小麦-长穗偃麦草7E抗赤霉病易位系培育

【目的】将二倍体长穗偃麦草7E染色体导入主栽小麦背景,培育小麦-长穗偃麦草7E染色体抗赤霉病易位系,为小麦抗赤霉病遗传改良利用外源优良基因提供新种质。【方法】利用中国春-长穗偃麦草7E代换系DS7E(7B)与扬麦16杂交的F_2种子进行~(60)Co辐射(30 000 rad)和种植,表现型选择收获存活M_1植株的种子,从M_2连续通过表型农艺性状选择、单花滴注法进行赤霉病抗性鉴定和长穗偃麦草7E染色体或染色体臂特异分子标记PCR扩增筛选,最后在M_4代对中选材料以长穗偃麦草基因组DNA为探针进行基因组原位杂交(genomic in situ hybridization,GISH)证实。【结果】M_1选择了赤霉病发病率不同的13个单株进行繁殖。利用前期开发的长穗偃麦草7E染色体和7EL、7ES特异标记检测13株的后代M_2单株,获得含有长穗偃麦草7EL片段7株和7ES片段14株;对21株M_2衍生的222个M_3植株进行特异标记检测,共选择含有长穗偃麦草7EL片段13株和7ES片段3株;利用来自12株M_3的后代(M_4)进行GISH,共9株M_3的后代具有小麦-长穗偃麦草易位染色体,体细胞染色体2n=42。2株M_3的后代显示附加2条长穗偃麦草染色体短臂,体细胞染色体2n=44。连续多年多途径的筛选,获得4份材料,3份材料均为长穗偃麦草7E染色体长臂易位系,命名为TW-7EL1、TW-7EL2和TW-7EL3。1份为7E染色体短臂附加系,命名为W-DA7ES,最后所获得的材料是源自M_1代2个单株。连续3年赤霉病抗性鉴定结果表明长穗偃麦草7EL易位系抗性高,发病率明显低于中国春和扬麦16,与苏麦3号相当,而7ES附加系的赤霉病抗性明显较低,发病率明显高于7EL易位系。【结论】通过赤霉病抗性鉴定、染色体特异分子标记筛选和GISH证实相结合培育了小麦-长穗偃麦草7EL抗赤霉病易位系,长穗偃麦草易位片段鉴定快速和准确。二倍体长穗偃麦草7E染色体长臂中含有抗小麦赤霉病的基因。     

普通小麦-十倍体长穗偃麦草衍生新品种抗赤霉病基因的分子鉴别

【目的】赤霉病(Fusarium head blight,FHB)是世界范围内严重危害小麦生产的病害之一。十倍体长穗偃麦草(Thinopyrum ponticum)具有优良的赤霉病抗性,普通小麦-十倍体长穗偃麦草衍生新品种西农509、西农511和西农529在田间展现出较强的赤霉病抗性。本文旨在对这3个品种的赤霉病抗性基因进行分子鉴别,为它们在小麦赤霉病抗性遗传改良中的应用提供理论依据。【方法】通过赤霉病菌(Fusarium graminearum)人工接种鉴定,明确3个小麦新品种对赤霉病的抗性水平。利用偃麦草E组染色体(臂)第1—7同源群的特异引物对3个普通小麦-十倍体长穗偃麦草衍生新品种及其主要亲本小偃693、小偃597和十倍体长穗偃麦草进行分子鉴定,确定其长穗偃麦草的遗传区段。利用与长穗偃麦草7EL染色体上抗赤霉病基因Fhb7紧密连锁的标记对实验材料进行分析,明确该抗性基因与Fhb7的关系。【结果】鉴定结果表明,西农509、西农529和西农511的赤霉病抗性与中抗对照品种扬麦158的抗性水平相当,表现为中抗。105个长穗偃麦草E基因组特异标记中有7个在3个新品种中均能扩增出长穗偃麦草的特异条带,其中5个标记定位于7EL染色体臂上,2个标记定位于7ES染色体臂上。利用97个定位于7E染色体的特异标记进一步对小偃693、小偃597和3个新品种的遗传片段进行鉴别,结果表明20个标记能在5个长穗偃麦草衍生品种(系)扩增出稳定的十倍体长穗偃麦草的特异条带,其中包括与Fhb7紧密连锁的Xsdau K8、Xsdau K144、Xsdau K27、Xsdau K99、Xcfa2040和Xsdau K116等6个标记(7EL 149.00—7EL 153.77),即表明该区段源自于十倍体长穗偃麦草,跨距约89 c M。然而,已报道的7EL染色体臂末端与Fhb7两侧紧密连锁的分子标记Xsdau K60(7EL 153.77)、Xmag1932(7EL 154.70)、Xcfa2240(7EL 156.27)、Xsdau K66(7EL 158.02)、Xsdau K71(7EL 158.97)和Xsews19(7EL 160.00)等在3个新品种及小偃597中均没检出长穗偃麦草的特异条带。【结论】普通小麦-十倍体长穗偃麦草衍生新品种西农509、西农511和西农529具有较好的赤霉病抗性,携带来自于十倍体长穗偃麦草7E染色体的遗传区段,然而该抗赤霉病基因不同于Fhb7。     

偃麦草基因组特异重复序列的分离与应用

【目的】偃麦草(Thinopyrum)是小麦(Triticum aestivum L.)的多年生野生近缘植物,具有许多可用于小麦品种改良的优异基因。利用基因组特异重复序列可以研究物种的进化关系、绘制染色体指纹图谱及检测外源染色质。克隆十倍体长穗偃麦草(Th.ponticum(Host)Liu and Wang)基因组特异重复序列,可用于鉴定和追踪导入到小麦背景中的偃麦草遗传物质。【方法】通过构建十倍体长穗偃麦草小片段质粒文库,并对文库进行高密度点杂交(Dot-blot hybridization)筛选,结合荧光原位杂交(fluorescence in situ hybridization,FISH)技术,获得偃麦草基因组特异的重复序列,分析其在不同基因组及染色体上的分布特点。利用Repeat Masker在小麦族重复序列数据库(Triticeae repeat sequence database,TREP)及NCBI Gen Bank对特异重复序列进行比对分析,并设计偃麦草基因组特异重复序列的PCR引物。通过FISH分析和特异PCR引物扩增,对小麦-偃麦草衍生后代进行鉴定和选择。【结果】获得7条偃麦草基因组特异的重复序列。FISH分析表明,其在十倍体长穗偃麦草和六倍体中间偃麦草所有染色体两臂上均呈弥散型分布,且在不加小麦封阻DNA的情况下,能明确区分八倍体小偃麦中的偃麦草和小麦染色体。将其应用到小麦-偃麦草代换系和易位系的分子细胞学检测中,特异重复序列同样可以在不加封阻的情况下分辨出偃麦草染色体及染色体片段,而且信号相比基因组原位杂交(genomic in situ hybridization,GISH)更加特异和清晰。基于偃麦草基因组特异重复序列开发了90对引物,通过在中国春、十倍体长穗偃麦草和八倍体小偃麦中的扩增产物比较分析,筛选出36对(40%)偃麦草基因组特异PCR标记;利用这些特异引物对109份小麦-偃麦草衍生材料进行扫描,发现10对扩增效果较好的特异引物,其检测效率为73.3%—95%。【结论】获得偃麦草基因组特异的重复序列,开发了特异PCR扩增引物,可应用于小麦背景下偃麦草遗传物质的高效检测和跟踪。     

普通小麦-长穗偃麦草异附加系的分子标记鉴定

利用分布于小麦7个部分同源群的1 246对引物对15个可能的小麦-长穗偃麦草二体异附加系的4个亲本的基因组DNA进行扩增。结果表明,186对SSR、209对EST-SSR和22对STS引物能够在长穗偃麦草中扩增出不同于其他3个小麦亲本的特异条带,其中18对引物可以在14个附加系中的1~7个附加系扩增出长穗偃麦草的特异带,说明它们可能添加长穗偃麦草的遗传物质。5个附加系(1-3、1-8、1-27、2-6和2-22)可能含有长穗偃麦草第7同源群染色体,4个附加系(5-10、5-20、6-23和6-18)可能含有长穗偃麦草第6同源群染色体。附加系1-13可能附加2条不同同源群的长穗偃麦草染色体。同时发现,小麦与长穗偃麦草杂交及其后代衍生过程中长穗偃麦草染色体间可能发生遗传重组。     

基于RNA-seq的百萨偃麦草染色体特异分子标记开发与应用

【目的】百萨偃麦草（Thinopyrum bessarabicum Löve, 2n = 2x = 14, JJ或E^bE^b）是小麦改良的重要亲缘物种,开发染色体特异分子标记对于加快其有利基因向小麦中的转移和应用有重要意义。【方法】利用百萨偃麦草分蘖期叶片RNA-seq获得的EST序列与节节麦D基因组序列进行比对,鉴定出4 957条没有相似性的序列作为筛选百萨偃麦草特异序列的基础序列。从这些基础序列中随机选择部分序列设计EST-PCR引物507对,通过在普通小麦中国春、百萨偃麦草和中国春-百萨偃麦草双二倍体中的扩增分析,筛选出百萨偃麦草基因组特异标记,然后在已经选育出的8个小麦-百萨偃麦草异染色体系中进行染色体定位,并探讨这些标记在小麦染色体工程中的应用潜力;根据谷类作物的共线性,以百萨偃麦草EST序列设计共线性引物100对,并比较这些引物的扩增和定位结果。【结果】在开发的507对引物中,204对（40.2%）在百萨偃麦草和中国春-百萨偃麦草双二倍体中具有特异扩增,多态率远高于利用小麦（12%）和百萨偃麦草（14%）EST设计的234对共线性引物产生的多态率,建立了高效开发小麦亲缘物种特异标记的新方法;利用8个中国春-百萨偃麦草异染色体系,共定位了198个百萨偃麦草特异标记,分别位于染色体1J（31）、2JS（15）、2JL（26）、3JS（20）、4JS（12）、4JL（12）、5J（27）、6JS（13）、6JL（22）和7JS（20）,其中189个是根据百萨偃麦草转录组序列设计;利用定位于1J和6J的特异标记确定了4个易位系的染色体身份,其中1个涉及1J的大片段易位,2个涉及6JS的不同区段易位,1个为小片段中间插入易位;利用这些易位系,将30个1J和12个6J特异标记分别定位于2个物理区段。【结论】通过RNA-seq结合与小麦基因组序列比对可以获得小麦亲缘物种相对特异的EST序列并据此开发引物,建立了开发外源染色体特异标记的新方法,开发的标记可应用于小麦异易位系鉴定和缺失物理图谱的绘制。     

应用基因组原位杂交技术鉴定抗黄矮病小麦新种质

利用生物素（biotin-16-dUTP）标记的中间偃麦草（Thinopyrum intermedium）基因组DNA作探针,以未标记的普通小麦中国春基因组DNA作封阻DNA（blocking DNA）,对3个抗黄矮病小麦新种质的体细胞染色体进行分子原位杂交。结果表明：抗性源于L1的抗黄矮病小麦新种质Yw642为小片段易位系,含40条小麦染色体和2条小麦-中间偃麦草易位染色体,易位的中间偃麦草染色体片段位于小麦染色体的端部;染色体配对和抗性分析表明该种质为纯合易位系。抗性源于无芒中4的小麦新种质Hw240和Yw060遗传构成不同：Yw060为易位系,含40条小麦染色体和2条小麦-中间偃麦草易位染色体;Hw240为代换易位系,含38条小麦染色体、2条中间偃麦草染色体和2条小麦-中间偃麦草易位染色体。在这2个种质中,易位的中间偃麦草染色体片段均位于小麦染色体端部。     

春小麦新种质的分子细胞遗传学鉴定

远缘杂交能把亲缘关系较远的种、属中有用基因引入栽培种,改良现有品种。利用形态学和分子细胞遗传学研究方法,对远缘杂交创制的5份普通小麦新种质进行分析鉴定。结果表明:5份材料为春小麦,株系4-11和4-30-32突出表现为大穗、多分蘖,2015-2016年分蘖数为别为18~20和16~17个,熟期正常。二者的千粒重平均为38和36g,籽粒蛋白质含量超过18%。株系6-30-31表现为矮秆(55cm),密穗有芒,平均穗长7cm,千粒重平均32.6g。株系5-6和5-19,穗型似小偃麦,无芒,千粒重平均为33.7和36.4g,籽粒蛋白质含量超过19%。5份材料根尖体细胞染色体数为42,减数分裂行为正常。分子标记和原位杂交检测表明,株系4-11和5-19带有中间偃麦草遗传成分,为小麦-中间偃麦草易位系,株系5-6为六倍体小偃麦,矮秆株系6-30-31带有E染色体组遗传成分。     

小麦-中间偃麦草代换系CH188分子细胞学鉴定

中间偃麦草在小麦遗传改良中具有重要的作用。通过对小麦-中间偃麦草代换系CH188进行农艺性状、细胞学和分子标记技术鉴定,以确定其优良农艺性状及导入小麦基因组中的外源染色体。结果表明,CH188平均穗长14.60 cm,株高70.60 cm,小穗数、千粒质量等优于主栽品种;根尖细胞的有丝分裂中期染色体数目为2n=42;基于中间偃麦草第6同源群Contig序列开发了160个STS标记,其中,8个可作为识别小麦-中间偃麦草代换系CH188中6St染色体特异标记,可以推测小麦-中间偃麦草代换系CH188是小麦的一对染色体被中间偃麦草6St染色体替代;其优良的农艺性状可能来源于中间偃麦草6St染色体。CH188具有优良的农艺性状,可能是一个小麦-中间偃麦草代换系。     

二倍体长穗偃麦草E组染色体研究进展

二倍体长穗偃麦草(Thinopyrum elongatum)E组染色体是组成偃麦草属(Thinopyrum)多倍体物种的基本染色体组,携带有对小麦遗传育种有益的基因,人们已从多方面解读了E组染色体与小麦及其他近缘种属的遗传进化关系。综述了二倍体长穗偃麦草E组染色体与比萨偃麦草和小麦的亲缘关系、生化标记、分子标记和高分子量谷蛋白研究的进展。     

携带抗秆锈病基因Sr25、Sr26小麦新种质的分子标记辅助选育

源于长穗偃麦草的抗病基因Sr25、Sr26对秆锈病强毒力小种Ug99及其变种具有良好抗性,本实验室前期在长穗偃麦草与普通小麦杂交后代中选育出两个携带Sr25、Sr26基因的八倍体小偃麦。本研究选用济麦22、泰农18、山农637等小麦品种(系)分别与两个八倍体小偃麦杂交、回交,利用与Sr25、Sr26紧密连锁的分子标记进行辅助选择,定向培育出9个含有Sr25或(和)Sr26的小麦新种质。这些新种质综合农艺性状优良,可以作为抗病新种质用于小麦抗病育种,以应对Ug99威胁。     

Structural Changes of 2V Chromosome of Haynaldia villosa Induced by Gametocidal Chromosome 3C of Aegilops triuncialis

Haynaldia villosa （2n=2X= 14, VV）, a relative of wheat, plays important roles in wheat improvement mainly owing to its disease resistance. Powdery mildew resistance gene Pm21 has been successfully transferred into wheat by Cytogenetic Institute, Nanjing Agricultural University, China, and is widely used in the current wheat breeding programs. In this research, our objective is to further transfer and utilize the beneficial genes such as eye-spot resistance, yellow rust resistance, and gene of the tufted bristles on the glume ridge （a remarkable morphology） mapped on 2V of Haynaldia villosa. A disomic addition line with gametocidal chromosome 3C ofAegilops triuncialis added in Norin-26 was crossed to the wheat-H, villosa disomic substitution 2V（2D） and the hybrid F1 was then self-crossed. Chromosome C-banding, genomic in situ hybridization （GISH）, and meiotic analysis in combination with molecular markers were applied to detect the chromosome variations derived from hybrids Fz and F3. To date, four translocations including one small segmental translocation T6BS·6BL-2VS, two whole arm translocations （preliminarily designed as T3DS·2VL and T2VS.7DL） and one intercalary translocation T2VS·2VL-W-2VL, one deletion Del. 2VS·2VL-, one monotelosomic Mt2VS, and one isochromosome 2VS·2VS line have been developed and characterized. One wheat SSR marker Xwmc25.120 tagging 2VS and one wheat STS marker NAU/STSBCD135-1 （2BL） tagging 2VL were successfully used to confirm the alien chromosome segments involved in the seven lines. The tufted bristles on the glume ridge appeared in lines T2VS-7DL, Mt2VS, 2VS-2VS as well as the parent DS2V（2D）, whereas in T3DS·2VL, this trait did not appear. The gene controlling the tufted bristles was located on 2VS. Gametocidal chromosome 3C ofAegilops triuncialis could successfully induce chromosome 2V structural changes.     

Identification of disease resistances in wheat-<Emphasis Type="Italic">Leymus multicaulis</Emphasis> derivatives and characterization of <Emphasis Type="Italic">L. multicaulis</Emphasis> chromatin using microsatellite DNA markers

To identify resistance to Fusarium head blight (FHB), cereal yellow dwarf virus (CYDV), stem rust (Sr), and powdery mildew (Pm) in 24 common wheat (Triticum aestivum)-Leymus multicaulis addition/translocation lines that were developed cytogenetically and to verify the authenticity of these lines using microsatellite (SSR) DNA markers. Resistance to FHB was identified in the wheat-L. multicaulis addition lines, Line 9 and Line 26, which both contained L. multicaulis-specific fragments as shown by SSR markers. The translocation line, Trans 1, and the addition lines, Line 5 and Line 29, have resistance to stem rust (IT 0). Resistance to CYDV was evaluated based on virus titers measured by enzyme linked immunosorbent assay (ELISA). The addition line, Line 23, showed low virus titer (0.15), indicating resistance to CYDV. The segregation distribution of CYDV resistance in 98 F₂ plants of Line 23/CS showed a significant deviation from 3:1. Inoculation with a set of 14 differential Blumeria graminis f. sp. tritici (Bgt) isolates did not detect powdery mildew resistance in translocation line Trans 1, addition line Line 9 and the amphiploid of wheat-L. multicaulis. However, Line 26 exhibited the resistance response pattern of Kavkaz, which carries Pm8, indicating that Line 26 most likely has the powdery mildew resistance gene Pm8 inherited from its parent lines Feng Kang 7 or Feng Kang 10. Twelve SSR markers, distributed on different homeologous chromosome groups of wheat, which distinguished L. multicaulis addition/translocation chromosomes, were used to verify the presence of L. multicaulis chromatin in the putative wheat-L. multicaulis addition/translocation lines. Of the 24 addition/translocation lines investigated using the 12 polymorphic SSR markers, 18 wheat-L. multicaulis derivatives showed the expected L. multicaulis-specific fragments, indicating that all of these 18 addition/translocation lines would most likely have the introgressed L. multicaulis chromosome(s). Chromosomal rearrangements also were detected in some of the wheat-L. multicaulis introgression lines.     

矮秆小麦XN0004的矮秆基因Rht21的染色体臂定位

XNOOO4是青431与小偃6号杂交选育的一个新的具有部分矮秆显性效应的小麦新矮源品系。和高秆亲本相比,其杂种F_1代的降秆作用平均为13.8％,对外源赤霉酸反应不敏感,在杂种小麦研究中,其配合力优良,增产显著,穗粒数增加,抗倒能力增强,收获指数提高等,无某些矮源对杂种F_1产生的不良效应,是杂种小麦比较理想的矮秆亲本,可作为常规育种的优良矮秆品种资源。用中国春缺—四体和双端体分析的方法,对XNOOO4丰矮秆显性基因进行了染色体定位,证明其矮秆显性基因位于2A染色体的短臂上,是一个不同于世界上已定位的20个Rht基因的新矮源,故暂定名为显性矮秆基因Rht21。     

Development of EST-PCR markers specific to the long arm of chromosome 6V of Dasypyrum villosum

Expressed sequence tags-derived polymerase chain reaction (EST-PCR) molecular markers specific for alien chromosomes can be used to not only monitor the introgressed alien chromatin in wheat background, but also provide the evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers specific for chromosome 6VL of Dasypyrum villosum, 297 primer pairs were designed based on the expressed sequence tags (EST) sequences, which were previously mapped in different bins of the long arms of wheat homoeologous 6AL, 6BL, and 6DL. By using the Triticum aestivum, D. villosum, T. durum-D. villosum amphiploid, and T. aestivum-D. villosum alien chromosome lines involving chromosome 6V, it was found that 32 (10.77%) primers could amplify specific bands for chromosome 6V, and 31 could be allocated to chromosome arm 6VL. These 6VL specific markers provided efficient tools for the characterization of structural variation involving the chromosome 6VL in common wheat background as well as for the selection of useful genes located on 6VL in breeding programs.     

Development of EST-PCR Markers for the Chromosome 4V of Haynaldia villosa and Their Application in Identification of 4V Chromosome Structural Aberrants

EST-PCR based molecular markers specific for alien chromosomes are not only useful for the detection of the introgressed alien chromatin in the wheat background, but also provide evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers on chromosome 4V of Haynaldia villosa, a total of 607 primer pairs were designed according to the EST sequences, which were previously located in 23 different bins of wheat chromosomes 4A, 4B and 4D. By using the Triticum durum-H, villosa amphiploid and T. aestivum-H, villosa alien chromosome lines involving chromosome 4V, it was found that 9.23% of the tested primers could amplify specific bands for chromosome 4V. Thirty and twenty-six specific markers could be assigned to chromosome arms 4VS and 4VL, respectively. These 4V specific markers provided efficient tools for the characterization of structural variation involving the chromosome 4V as well as for the selection of useful genes located on chromosome 4V in breeding programs.     

利用转录组数据开发意大利蜜蜂的SSR分子标记

意大利蜜蜂(简称意蜂)是自然界重要的授粉昆虫,广泛用于世界各国的养蜂生产,具有很高的生态和经济价值。为了开发意蜂的SSR分子标记,利用MISA软件对基于前期获得的意蜂幼虫肠道转录组数据组装得到的12 115条unigenes进行搜索,共预测出分布于2 149条unigenes的6 312个SSR位点,其中主要的重复类型为二核苷酸重复(54.42%)和三核苷酸重复(32.49%),主要基元为AT/AT(31.2%)和AG/CT(18.6%)。进一步利用软件设计出18 444对特异性引物,随机选取24对引物对国内3个不同来源的意蜂幼虫样品进行SSR位点扩增,有22对成功扩增出目的片段。研究开发出的SSR分子标记可用于意蜂的种群遗传和分子进化等研究,结果表明利用转录组数据鉴定非模式生物SSR位点的方法可行且高效。     

提莫菲维小麦与光稃野燕麦易位系TGF314的顺序C-分带-基因组原位杂交鉴定

采用顺序C-带-基因组原位杂交（GISH）技术对提莫菲维小麦与光稃野燕麦远缘杂交后代中的外源遗传物质进行鉴定。GISH分析结果表明，在根尖细胞有丝分裂中期染色体具有2对清楚的杂交信号，说明该后代中可能含有稳定的外源遗传物质，且以易位的染色体片段形式存在。对该后代进行顺序C-带-GISH分析将杂交信号分别定位于提莫菲维小麦的7A和6G染色体短臂上，提莫菲维小麦的7A和6G染色体中含有光稃野燕麦的遗传物质。