【作者】 刘爱峰；

【导师】 王洪刚；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 长穗偃麦草（Elytrigia elongata，2n=70，StStStStEeEeEbEbExEx）是小麦的野生近缘种，具有许多优良性状，是小麦的三级基因库和小麦性状改良的优异外源基因供体之一。小偃麦种质系山农87074来源于长穗偃麦草（2n=70）与普通小麦品种鲁麦5号和济南13的复合杂交（长穗偃麦草／鲁麦5号／／济南13）后代，山农19、山农20和山农122是利用十倍体长穗偃麦草和普通小麦直接杂交创制的小偃麦中间类型。本研究综合利用抗性接种鉴定、细胞学、生物化学、SSR分子标记和基因组原位杂交（GISH）等方法技术，对山农87074衍生的10个种质系山农87074—513、—519、—526、—541、—551、—554、—555、—557、—565和—576和3个小偃麦中间类型山农19、山农20、山农122进行了鉴定；对小偃麦种质系的抗病基因进行了染色体定位和SSR分子标记分析；利用回收特异PCR扩增片段法回收克隆了长穗偃麦草的特异SSR片段，并制备成特异探针应用于基因组原位杂交。获得以下主要结果：1、利用细胞学方法对山农87074的10个衍生种质系和3个小偃麦中间类型进行了鉴定，结果表明：10个种质系均为小偃麦异染色体系，其中山农87074—519是双体异附加系（2n=44，PMC MI染色体基本构型为22Ⅱ）：山农87074—513、—526、—541、—551、—554、—555、—557、—565和—576等9个种质系为双体异代换系（2n=42，PMC MI染色体基本构型为21Ⅱ）；3个小偃麦中间类型山农19、山农20和山农122为新型八倍体小偃麦，它们分别携带有长穗偃麦草的1个染色体组。经田间试验和人工接种鉴定证明，这些八倍体小偃麦或小偃麦异染色体系综合农艺性状较好，具有对小麦条锈病或白粉病良好的抗性，或多花多实等优良特点，在小麦遗传改良中具有重要利用价值。2、分别以假鹅冠草（2n=14，StSt）和二倍体长穗偃麦草（2n=14，EE）总基因组DNA为探针对异附加系山农87074—519的根尖细胞染色体进行了原位杂交，鉴定结果显示，在以假鹅冠草总基因组DNA为探针的根尖细胞44条染色体中有2条出现黄绿色杂交信号，而且杂交信号遍布整条染色体；以二倍体长穗偃麦草总基因组DNA为探针的根尖细胞染色体未观察到杂交信号。表明山农87074-519附加的2条染色体来自长穗偃麦草的St染色体基组，是一个附加了长穗偃麦草1对St染色体的双体异附加系。利用小麦SSR引物进行的SSR—PCR扩增鉴定证明，在170对小麦SSR引物中有1对引物BARC165—5AL能在山农87074—519与其亲本间揭示特异性差异，特异片段大小约270bp，记为BARC165₂₇₀，它可用于山农87074—519的鉴定。利用回收特异PCR扩增片段法回收克隆长穗偃麦草的DNA特异重复片段BARC165₂₇₀，经测序分析其碱基序列全长为268bp，命名为BARC165₂₆₈，并将该序列提交Genbank注册（Genbank登录号EF397435）。将该重复序列制备为探针进行原位杂交鉴定，结果显示，山农87074—519的根尖细胞染色体出现很强的黄色点状杂交信号，而在普通小麦中没有观察到杂交信号。结合以假鹅冠草（2n=14，StSt）总基因组DNA为探针的GISH鉴定结果，推测特异重复序列BARC165₂₆₈是St染色体基组的特异重复序列，可用于检测小麦遗传背景中的长穗偃麦草遗传物质。应用SDS-PAGE技术对山农87074—519的鉴定表明，其附加的染色体上没有编码长穗偃麦草特异亚基带型的基因位点。3、利用SSR分子标记技术对9个异代换系山农87074—513、—526、—541、—551、—554、—555、—557、—565和—576进行了鉴定分析。结果表明，从592对小麦SSR引物中获得9对引物能在异代换系中扩增出长穗偃麦草的特异带，进一步证明它们携带有长穗偃麦草遗传物质。9对特异引物扩增的SSR分子标记中，标记BARC066₂₃₀和BARC023₂₇₀可用于鉴定山农87074—513；BARC148₂₄₀、BARC066₂₃₀、BARC168_150／180、BARC232₄₅₀、BARC195₂₀₀和BARC023₂₇₀等可用于87074—526的鉴定；BARC148₂₄₀、BARC099₁₅₀、BARC232₄₅₀、BARC023₂₇₀等可用于山农87074—541的鉴定；BARC148₂₄₀、BARC099₁₅₀、BARC040₁₈₀、BARC023₂₇₀等可用于鉴定87074—551；BARC148₂₄₀、BARC040₁₈₀、BARC023₂₇₀等可用于鉴定山农87074—554；BARC148₂₄₀和BARC232₄₅₀可用于山农87074—555的鉴定；BARC066₂₃₀、BARC040₁₈₀、BARC023₂₇₀等可用于山农87074—557的鉴定；BARC100₁₅₀、BARC040₁₈₀、BARC232₄₅₀等可用于山农87074—565的鉴定；BARC148₂₄₀、BARC066₂₃₀、BARC023₂₇₀等可用于鉴定山农87074—576。利用A-PAGE和SDS-PAGE技术对9个异代换系的鉴定结果表明，它们被代换的染色体不属于第1部分同源群染色体。进一步利用一套中国春缺体—四体系对9个异代换系中代换染色体的同源群归属进鉴定结果表明，定位于2B染色体的引物SWES240能扩增出1条大小约400bp的差异带，此特异带在长穗偃麦草、二倍体长穗偃麦草、山农87074—513、—526、—541、—551、—554、—555、—557和—576、中国春2B缺体2D四体系（N2BT2D）中均不存在，而在假鹅冠草、中国春、其余22个中国春缺体-四体系、普通小麦亲本鲁麦5号和济南13、山农87074—565中均出现。推断山农87074—513、—526、—541、—551、—554、—555、—557和—576等8个异代换系染色体基组中的1对小麦2B染色体被1对长穗偃麦草的2E染色体所代换，它们为2B／2E双体异代换系。4、PMC MI染色体构型分析结果表明，山农20和山农122的染色体基组构成相同，它们与前人选育的小偃68的染色体基组可能为同一类型；山农19的染色体基组构成不同于山农20、山农122、和前人选育的小偃68、小偃693和小偃7430的染色体基组。分别以十倍体长穗偃麦草、二倍体长穗偃麦草和假鹅冠草总基因组DNA作探针进行GISH鉴定证明，山农19附加的长穗偃麦草染色体组为St；山农20和山农122附加的长穗偃麦草染色体组可能为E基组。综合上述结果，可将山农19的染色体基组确定为ABDSt，山农20和山农122的染色体基组记为ABDE。5、综合运用抗性接种鉴定、细胞学分析、SSR分子标记和GISH等技术在山农87074的衍生种质系中鉴定了来源于十倍体长穗偃麦草的3个抗性基因，即抗小麦条锈病基因YrSt和Yr2E以及抗小麦白粉病基因Pm2E，并初步将YrSt定位在偃麦草的St染色体上，将Yr2E和Pm2E定位在偃麦草的2E染色体上，推测它们可能为新的抗病基因。以种质系山农87074—557／辉县红的F₂分离群体为材料，应用SSR分子标记技术对山农87074—557中的抗病基因进行了标记分析。592对小麦SSR引物中有2对引物Xgwm344和Barc074与山农87074—557中的偃麦草染色体2E有连锁关系，其中标记Xgwm344_120／150与抗白粉病基因Pm2E和抗条锈病基因Yr2E连锁，标记Barc074₁₅₀与抗叶锈病基因连锁。更多还原

【Abstract】 Elytrigia elongata is a perennial partial auto-allo-decaploid species （2n=10x=70, previously designated with genomes StStStStEeEeEbEbExEx）, that is a potentially useful source of a number of genes for wheat improvement, including rust resistance, powdery mildew resistances, low-temperature and drought tolerance, high protein, an increased number of florets per spike. Tritielytrigia Shannong87074, Shannongl9, Shannong20 and Shannong122 all are novel germplasm created by the hybridization between decaploid Elytrigia elongata and common wheat. Shannong87074 is a progeny from hybrid cross ofElytrigia elongata and common wheat Lumai5 and Jinanl 3, the ten lines Shannong87074-513,-519,-526,-541,-551, -554,-555,-557,-565,576 all are derived from Tritielytrigia Shannong87074; Shannongl9, Shannong20 and Shannong122 all are octaploid Tritielytrigia types. The inoculation assessment, cytological analysis, biochemistry assay, simple sequence repeat（SSR） molecular marker technique, and genomic in situ hybridization（GISH） were employed in the identification of Tritielytrigia germplasm; The specific repeat sequence of decaploid Elytrigia elongata amplified by SSR was retrieved and cloned, and was prepared as the specific probe used in the GISH. The results are as following:1. The cytological identification indicated that Shannong87074-519 was a alien disomic addition line, with 44 chromosomes in root tip cells and basic chromosomal configuration of PMC MI 2n=22Ⅱ, Shannong87074-513,-526,-541,-551,-554,-555,-557,-565,576 all were alien disomic substitution lines, with 42 chromosomes in root tip cells and basic chromosomal configuration of PMC MI 2n=21Ⅱ, and the three Tritielytrigia interspecies Shannongl 9,Shannong20 and Shannong 122 all were novel octaploid Tritiielytrigia containing a genome of Elytrigia elongata. The morphological characterization and disease resistance evaluation showed that they appeared well agronomic characteristic, powdery mildew resistance or yellow rust resistance, more florets per spike. They will play an important role in the wheat genetic improvement.2. The GISH identification of the addition line Shannong87074-519 with root tip cells was conducted by using Ps.strigosa（2n= 14,StSt） and E.elongata（2n=14,EE） total genomic DNA as probe respectively. It showed that two chromosomes with green-yellow hybridization signal were observed among the 44 chromosomes, with the signal covering the whole chromosome in the GISH pattern with the Ps.strigosa as probe; no signal was observed with the E.elongata as probe. It indicated that Shannong87074-519 was one alien disomic addition line with two added chromosomes belonging to genome St of Elytrigia elongata.The SSR technique was further applied to verify Shannong87074-519, with 170 primer pairs testified. The results indicated that Shannong87074-519 contained genetic material transferred from Elytrigia elongata; the primer pairs BARC165 on chromosome 5AL could show the diversity between Shannong87074-519 and its parents, and the specific segment of Elytrigia elongata could be stably amplified in its DNA PCR products, whose size was about 270bp, assigned as BARC165₂₇₀ temporarily. The molecular maker BARC165₂₇₀ could be used in the verification of Shannong87074-519. Subsequently, the specific repeat segment of Elytrigia elongata was retrieved and cloned. DNASTAR software analysis showed that the nucleotide sequence length of specific segment is 268bp, then the marker was designated as BARC165₂₆₈, and the repeat sequence was submitted to the genbank with the accession number EF397435. BARC165₂₆₈ was prepared as specific probe in the GISH identification, strong yellow dot hybridization signal was observed in chromosome of Shannong87074-519, but not in the common wheat. Considering the GISH pattern by the probe St, it was inferred that the specific repeat sequence likely concentrated on chromosome St, and it was the specific repeat sequence of genome St. The repeat sequence could be used as a valuable marker to identify the germplasm of Elytrigia elongata in common wheat background.The identification of Shannong87074-519 by SDS-PAGE showed that no specific locus coded the Elytrigia elongata HWM-GS located on the added chromosome St.3. 592 primer pairs were amplified in the identification of the nine alien substitutions Shannong87074-513,-526,-541,-551,-554,-555,-557,-565,-576 by SSR molecular technique. Nine primer pairs among them could amplify the specific band of Elytrigia elongata in PCR products of the nine substitutions, which indicated the specific difference between the substitutions and their parents, and further verified that the substitutions were the progeny from Elytrigia elongata.The nine primer pairs could be used as molecular markers in the identification of the nine alien substitutions, such as:BARC066₂₃₀ and BARC023₂₇₀ of Shannong87074-513, BARC148₂₄₀, BARC066₂₃₀, BARC168_150/180, BARC232₄₅₀, BARC195₂₀₀ and BARC023₂₇₀ of Shannong87074-526, BARC148₍2400, BARC099₁₅₀, BARC232₄₅₀ and BARC023₂₇₀ of Shannong87074-541, BARC148₂₄₀, BARC099₁₅₀, BARC040₁₈₀ and BARC023₂₇₀ of Shannong87074-551, BARC148₂₄₀, BARC040₁₈₀ and BARC023₂₇₀ of Shannong87074-554, BARC148₂₄₀ and BARC232₄₅₀ of Shannong87074-555, BARC066₂₃₀, BARC040₁₈₀ and BARC023₂₇₀ of Shannong87074-557, BARC100₁₅₀, BARC040₁₈₀ and BARC232₄₅₀ of Shannong87074-565, BARC148₂₄₀, BARC066₂₃₀ and BARC023₂₇₀ of Shann0ng87074-576.The identification of the nine alien substitutions by A-PAGE and SDS-PAGE showed that the substitutions contained genetic material of Elytrigia elongata, and the substituted chromosome did not belong to the first homologous group. One set of Chinese Spring nullisomic-tetrasomic lines were utilized to further verify the homologous substitution chromosome. The results showed that, the primer pair SWES240 on 2B chromosome could amplify one specific segment in the PCR products to distinguish the differences among the tested materials, with the segment size about 400bp;The specific band could not be amplified in the PCR products of Elytrigia elongata, E.elongata（2n=14）, substitutions Shannong87074-513,-526,-541,-551,-554,-555,-557,-576, and the 2B nullisomics 2D tetrasomics（N2BT2D） of Chinese Spring, but appeared in Ps.strigosa, Chinese Spring, the other 22 Chinese Spring nullisomic-tetrasomic lines, common wheat Lumai5 and Jinanl3, substitution line Shannong87074-565. It concluded that the 2B chromosomes of the eight lines Shannong87074-513,-526,-541,-551,-554,-555,-557,-576 were substituted by 2E chromosome of Elytrigia elongata, the eight lines were alien disomic substitutions with 2B/2E.4. The chromosomal pairing behavior of PMC MI of the three octaploid Tritielytrigia was analyzed and the results showed that Shannong20 and Shannong122 had the same chromosomal genomes, and belonged to the same type as xiaoyan68（previously designated with the basic genomes ABDE2 ）, however, Shannongl 9 was the other type different from Shannong20, Shannong122, and the former xiaoyan68,xiaoyan693 and xiaoyan7430. The GISH patterns with Elytrigia elongata（2n=70）, E. elongata（2n=14,EE） and Ps.strigosa（2n=14,StSt） total genomic DNA as probe respectively indicated that Shannongl9 was added by one set St genome, Shannong122 probably was added by one set E genome. The basic chromosomal genome of Shannongl9 could be designated as ABDSt, and that of Shannong20 and Shannong 122 was assigned as ABDE temporarily.5. The inoculation assessment, cytological analysis, SSR molecular marker technique, and GISH identification were used in the identification and chromosomal location of resistant genes. Three resistant genes derived from decaploid Elytrigia elongata, YrSt andYr2E with the yellow rust resistance, and Pm2E with the powdery mildew resistance, were located on St chromosome and 2E chromosome respectively. It could be inferred that the three resistant genes likely all were novel genes.The F₂ segregating population of alien substitution line Shannong87074-557 and common wheat huixianhong was employed to further analyze the dominant resistant gene on the substitution chromosome by SSR technique. Two primer pairs that linked to the substitution chromosome were singled out from 592 SSR primer pairs of wheat, one was Xgwm344_120/150 on the 7B chromosome of wheat, it linked to the powdery mildew resistant gene Pm2E and yellow rust resistant gene Yr2E; the other was Barc074₁₅₀ on the 5BL chromosome, it linked to the leaf rust resistant gene.更多还原