【作者】 杨克强；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2003， 博士

【摘要】 无核葡萄具有重要的商业价值，葡萄无核育种已成为当今葡萄育种的重要方向。近年的研究表明，葡萄无核性状由主效基因和一些微效基因共同作用而控制。本研究以红地球、红光无核、无核白及红地球×红光无核F₁代163株杂种为试材，对葡萄无核主效基因进行了定位与作图研究，对与无核主效基因紧密连锁的特异标记的DNA序列在GenBank中进行了相似性分析和开放阅读框架（ORF）分析，还进行了Southern杂交分析，主要结果如下。 1．葡萄无核基因连锁的SCAR标记的获得 以UBC-269₄₈₄和GSLP1₅₆₉的序列为支点，设计合成了包括UBC-269和GSLPI在内的9条引物，用有核亲本红地球和无核亲本红光无核的DNA为模板，对这9个引物进行筛选，结果序列长度均为18个核苷酸的GSLP1、39970524-5号引物和39970524-6号引物在无核亲本红光无核上扩增出了特异标记。GSLP1扩增出的特异标记为GSLP1₅₆₉，39970524-5号引物扩增出的特异标记为39970524-5-564，39970524-6号引物扩增出的特异标记分别为39970524-6-1538和39970524-6-1200。用这3个特异引物在红地球、红光无核、无核白和红地球×红光无核杂交组合F₁代163株杂种的DNA样上进行PCR扩增，结果4个特异标记在F₁群体中与无核主效基因共分离。这4个特异标记也出现于本研究所用组合中无核基因原始供给者无核白上。这些标记和葡萄的无核主效基因相连锁。 2．葡萄无核基因的定位与作图 用QTXb17遗传作图软件，对葡萄无核主效基因S定位与作图，特异标记GSLP1₅₆₉、39970524-5-564、39970524-6-1538和39970524-6-1200与无核主效基因S连锁紧密。当P=0.01时，LOD值在32.7—46.4之间，置信界限在0.2—9.9之间。这4个特异标记和无核主效基因S处于在同一连锁群，位于无核主效基因S的两侧，覆盖基因组12.3cM。特异标记39970524-5-564距S基因0.6cM，特异标记GSLP1₅₆₉距S基因1.2cM，特异标记39970524-6-1538距S基因4.9cM，特异标记39970524-6-1200距特异标记n.lcM。 3.葡萄无核基因特异标记DNA序列的相似性分析 与葡萄无核基因相连锁的特异标记39970524一5一564和39970524一6一1538的DNA片段回收、克隆、测序，特异标记39970524一5一564长度为564bp，39970524一6一1538长度为1538bp。这两个序列已登录GenBank，序列号分别为AY327513和AY327514。 在GenBanLk中，用BLAST对39970524一5一564和39970524一6一1538特异标一记的DNA序列进行了相似性分析，这2条DNA序列和GenBallk中其他生物的DNA序列同源性较小，为葡萄（巧tis vin沙ra）基因组所特有。39970524一5一564特异标记DNA序列中第38一59碱基间的22个碱基和拟南芥的2条编码MAP3K（受分裂素激活的蛋白激酶激酶激酶激酶）有关的核酸序列有相似性，39970524一5一 564特异标一记DNA序列中第38一59碱基间的22个碱基同时还和拟南芥第3条染色体的1条DNA序列有相似性。39970524一5一564特异标记这22个碱基所提供信息的实际意义有待进一步研究。39970524一6一1538特异标记的DNA序列和拟南芥的DNA序列没有相似性，但和水稻第6条染色体的DNA片段有23个碱基的相似性。 4.葡萄无核基因特异标记DNA序列开放阅读框架分析 在GenBank中，对 39970524一5一564和39970524一6一1538特异标记DNA序列的开放阅读框架分析表明，39970524一5一564在第405一536碱基间存在一个开放阅读框架，共包含1犯个碱基，可编码44个氨基酸。但在BLASTp中没有检测到与其同源的蛋白序列。 39970524一6一1538的序列最大的一个开放阅读框架在第1168一704碱基间，序列长度为465bp，可编码154个氨基酸。将这个开放阅读框架所编码的氨基酸序列，在BLASTP中和拟南芥的功能蛋白序列进行相似性比较，这一氨基酸序列和13个拟南芥的蛋白序列有部分相似性，相似位点在39一42个氨基酸间，E值在0.003一0.032间。这13个蛋白中，2个蛋白和胚胎发育后期丰富蛋白有关;1个蛋白和种子贮藏物及油脂转移蛋白酶抑制因子有关;还有一些分别和富脯氨酸、富亮氨酸及胞壁脂质体蛋白有关。由于39970524一6一1538的这个开放阅读框架所编码的序列和拟南芥蛋白序列的同源性偏小（E值0.003一0.033），所以这个开放阅读框架的实际功能还需进一步验证。 5.葡萄无核基因特异标记DNA序列酶切位点和Southem blot分析 用Wingene23 1 DNA分析软件对39970524一5一564和39970524一6一1538序列的限制性内切‘酶酶切位点进行了分析，分别有30和130个可识别6个碱基及其以上序列酶切位点的限制性内切酶在39970524一5一564和39970524一6一1 538的DNA序列上存在酶切位点;EcoRI和Hindlll在39970524一5一564特异标记上没有酶切位点，而EeoRI在39970524一6一1538第135个碱基处有一个酶切位点，Hindlll在39970524一6一1538上没有酶切位点。 用39970524一5一564DNA作探针对葡萄基因组DNA作Southem杂交，结果在无核亲本红光无核及无核基因供给者无核白和无核杂种上杂交带出现，而且呈单拷贝，而在有核亲本红地球及有核杂种上杂交带未出现，进一步证明了39970524更多还原

【Abstract】 Seedless grapes have high commercial value and seedlessness for table grapes and raisins is one of the principle objectives of Vitis vinifera grape breeders. Recent studies have shown that the seedless trait in grapes is controlled by one major and some minor genes. The position and mapping of the major gene for seedlessness was studied in this research using Red Globe, Flame Seedless, and Thompson Seedless, together with Fl progenies from Red Globe X Flame Seedless crosses. The sequences of marker linked to the major seedlessness gene were analyzed by BLAST in GenBank and the open reading frame (ORF) of these sequences was also found in GenBank. 39970524-5-564 DNA labeled with a DIG DNA Labeling and Detection Kit was used as a probe against Southern blot with genomic DNA from grapes. The main results were as follows.1. Identification of SCAR markers linked to grape seedlessness geneNine primers (including UBC-269 and GSLP1) were designed and synthesized based on DNA sequences of UBC-2694g4 and GSLP1569. The template DNA from Red Globe (seeded paternal parent) and Flame Seedless (seedless maternal parent) were screened using these primers. For Flame Seedless, GSLP1 yielded specific marker GSLPlseg; No.39970524-5 primer yielded specific marker 39970524-5-564; and No.6 primer yielded specific marker 39970524-6-1538 and 39970524-6-1200. GSLP1, No. 39970524-5, and No. 39970524-6 primers were used specifically to screen template DNA from the experimental plant materials: results showed that the specific markers GSLPlseg, 39970524-5-564, 39970524-6-1538 and 39970524-6-1200 were co-segregating with the major seedlessness gene. All these specific loci were also present in Thompson Seedless which was the initial donor of the seedlessness gene. It suggests that these SCAR markers are linked to a major grape seedlessness gene S .2. Position and mapping for grape seedlessness geneMarker order and map distance were estimated using the software ’QTXbl7’. This showed that GSLP1569, 39970524-5-564, 39970524-6-1538 and 39970524-6-1200 wereIVtightly linked to gene S. When P=0.01, confidence limits for map distance ranged from 0.2 to 9.9; standard errors of map distance were from 0.6 to 1.9; LOD for linkage were from 32.7 to 46.4. These markers and the gene S were found to be in the same group. The markers were either sides of gene S, covering 12.3 cM of the grape genome. The genetic distances between gene S and 39970524-5-564, GSLP1569, 39970524-6-1538 and 39970524-6-1200 were 0.6 cM, 1.2 cM, 4.9 cM and 11.1 cM respectively.3. Comparative analysis of the DNA sequences of specific markers linked to grape seedlessness geneDNA fragments of 39970524-5-564 and 39970524-6-1538 derived from Flame Seedless were retrieved from agarose gel and ligated into the pGEMB-T Easy Vector System, and then transformed into E. coli DH5-a. Sequencing of positive clones was carried out and the result showed that the 39970524-5-564 was 564bp and the 39970524-6-1538 was 1538bp. The sequences data had submitted to GenBank. The GenBank accession number for 39970524-5-564 is AY327513 and 39970524-6-1538 is AY327514.It has been suggested that the sequences of 39970524-5-564 and 39970524-6-1538 are unique to the grape (Vitis viniferd) genome. But, these sequences were extensively compared by BLAST in GenBank and a small part was found to be homologous. 22 base pairs from the sequence of 39970524-5-564 (from 38 to 59) matched two nucleic acid sequences coded MAP3P from Arabidopsis thaliana and also matched the sequence for Arabidopsis thaliana BAG T17H7 from Chromosome 1. Further study is needed to determine the significance of the 22 base pairs from the sequence of 39970524-5-564. The sequence of 39970524-6-1538 was not homologous with Arabidopsis thaliana, but it matched rice [Oryza sativa (Japonica cvs)] genomic DNA from chromosome 6.4. Finding ORF’s of the DNA sequences of specific markers linked to grape seedlessness geneORFs of the sequences of markers linked to gene S were found in GenBank. There was an ORF in the sequen更多还原