【作者】 陈晓流；

【导师】 束怀瑞； 陈学森；

【作者基本信息】 山东农业大学 ， 果树学， 2003， 博士

【摘要】 欧洲甜樱桃（P.avium L）．是重点发展的早熟果树，其自交不亲和性是影响生产的重要因素。现有主栽品种多数不知道S基因型。确定S基因型，对配置授粉树、选配杂交亲本、探讨自交不亲和的分子机制及选育自交亲和品种都非常重要；中国樱桃（P.pseudocerasus L.）具有自交亲和、果实发育期短、部分类型树体矮化等特殊的重要资源性状，对培育自交亲和、早熟、矮化的优良品种有重要价值。本研究主要利用S基因特异PCR、田间杂交座果率调查、花粉管生长观察和等电聚焦凝胶电泳等手段，克隆了甜樱桃品种的S基因，确定了山东省主栽品种的S基因型，探讨了甜樱桃自交不亲和及中国樱桃自交亲和的机制，建立了确定S基因型的技术体系，可直接用叶片DNA，根据S基因特异PCR片段大小来确定S基因型。主要结论如下： 1．根据蔷薇科S基因核酸序列的高度保守区C2和RC4区的序列设计引物PruC2和PruC4R，对叶片基因组DNA做S基因特异PCR扩增，克隆测序各S基因的扩增片段并在genebank上搜索。结果证明，在电泳中显示的相同大小的PCR片段，是同一种S基因。各S基因扩增片段大小分别是：S₁为676-677bp，S₃为761-762bp，S₄为943-944bp，S₆为456bp。首次阐明部分S基因扩增片段的核酸序列及大小。 2．建立了一套技术体系来鉴定基因型未知品种的S基因型。用PruC2和PruC4R专一引物对叶片DNA做PCR扩增，根据扩增片段的大小来确定该片段是那种S基因，确定该品种的S基因型。依据该方法，对现有主栽品种确定了S基因型。红灯、早红宝石、红艳、先锋是S₁S₃，抉择、那翁、红丰、外引7号为S₃S₄，大紫是S₁S₆，长把红是S₁S₄，养老是S₃S₆，斯太拉是S₃S₄。 3．田间杂交座果率表明，S基因型相同的品种间杂交，表现杂交不亲和，不能互为授粉树。不同S基因型的品种间杂交，表现杂交亲和，但亲和花粉所携带的S基因的种类和数量不是影响座果率高低的因素。 4．通过分析现有品种的S基因型，发现各S基因出现的频率不同，以S₃频率最高，为42.31％；S₁和S₄中等，分别为23.08％、26.92％，S₆最小，为7.69％；S₂和S₅没有出现。因此，把S₃称为高频S基因，S₁和S₄为中频S基因，S₆为较低频S基因，S₂和S₅为低频S基因。 5．樱桃参试品种用RAPD聚类分析方法，分成8类。同一类中的品种亲源关系较近，都共有一个相同的S基因，甚至S基因型相同，是S基因的遗传造成。但遗传距离较远的品种，多共有高频的S₃基因，不只是遗传因素所致，还可能与S₃基因在育种中有选择优势有关。 6．甜樱桃品种抉择、早红宝石、养老等自花授粉及杂交授粉表明，授粉后24-30h，亲和及不亲和花粉管生长没有差异，都能萌发并到达花柱上、中部；授粉后48-77h，亲和及不亲和花粉管生长表现明显差异，亲和花粉管不断延伸，进入子房。而不亲和花粉管进入花柱中、下部，在中、下部生长受抑制，停止生长，有的表现顶端膨大、 陈晓流：樱桃S基因型及自交（不）亲和机制研究破裂，但多数形态正常。 7．甜樱桃自交不亲和的机制是石基因在花柱表达产生 S－RNase的量很丰富，使卜RNase能够抑制自体花粉管生长，使不亲和花粉管在花柱中、下部生长受抑制而停止生长，不能进人子房完成授粉受精的过程。 8．首次阐明中国樱桃自交亲和的机制是：花柱中可溶性蛋白含量明显比SI的甜樱桃的低，RT－PCR显示S基因在花柱中表达，但IEF－PAGE上分离到的S—RNase的量很少，即S基因表达产生的S－RNase量太少，使S－RNase不能抑制自体花粉管的生长，导致自交亲和，自交座果率达25％。 9．RAPD聚类分析和S基因的核酸序列比较都表明，中国樱桃和酣樱桃的遗传距离很远，把它们划归两个类。但这两个种的部分品种间杂交，表现出种间杂交亲和。 10．对叶片基因组 DNA做 S基因特异扩增，甜樱桃各品种得到两条不同的扩增带，说明基因组中有两种不同的s基因；中国樱桃只得到一条扩增带，说明基因组中有一种S基因。 11．把中国樱桃的 S基因型定为 S；m，SM表示花柱部分的突变使 S基因在花柱中不表达或表达水平低。 12．中国樱桃和甜樱桃基因组中 S基因比花柱中相应的叩NA片段长，表明樱桃 S基因中有内含子。更多还原

【Abstract】 Sweet cherry (P. avium L) as a kind of fruit tree is planted in great area and its fruit is early ripe. The self-incompatibility of sweet cherry is the important factor affecting the production. Most of varieties are unknown the S-genotypes, so identification of the S-genotypes is very important for choosing the pollinators, breeding parents, studying the molecular mechanism of SI and breeding self-compatible varieties. Chinese cherry (P. pseudocerasus L.) has very important resource properties, such as self-compatibility, short development period of fruit and compact type which are all of significant value for breeding self-compatible excellent varieties. Our study mainly utilized the methods such as S gene specific PCR techenique, identification of fruit setting percentage in field and isoelectric focusing to clone the S genes of sweet cherry of which S genotypes are unknown, to identify the variety’s S genotype, to study the mechanism of sweet cherry’s self-incompatibility and Chinese cherry’s self-compatibility. As the result, by using leaf genomic DNA we set up the technique system which could identify the S genotypes based on the length of amplified bands in S gene specific PCR. The main results are as follows:1. We designed a pair of specific primers PruC2 and PruC4R based on the very conserved nucleic acid region C2 and RC4 of rosaceous S gene to do S gene specific PCR on leaf genomic DNA, cloned and sequenced the amplified bands of S genes and compared the sequences on GeneBank. It was proved that the same length of amplified bands was the same kind of S gene. The lengths of S gene amplified bands were that S1 was 676-677bp, S3was 761-762bp, S4was 943-944bp, S6was 456bp respectively. The sequences and lengths of S gene amplified bands were made clear for the first time.2.We set up the technique system to identify the S genotypes of unknown genotype’s varieties by using the PruC2 and PruC4R specific primers to do specific PCR on genomic DNA based on the lengths of amplified bands. We identified the S genotypes of mainly planted varieties, for example ’Hongdeng’ ’Early ruby’ ’Hongyan’ and ’Van’ all were S1S3 ’Jueze’ ’Napoleon’ ’Hongfeng’ and ’Waiyin 7’ all were S3S4; ’Black tartarin’ was S1S6; ’Changbahong ’ was S1S4 and ’Elton’ was S3S6.3. The fruit setting percentage showed that the varieties with the same kind of S genotype could not be used as pollinators for each other because the hybridizing between them cross-incompatible; the varieties of different S genotypes cross-compatible; the kinds of S genes in compatible pollens didn’t affect the fruit setting percentage.4. By analyzing the S genotypes of planted varieties we found that the occuringfrequency of S genes were different. It was showed that the frequency of S3 was the highest for 42.31%; the frequencies of S1 and S4 were the secondary for 23.08% and 26.92%; the frequency of S6 was lower for 7.69%; S2 and S5 didn’t occur0 For this reason we called S3 the high frequency S gene, S1 and S4 the middle frequency S gene, S6 lower frequency S gene, S2 and S5 the low frequency S gene.5. We divided the tested cherry varieties into 8 types by using RAPD analysis. The varieties in the same kind type had the same kind of S gene or the S genotype because of the heredity of S gene. But most of the varieties in distant relationship each other had the same high frequency S3 gene not only due to genetic reason likely due to S3 having the advantage of selection in breeding.6.The self-pollination and cross-pollination tests on the sweet varieties, for example, ’Jueze’, ’Early ruby’ ’Elton’ et al. showed that the growth of compatible and incompatible pollen tubes had no difference during 24-3 Oh after pollination, and they all could germinate and arrive at the top and middle part of style. But the growth of them had obvious difference during 48-77h after pollination, it was observed that the compatible pollen tubes could arrive at ovary but the incompatible pollen tubes could arrive at the middle and base part of style and the g更多还原