【作者】 戴秀梅；

【导师】 李加纳； 柴友荣；

【作者基本信息】 西南大学 ， 作物遗传育种， 2008， 博士

【摘要】 小麦（Triticum aestivam）是世界主要粮食作物之一,白粉病（Blumeria graminisf.sp.tritici）是世界小麦产区的主要病害,严重影响产量和品质。选育和推广抗白粉病的新品种是防治白粉病的有效而安全的途径。从小麦近缘物种中转移抗白粉病基因到小麦遗传背景中,创建新的抗白粉病材料是抗病育种的一个重要内容。同时,筛选与抗白粉病基因连锁的分子标记并应用于新品种、新材料选育中,对提高优良性状聚合效率和加快抗病育种进程具有重要意义。1.选育并鉴定了一个新型的小麦6V/6B异代换系010714贵农21是多抗优良的小麦品系,尤其是对白粉病免疫。它是普通小麦-簇毛麦的6V/6A异代换系,其6V染色体来自于簇毛麦（Haynadia villosa）。硬偏麦六倍体（Hexaploid of Ae.ventricosa-T.durum）来自于硬粒小麦（Triticum durum）与偏凸山羊草（Aegilops ventricosa）杂交的后代。二者均有稳定而卓越的抗白粉病性。抗病种质010714为贵农21和硬偏麦六倍体杂交后连续自交,在F₇代选出的能稳定遗传、具优良抗白粉病性,且农艺性状接近于育种目标的株系。对抗病种质010714的遗传背景进行细胞学鉴定,分别以簇毛麦和偏凸山羊草的基因组总DNA为探针,中国春基因组总DNA为封阻剂,对010714根尖细胞分裂中期相的染色体组进行原位杂交,结果表明010714染色体组中有1对染色体能与簇毛麦基因组DNA探针杂交,010714染色体组与偏凸山羊草基因组DNA探针无杂交信号。说明010714带有一对来自于簇毛麦的染色体,没有来自偏凸山羊草的M^V染色体或染色体大片段。核型分析结果表明,010714中来自簇毛麦的染色体臂比值为1.20,与簇毛麦6V染色体臂比值1.12类似。同时在010714中只有一对臂比值为1.85的随体染色体,与1B染色体类似,缺少臂比值大于2.10的6B随体染色体。C-带分析表明,能与簇毛麦基因组DNA杂交的染色体带型特征与簇毛麦6V染色体C-带带型一致,普通小麦染色体不具有这一带型。因此可推断,010714为一个6V/6B代换系,是一个不同于贵农21的6V/6A异代换系的新型育种材料。010714的优良抗白粉病性与6V染色体有关。2.获得了与010714抗白粉病基因连锁的RAPD分子标记RM874C050S具有温光敏核不育性和优良的农艺性状,但不抗白粉病。本研究以C050S作为母本,将010714作为父本,进行杂交,在后代中进行抗白粉病基因标记筛选和转化,并利用所得分子标记进行分子标记辅助选择,以便真正将010714的抗白粉病基因应用于小麦新材料新品种选育中。以010714为父本与C050S杂交的F₁代植株表现为抗病,说明010714所携带的抗白粉病性状为显性遗传。F₁自交得到的F₂分离群体,其抗病:感病植株比例为139:96。010714的抗白粉病性呈单基因显性遗传模式,其抗白粉病基因与6V染色体有关,由于6V在普通小麦遗传背景下传递率低于50%,致使C050S×010714的F₂群体出现偏离孟德尔模式3:1的分离比例。由引物S2018扩增出的长度为874bp的特异标记RM874可以作为010714、贵农21抗白粉病基因的RAPD分子标记。3.基于单一PCR条带的大量测序和多重比对,成功地将含有大量重复序列的RM874标记转化为SCAR标记SM142RM874稳定性欠佳,有待于转化成更稳定和特异的SCAR标记。测序表明,RM874单一条带包含有一系列等长的、来自于重复序列的同源序列片段。在已经测序的克隆子中,克隆子序列RS874与抗白粉病基因一起沿簇毛麦→贵农21→010714→C050S×010714的F₂群体的传递路线。将RM874条带中能在簇毛麦、010714和C050S×010714的F₂群体中均有出现的序列RS874与GenBank中已登录序列进行广泛比对,结果表明010714与抗白粉病基因连锁的RAPD分子标记RM874所包含的重复序列家族与一粒小麦、圆锥小麦、普通小麦、大麦甚至水稻的对应序列具有同源性。RM874及其同源序列为类似gypsy逆转录转座子中gag-pol编码区的部分序列,很可能在转座过程中大量遗留于不同染色体的不同位置,构成了重复序列家族,GISH表明它们在010714染色体组中呈散在分布。RM874为簇毛麦特有的序列,有别于其他近缘物种。010714为6V/6B代换系,所以在010714中RM874为6V染色体特异的RAPD标记。通过RS874与其他RM874序列、一粒小麦、圆锥小麦、普通小麦、大麦和水稻对应序列进行比对,设计出SCAR引物组合05-116/05-117,能扩增出长度为142bp的特异条带,命名为SM142,SM142与抗白粉病基因具有连锁关系,但未呈现共分离的完全连锁。4.利用SM142标记的辅助选择,实现了抗白粉病性与TGMS不育性的聚合以C050S×010714的F₂代单株和F₅代单株为RM874和SM142与抗白粉病基因连锁性的验证群体,结果表明RM874和SM142与抗白粉病基因间存在连锁关系,但SM142稳定性好于RM874,二者均可用作C050S×010714杂种后代抗白粉病性的分子标记。利用SCAR标记SM142,进行抗白粉病性分子标记辅助选择,结合传统选育方法,在部分C050S×010714的F₅代群体中选育到2个抗白粉病性得到改良、不育度和株高基本符合育种要求的株系。证明SM142在育种实践中具有一定实用价值。C050S×010714杂种后代出现少量具有SM142但不带抗白粉病基因的现象,是因为个别后代植株发生了易位,出现了感病的6V长臂易位系和抗病的6V短臂易位系,前者SM142与抗白粉病基因不连锁,而后者SM142与抗白粉病基因连锁,说明抗白粉病基因位于6V染色体的短臂上。对于010714和杂种后代,以及其他携带来自于簇毛麦6V染色体的小麦材料来讲,RM874和SM142可作为6V染色体特异的分子标记。对于携带来自于簇毛麦其他染色体的小麦材料而言,RM874有可能在一定程度上作为跟踪簇毛麦遗传物质的分子标记。更多还原

【Abstract】 Wheat（Triticum aestivam） is an important grain crop worldwide.Powdery mildew（PM,caused by Blumeria graminis f.sp.tritici） is a major epidemic disease of wheat,causing serious reduction of yield and quality.Breeding and extension of powdery mildew resistant varieties is an efficient and safe strategy to control this disease,lntrogression of powdery mildew resistance genes from relative species into common wheat genetic background is a crucial for disease resistance breeding in wheat. At same time,identification of molecular markers linked with resistance genes and application of them in the selection of cultivars and stocks,will promote the pyramiding efficiency and comprehensive elite traits and the disease resistance breeding steps.1.The 6V/6B alien substitution line 010714 of wheat was bred and identifiedGuinong 21 is an elite wheat line with multiple resistances especially with to powdery mildew.It is a 6V/6A substitution line in which the 6V chromosome was introgressed from Haynadia villosa.Hexaploid of Ae.ventricosa-T,durum is an offspring of T.durum and Aegilops ventricosa.Both of them have good resistance to powdery mildew.Bridge-cross between Guinong 21 and hexaploid of Ae.ventricosa-T. durum was made and a PM-resistant intermediate line 010714 was selected from the F₇ generation.It is relatively stable,highly resistant to PM,and has comprehensive agronomic traits near general common wheat lines.Cytological identifications of 010714 were carried by Genome in Situ Hybridization（GISH）,karyotype investigation and C-band analysis.Using Digoxigenin-11-dUTP labeled total genomic DNA of H.villosa and A.ventricosa as the probes.The results revealed that line 010714 has a pair of alien chromosomes from H.villosa and no chromosome or mega fragment from A.ventricosa.Karyotype analysis suggested that the arm ratio of the chromosome from H.villosa is 1.20, simility to the 1.12 of 6V.In 010714,there is only a pair of 1B-like satellite chromosomes with an arm ratio of 1.85.But it lacks another satellite chromosome,6B, whose arm ration is 2.10.C-band analysis showed the substitution chromosome is the 6V chromosome.Is definite that 010714 is a 6V/6B substitution line which is a new material,and its resistance to PM is correlated to the alien chromosome.2.The RAPD marker RM874 was identified,with linkage to the PM-resistance in 010714C050S has the thermo-photo-sensitive genic male sterility（TGMS） and good agronomic traits but is highly susceptible to many diseases especially PM.This study used 010714 as the male parent and C050S as the female parent to make a cross,and identified the molecular markers with linkage to the PM-resisntance.The F₁ plants are resistant to PM,indicating that the PM-resistance of 010714 is a dominant trait.The F₂ plants segregated for a ratio of 139 resistant over 96 susceptible ones.The PM resistance is a dominant trait determined by a single gene.This segregation ratio did not follow the typical Mendelian ratio 3:1,because in 6V substitution line,the transmission rate of the 6V chromosome through gametes was significantly lower than that of common wheat chromosomes.RAPD primer S2018 amplified a specific band of about 874 bp,named RM874 that is a specific RAPD marker for powdery mildew resistance in GN21 and 010714.3.Based on batched sequencing within single PCR bands and multi-alignments, the repeat-containing RM874 marker was successfully converted into SCAR marker SM142RM874 showed a little instability and needed to be converted into a more stable and specific SCAR marker.Sequencing results indicated that RM874 single band contained a family of homologous repetitive sequences with equal lengths.Among the sequenced clones,sequence F2R874-7 from the F₂ resistant pool was completely identical to sequence PR874-910 from 010714 and sequence Hv874-7 from H.villosa. Then the specific F2R874-7/PR874-910/Hv874-7 fragment was named as RS874. RS874 could be traced together with the PM-resistance from H.villosa to Guinong 21, then to 010714,and finally to the F₂ plants of cross C050S×010714.NCBI BLASTn revealed that,in local alignments,RS874 had 58.1%to 72.1%identities to LTR-retrotransposon like repetitive regions from T.monococcum,T.turgidum, common wheat,Hordeum vulgare and Oryza sativa.RM874 and its homologous sequences were in the GAG-POL coding regions of Ty3-gypsy retrotransposons.The family sequences from RM874 were dispersive in 010714’s chromosomes.Based on multi-alignments of RS874 with other 43 RM874 sequences and homologous sequences from T.monococcum,T.turgidum and common wheat,a pair of SCAR primers 05-116/05-117were designed.This SCAR primer pair specifically amplified a 142-bp fragment from 010714 and F₂ resistant pool,but not from C050S and F₂ susceptible pool.This SCAR marker was named SM142.SM142 is linked with the PM resistance gene but did not show cosegrating/complete linkage with the PM resistance gene.4.Based on SM142-assited selection,the PM resistance and the TGMS were successfully combinedF₂ and F₅ individual plants from the C050S×010714 were examed for the presentation of RAPD marker RM874 and SCAR marker SM142.The results showed that the markers are linked to the PM resistance,and SM142 is more stablie than RM874.Both of them can be applicated as the markers of PM resistance in MAS.Two TGMS lines with elite PM resistance and other agronomic traits were selected from F₅ population by SM142-assisted selection and traditional breeding.It proved the value of SM142.Because of translocation between 6V chromosome and common wheat’s chromosome,the linkage between the marker and the PM-resistance has been disrupted in several progenies of cross C050S×010714.The results of GISH show that the individuals having SM142 but no PM resistance are 6VL translocation lines,while the individuals that having both SM142 and resistance are 6VS translocation line. These suggested that the PM resistance gene is located at the short arm of 6V chromosome.For 010714,its hybrid offsprings,and other wheat materials with 6V chromosome from H.villosa,RM874 and SM142 can act as 6V-specific PCR marker. For wheat stocks harboring other chromosome from H.villosa,RM874 might be capable to certain extent for tracing the alien genetic substances as well.更多还原