快速构建物种基于FISH的分子核型。     

顶芒山羊草特异寡核苷酸探针开发和oligo-FISH核型构建

栽培小麦近缘物种顶芒山羊草(Aegilops comosa, 2n=2x=14, MM)是小麦改良的三级基因库。为准确鉴定顶芒山羊草M基因组染色体或染色体区段,本研究利用二代测序获得顶芒山羊草M基因组序列信息,从中鉴定出16条可能的特异卫星重复序列。根据这些序列设计12个寡核苷酸(oligo)探针进行oligo-FISH,结果表明,其中10个探针可在顶芒山羊草染色体上产生明显的杂交信号。对探针特异性分析发现, 5个探针仅在顶芒山羊草染色体上产生杂交信号,在小麦染色体上未观察明显杂交信号,可作为顶芒山羊草特异探针鉴定小麦背景中的顶芒山羊草染色体。选择在顶芒山羊草染色体上信号分布丰富的3个探针(oligo-pAc89、oligo-pAc148、oligo-pAc225)组成探针套ONPS#AC1,结合利用本实验室根据小麦D亚基因组开发的寡核苷酸探针库,构建了顶芒山羊草的oligo-FISH核型。本研究构建的FISH核型可以准确识别顶芒山羊草各条染色体,为挖掘、转移和利用顶芒山羊草优异基因提供了快速准确的鉴定手段。     

普通小麦贵紫麦1号染色体的FISH核型分析

采用FISH(荧光原位杂交)技术,分析普通小麦贵紫麦1号染色体的FISH核型特点,为贵紫麦1号在小麦新品种选育上的应用提供参考。结果表明,贵紫麦1号包括21对染色体,pAs 1红色探针信号主要分布在A组和D组染色体上,pSc 119.2-1绿色探针信号则主要分布在B组染色体上;根据贵紫麦1号染色体上这2种探针的分布特征,可以准确辨识每条染色体。贵紫麦1号与硬粒小麦(AABB)在2A、5A、2B、3B及5B染色体上表现出pSc 119.2-1信号分布差异,与节节麦(DD)在染色体1D、4D及5D上也存在信号分布差异,与中国春(AABBDD)在2A、5A、6B、1D及7D染色体上的信号也各有不同,说明DNA重复序列在不同小麦材料之间具有多态性。     

野大麦的分子核型分析

以野大麦(Hordeum brevisubulatum L.)为材料,采用荧光原位杂交技术,以5 SrDNA、45 SrDNA及重复序列pAs 1和pSc 119.2为探针,与野大麦染色体标本进行原位杂交,通过观察探针在染色体上标记的位置、标记信号的强弱,对野大麦染色体组进行核型分析。结果表明,5 SrDNA位于第10号和第14号染色体的短臂上;45 SrDNA位于第6号、第10号、第11号、第12号、第13号、第14号染色体的短臂上;pAs 1 DNA的荧光信号大部分出现在染色体的端部,部分出现在着丝粒区域;pSc 119.2的荧光信号出现在第7号和第14号染色体短臂的顶端,第10号染色体一条短臂的顶端,另一条染色体可能顶端缺失导致没有出现pSc 119.2荧光信号。核型公式为2 n=4 x=28=28 m(12 SAT),核型类型属于1 A型,核型不对称系数为57.227%。     

利用六倍体小黑麦×普通小麦培育伴随易位的非整倍体

用核型和GiemsaC—带技术对六倍体小黑麦×普通小麦的杂种后代（F4）83C3796和83C3804混系今的非整倍体进行了分析。在74个随机抽样的细胞中，染色体数从35条到43条都有分布，比率分别是4．05％、5．40％、2．7O％、9．46％、9．46％、12．16％、12．16％、41．89％和2．70％．发生小麦染色体丢失的植株频率超过55．39％，2n－13植株出现的比率最低（占2．70％）．并从一般核型中观察到染色体游离片段和染色体“断裂一融合”过程．C一带分析观察到高频率的小麦一黑麦染色体易位，都属罗伯逊易位．易位包含了小麦的A、B、D组染色体和黑麦所有14条染色体臂，其中IRS易位频率最高（占48．3％），且全为纯合易位，其它易位染色体纯合的程度各自不同．研究中还观察到在一个细胞中有多个小麦一黑麦染色体易位共存的现象．文中还讨论了易位非整倍体在小麦育种上的利用问题。     

棉属D染色体组的核型变异和进化

本文报道了棉属 D 染色体组11个种的染色体数目和核型,并采用了核型对称系数、异质系数和重合率等新的计算方法进行了分析。根据核型同质程度,可将11个种分为6个亚组,即1—D_1、D_8和 D_6;2—D_(3-k)、D_(3-d);3—D_(2-1)、D_(2-2);4—D_4和 D_7;5—D_5;6—D_9。其中,D_6是 D 组的最原始种。根据核型比较分析,结合地理分布、     

棉花Oligo-FISH技术建立及其初步应用

【目的】荧光原位杂交技术可以实现DNA序列直观准确的染色体定位,是基因组深入研究的重要技术之一。染色体特异探针的获得是该技术应用的关键。本研究旨在建立棉花寡核苷酸荧光原位杂交技术。【方法】利用已经公布棉花基因组序列数据,采用生物信息学方法获得染色体特异的寡核苷酸库,随后用乳化聚合酶链式反应方法标记成荧光探针,在棉花有丝分裂中期染色体上进行原位杂交。【结果】建立了一套棉花寡核苷酸荧光原位杂交技术体系。【结论】该体系可用于棉花单染色体识别鉴定。     

二倍体野燕麦染色体的C-带分析

采用C-带技术对二倍体野燕麦根尖细胞染色体进行了带型分析,以研究该野燕麦染色体的C-带特点.结果表明:二倍体野燕麦具有7对染色体,其上共有35条带,其中长臂具有带纹19条,包括13条中间带(I),6条末端带(T);短臂具有8条带纹,包括2条中间带(I),6条末端带(T),另外还有l条随体带(S)以及7条着丝点带(C),其带型公式为2 n=14=2CIT+ +2 CIT+8 CI+ T+2 CI+ T+S.二倍体野燕麦染色体组成为CC,核型为2A,属于较对称核型,进化指数为7.     

应用SSR分子标记分析小麦品种(系)的遗传重组

为了解小麦品种形成中亲本基因组的遗传重组和遗传保留区段的分布特点,对周麦18和百农AK58及其衍生品系共23个材料进行了全基因组SSR扫描分析。遗传重组分析表明,单交组合的平均重组数(12.3)低于回交组合(13.9);染色体4A、5A、7A、1B、3B、4B、7B、1D、2D、3D、5D、6D和7D重组发生较多,其余染色体重组相对较少,染色体的中间区段与远端区段重组数相当,分别为6.1和6.0。子代间基因组比较发现,一些染色体区段成为重组的多发区,如5D的gwm358–wmc357、6D的cfd49–barc196、7A的wmc158–barc23和7B的gwm274–gwm146区段,分别有35、19、15和14次重组。分析亲本传递给子代的染色体区段,发现子代继承亲本的遗传区段在14~29个,每个区段涉及2~8个多态性位点,大的遗传区段主要分布于4A、5A、5B、5D和7D染色体。以上基因组区域的关联性状是进一步研究的重点。     

A new secondary reciprocal translocation discovered in Chinese wheat

A new wheat-rye secondary reciprocal translocation involving T1RS·7DL and T7DS·1BL was detected by chromosome C-banding and genomic in situ hybridization (GISH). The meiotic configuration analysis combined with C-banding and GISH on F₁ hybrids of this newly discovered translocation with T1RS·1BL and Chinese Spring Dt7DS indicated that the new translocation probably resulted from a secondary reciprocal translocation between the primary translocation T1RS·1BL and 7D in the progenies of Aifeng3//Mengxian201/Neuzucht. On the basis of the cytological analysis of progenies and recombinant inbred lines (RILs) (derived from a cross between T1RS·7DL, T7DS·1BL and T1RS·1BL), the translocation chromosomes T1RS·7DL and T7DS·1BL transmitted readily, and appeared in most of the progenies.     

十个八倍体小偃麦的细胞学鉴定和染色体构成分析

培育新的八倍体小偃麦,对于利用偃麦草遗传物质进行小麦的遗传改良具有重要意义。本研究利用细胞学和基因组原位杂交技术,对从中间偃麦草与小麦品种烟农15杂交后代选育出的10个八倍体小偃麦山农TE256、山农TE259、山农TE261、山农TE262、山农TE263、山农TE265、山农TE266、山农TE267-1、山农TE270和山农TE274进行了细胞学鉴定和染色体构成分析。结果表明,10个八倍体小偃麦绝大多数单株根尖细胞的染色体数目为2n=56,个别单株含有54或55条染色体;大多数2n=56单株的花粉母细胞在减数分裂中期I的染色体构型为2n=28II,少数花粉母细胞存在单价体、三价体或四价体,后期I染色体可均等分向两极,仅有极少数细胞出现染色单体提前分离等现象;10个八倍体小偃麦均含有普通小麦的全套染色体和中间偃麦草的1个混合染色体基组,其中间偃麦草染色体是由来自中间偃麦草3个不同染色体基组的染色体构成的混合染色体基组,其染色体构成分别为2St+8J~S+2J+2J-St、2St+8J~S+4J、2St+8J~S+2J+2J-St、2St+8J~S+2J+2J-St、2St+8J~S+2J+2J-St、6St+4J~S+2J+2J-St、4St+6J~S+2J+2J-St、2St+8J~S+4J、2St+8J~S+4J和4St+6J~S+4J,与目前已报道的八倍体小偃麦均有所不同。研究结果可为这些新型八倍体小偃麦的研究和有效利用提供参考依据。     

Effects of substituted D genome chromosomes on photosynthetic rate of durum wheat (Triticum turgidum L. var. durum)

Summary A durum wheat cultivar Langdon (LDN) and fourteen disomic D genome chromosome substitution lines of Langdon, where A or B genome chromosomes were replaced with homoeologous D genome chromosomes of Chinese Spring (CS), were used to assess the compensatory effect of the D genome chromosomes on photosynthetic rates at tetraploid level. The LDN 1D(1B) and LDN 3D(3B) lines showed significantly higher photosynthetic rates than Langdon, whereas LDN 1D(1A) and LDN 3D(3A) lines were not greatly different from Langdon. It appears that chromosomes 1B and 3B decrease photosynthesis. This suggests the differentiation of the effects on the photosynthesis within the first and third homoeologous groups. Substitution with the 2D chromosomes did not compensate the effects of either 2A or 2B chromosomes as it reduced photosynthetic rate compared to plant with either chromosomes 2A or 2B. Tetra CS had a higher photosynthetic rate than CS and Penta CS. The photosynthetic rate of CS was similar to that of Penta CS, which lacked one set of D genome. The results suggest that it may be possible to increase photosynthesis, if both sets of the D genome were entirely removed from hexaploid wheat. However, it is difficult to conclude that the lower rate of photosynthesis of the hexaploids was mainly attributable to D genome chromosome effects, because we did not find a dose dependent effect of D genome. Homoeologous differentiation of chromosomes may be involved in photosynthesis.     

普通小麦中国春-百萨偃麦草异染色体系的分子标记分析

综合利用HMW-Glu亚基、STS、SSR和RFLP等分子标记对普通小麦中国春、百萨偃麦草、中国春-百萨偃麦草双二倍体和11个中国春-百萨偃麦草异染色体系进行了分析。结果表明，14对SSR、10对STS引物和6个RFLP标记可以特异追踪百萨偃麦草染色质。C7-17及其后代株系C7-17-2等编码百萨偃麦草特异HMW-Glu亚基，添加染色体涉及与小麦第１部分同源群染色体部分同源的1J；1对STS、3对SSR和1个RFLP探针可以特异追踪二体附加系CH05中的百萨偃麦草染色体，并揭示最初根据分带核型确定的J3与小麦第2部分同源群染色体具有较高的部分同源性；2对STS、1个RFLP探针和1对SSR可以追踪CH09的外源染色体，并揭示最初确定的J7与小麦第3部分同源群染色体具有较高的部分同源性；1对STS和1个RFLP探针在CH03、CH04和CH34中具有相同的多态，3个附加系可能添加了相同染色体，最初确定的J₁、J₂和J_?与小麦第7部分同源群染色体具有较高的部分同源性；3对SSR引物可以特异追踪CH12中附加的大片段易位染色体和CH11中的小片段易位染色体，推测易位可能涉及同一条百萨偃麦草染色体。发现13个标记（5个STS、3个RFLP探针和5个SSR）可以追踪未涉及到的4J和5J等染色体。     

A Robertsonian translocation from Thinopyrum bessarabicum into bread wheat confers high iron and zinc contents

Development of wheat–alien translocation lines has facilitated practical utilization of alien species in wheat improvement. The production of a compensating Triticum aestivum–Thinopyrum bessarabicum whole‐arm Robertsonian translocation (RobT) involving chromosomes 6D of wheat and 6E^b of Th. bessarabicum (2n = 2x = 14, E^bE^b) through the mechanism of centric breakage–fusion is reported here. An F₂ population was derived from plants double‐monosomic for chromosome 6D and 6E^b from crosses between a DS6E^b(6D) substitution line and bread wheat cultivar ‘Roushan’ (2n = 6x = 42, AABBDD) as female parent. Eighty F₂ genotypes (L1–L80) were screened for chromosome composition. Three PCR‐based Landmark Unique Gene (PLUG) markers specific to chromosomes 6D and 6E^b were used for screening the F₂ plants. One plant with a T6E^bS.6DL centric fusion (RobT) was identified. A homozygous translocation line with full fertility was recovered among F₃ families and verified with genomic in situ hybridization (GISH). Grain micronutrient analysis showed that the DS6E^b(6D) substitution line and T6E^bS.6DL stock have higher Fe and Zn contents than the recipient wheat cultivar ‘Roushan’.     

Responses to salt stress controlled by the homoeologous group 5 chromosomes of hexaploid wheat

The responses to salt stress in NFT (nutrient film) hydroponics of ‘Chinese Spring’ wheat and a number of its aneuploids involving the chromosomes of homoeologous group 5 were studied. This showed that the absence of chromosome 5D allowed plants to survive better than in the euploid condition. Much of this response could be related to the effects of Vrn3, which conditions the spring habit of ‘Chinese Spring’. The ability to survive relatively high levels of stress was promoted by the group 5 homoeologue from Thinopyrum bessarabicum.     

普通小麦中国春–簇毛麦易位系T1DL•1VS和T1DS•1VL的农艺和品质特性

为明确普通小麦中国春–簇毛麦整臂互补易位系T1DS?1VL和T1DL?1VS对农艺特性和加工品质的效应, 于2008-2010年在陕西杨凌连续2个生长季对这2个易位系和CS的主要农艺性状和加工品质性状进行了研究, 并采用SDS-PAGE法对簇毛麦高分子量麦谷蛋白亚基(HMW-GS)基因进行了进一步的染色体臂定位。结果表明, 这2个易位系的抽穗期和成熟期相同, 但比CS晚1~2 d, T1DS?1VL其他农艺性状与中国春相似, T1DL?1VS的春季单株分蘖、千粒重和单株粒重显著高于中国春;2个易位系的籽粒蛋白质含量与中国春无显著差异, T1DS?1VL的Zeleny沉淀值、面团形成时间、稳定时间和粉质仪质量指数显著降低;相反, T1DL?1VS的这些性状值显著提高。说明T1DS?1VL易位系对小麦的面团强度有显著的负向效应, 而T1DL?1VS易位系显著增强面筋强度。SDS-PAGE分析结果表明, 簇毛麦的1VS和1VL上均含有簇毛麦的HMW-GS基因Glu-V1, 但该基因在T1DL?1VS中的表达可能强于在T1DS?1VL中。     

Genetic segregation and the detection of spontaneous wheat-alien translocations

Robertsonian translocation between homoeologous chromosomes is one means for introducing alien genes from related species into common wheat. This study was undertaken to determine if selection for normal segregation of an alien trait could be used to identify favourable translocations. Agrotana (an octoploid agrotriticum, probably derived from Thinopyrum ponticum) carries resistance to the wheat curl mite (Eriophyes tulipae Keifer) which is the vector of wheat streak mosaic virus. Agrotana was crossed with the winter durum Michurinka (Triticum turgidum L. em. Bowden), and resistant F₂ plants were backcrossed four times with Norstar winter wheat (T. aestivum L. em. Thell.). During the later stages of backcrossing and selfing, resistant progenies were tested for their segregation ratios; selection was made for high and stable rates of transmission of mite resistance in segregating male gametes and zygotes. The 49 descendant gametes were tested for Mendelian-like inheritance of resistance. Two lines were identified with segregations that approached Mendelian ratios (1:1 male transmission in a testcross, 3:1 in F₂ and 1:2:1 among F₃ families). Contrary to expectations, selection for stable F₂ ratios (3:1) proved more efficient than selection for normal male transmission (1:1) as a means of identifying these stocks. Chromosome painting and PCR (polymerase chain reaction) analysis indicated the recovery of two independent Robertsonian translocations, both of which probably involved the reunion of the short arm of a group 6 chromosome of Thinopyrum ponticum and the long arm of chromosome 6D. In conclusion, selection for Mendelian ratios from among backcross derivatives of an interspecific hybrid was successful in isolating desirable translocations; cytogenetic methods were only used to characterise desirable stocks once these were identified. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inheritance of traits associated with seed size in groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

A new wheat-Thinopyrum substitution line AS1677, developed from a cross between wheat line ML-13 and wheat-Thinopyrum intermedium ssp. trichophorum partial amphiploid TE-3, was characterized by fluorescence in situ hybridization (FISH), sequential Giemsa-C banding, genomic in situ hybridization (GISH), seed storage protein electrophoresis, molecular marker analysis and disease resistance screening. Sequential Giemsa-C banding and GISH using Pseudoroegneria spicata genomic DNA as probe indicated that a pair of St-chromosomes with strong terminal bands were introduced into AS1677. FISH using pTa71 as a probe gave strong hybridization signals at the nuclear organization region and in the distal region of the short arms of the St chromosome. Moreover, FISH using the repetitive sequence pAs1 revealed that a pair of wheat 1D chromosomes was absent in accession AS1677. Seed storage proteins separated by acid polyacrylamide gel electrophoresis (APAGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that AS1677 lacked the gliadin and glutenin bands encoded by Gli-D1 and Glu-D1, further confirming the absence of chromosome 1D. The introduced St chromosome pair belonging to homoeologous group 1 was identified by newly produced genome specific markers. AS1677 is a new 1St (1D) substitution line. When inoculated with stripe rust and powdery mildew isolates, AS1677 expressed stripe rust resistance possibly derived from its Thinopyrum parent. AS1677 can be used as a donor source for introducing novel disease resistance genes to wheat in breeding programs aided by molecular and cytogenetic markers.     

干旱胁迫下小麦脯氨酸积累相关基因的染色体定位

对于旱胁迫和对照条件下中国春-Hope代换系和中国春-长穗偃麦草代换系的叶片脯氨酸含量进行了测定，通过方差分析表明：普通小麦可能在4B、5A和5D染色体上有控制干旱胁迫下脯氨酸积累的基因存在，6B和6D染色体上可能有抑制脯氨酸积累的基因存在。在测定中国春-长穗偃麦草代换系的叶片脯氨酸含量后，确定小麦5A和5D染色体上有