【作者】 高启明；

【导师】 李疆；

【作者基本信息】 新疆农业大学 ， 果树学， 2011， 博士

【摘要】 扁桃(Amygdalus communis L.)为世界著名干果及木本油料树种,具有较高的营养价值和药用价值。我国新疆是世界上32个扁桃产地之一。扁桃是一种配子体型自交不亲和果树,生产栽培中必须配置授粉品种或通过人工授粉等措施来保证高产稳产。配置授粉品种时,首先要考虑品种间的亲和性,而品种间的亲和性取决于两个品种的S-基因型,S-基因型相同则杂交不亲和,否则杂交亲和。本研究以新疆扁桃24个栽培品种为试材,以S-基因的分离鉴定和S-基因型的确定为目标,利用田间授粉试验、花柱离体培养以及S-等位基因特异PCR等方法,开展了分子生物学和杂交亲和力的研究,为扁桃丰产栽培和遗传改良提供重要的科学依据,为全面开展扁桃及其它果树的自交不亲和性研究提供理论依据和实践指导。主要研究结果如下：1田间授粉试验表明,在‘纸皮’、‘双果’、‘麻壳’、‘克西’和‘267’等五个新疆扁桃品种中,‘双果’表现出一定的自交亲和性,其自花结实率为29.05%,‘纸皮’自花结实率为12.76%,‘麻壳’、‘克西’和‘267’的自花坐果率低于5%,表现出典型的自交不亲和性；自然条件下,‘纸皮’坐果率最高,达到了37.53%,‘克西’的自然授粉坐果率最低,只有4.3%；人工异花授粉条件下,各品种坐果率均有显著提高,其中‘双果’ב纸皮’授粉组合的坐果率高达68.09%。用不同品种的花粉给‘纸皮’授粉时,均能获得较高的坐果率,其中以‘麻壳’花粉授粉的坐果率为最高,达63.43%,在用不同品种的花粉给‘267’进行授粉时,则获得了较低的坐果率,仅‘纸皮’花粉授粉的坐果率达到了36.25%,‘克西’花粉授粉的坐果率仅为16.18%。2花柱离体培养试验表明,‘纸皮’、‘双果’、‘麻壳’、‘克西’和‘267’等五个品种自交时,长出花粉管的花柱较少,比率低。‘双果’自交组合在1/3、1/2及花柱基部三个花柱切口处均有花粉管长出,表现出一定的自交亲和性；‘纸皮’和‘克西’两个自交组合在任何花柱切口处均没有花粉管长出,‘麻壳’和‘267’两个自交组合仅在花柱1/3切口处有花粉管长出,表现出典型的自交不亲和性。五个品种相互异交时,大部分杂交组合长出花粉管的花柱数均高于自交组合,这说明科学的配置主栽品种与授粉树,能有效的提高扁桃的坐果率。当母本相同,父本不同时,授粉亲和性表现差异很大,说明扁桃在异交时,对授粉品种有很强的选择性。因此生产中应选择适宜的授粉品种,同时注意主栽品种和授粉树的合理搭配。在选取授粉树和主栽品种时,还要考虑授粉树的经济价值。‘纸皮’、‘双果’、‘麻壳’三个品种可互作授粉树,相互异交亲和性高,作为授粉树也有较高的经济价值。3四种方法提取扁桃基因组DNA试验表明,改良CTAB法提取的DNA质量最好,OD26o/OD280比值接近于1.8,在电泳图上没有拖尾现象,说明提取的DNA较纯,RNA较少,多糖、蛋白、酚类等杂质污染也较少,DNA沉淀呈透明胶状,没有酚类氧化或其它色素造成的污染。其次是改良SDS法和CTAB法；而采用SDS法提取的DNA,纯度较差,OD260/OD280小于1.8,样品中存在蛋白质或酚污染。4不同的引物组合对参试扁桃品种的扩增系数和扩增成功率差异很大,其中引物组合AmyC5R+AS1Ⅱ扩增效果最好。5 AmyC5R+AS1Ⅱ、PruC2+PruC4R、PaF+PaR等8对引物组合的扩增结果表明,要对新疆扁桃品种资源的S-基因型进行全面、准确鉴定,除了采用不同的引物组合外,还需要根据新疆扁桃的S-基因序列进一步开发、设计新的专用引物。6利用引物组合AmyC5R+AS1Ⅱ对18个新疆扁桃品种的基因组DNA进行S-基因特异性PCR,共扩增出32条清晰可辨的片段,克隆测序后,通过DNAMAN多重序列比对,发现不同品种位于相同位置上的带,核酸序列相似性在99%以上,可以认为是同一基因片段,也就是说32条基因片段共包括11个核苷酸序列,大小分别为1688 bp (Sn)、1404 bp (S19)、1330 bp (S61)、1222 bp (S24)、1141 bp (S25)、1087 bp (517)、979 bp (S10)、785 bp (S63)、777 bp (S6)、690 bp (S50)、602 bp (S15)。7根据S-基因的鉴定结果及参试扁桃品种的杂交授粉试验结果,确定了10个扁桃品种的S-基因型,分别为‘阿买提’(S15S17S63)、‘红宝石’(S15S17)、‘莎舟’(S15S17)、‘鹰嘴’(S10S24)、‘矮丰’(Sn1S25)、‘扁石头’(Sn1S25)、‘桃扁桃’(S6n1)、‘开心果’(S6S61)、双果’(S63Sn1)、‘纸皮’(S50S61)。更多还原

【Abstract】 Almond is the famous dried fruits and oil trees in the world, which has a higher nutritional and medicinal value. Xinjiang in China is one of 32 almond origins all over the world. Almond is a gametophytic self-incompatible fruit trees, so it must configure pollination varieties or through artificial pollination to ensure high and stable yield in the aspects of production and cultivation. The first to consider is the affinity between varieties when configuring pollination varieties, and affinity between the two varieties depends on their S-genotype, hybrid incompatibility when 5-genotypes is the same, or hybridization affinity. This study take 24 almond cultivars in Xinjiang as materials, taking isolation and identification of the 5-gene and determine of S-genotypes as targets, using field pollination, style cultivated in vitro and 5-allele specific PCR methods, launched the research of molecular biology and the affinity of hybrid. The aims are to provide important scientific basis of almond cultivation and genetic improvement, provide theoretical basis and practical guidance for carrying out a comprehensive self-incompatibility in almond’s another fruit trees. The main results are as follows:1 Field pollination experiment showed that in the five Xinjiang almond varieties, which was ’Zhipi’,’Shuangguo’,’Make’,’Kexi’and’267’,’Shuangguo’showed some self-compatibility and its setting rate of self-flower was 29.05%, setting rate of self-flower of’Zhipi’was 12.76%, those whom of ’Make’,’Kexi’and’267’was less than 5%, which indicated typical self-incompatibility. Under natural conditions, the setting rate of ’Zhipi’was the highest, which reached to 37.53%, setting rate of natural pollination of’Kexi’was the lowest, only 4.3%; Under the conditions of artificial cross-pollination, the fruit setting rates of all varieties were significantly increased, the fruit setting rates of pollination combination of’Shuangguo’x’Zhipi’ was up to 68.09%. When using pollens of different cultivars pollinated to the’Zhipi’, all could obtain a higher setting rate, the fruit setting rate pollinated using pollen of’Make’was the highest, up to 63.43%; When using pollens of different cultivars pollinated to the’267’, which obtained lower setting rates, only the fruit setting rate pollinated using pollen of ’Zhipi’was up to 36.25%, those whom of’Kexi’was only 16.18%.2 In vitro culture of style showed, when the five cultivars of ’Zhipi’,’Shuangguo’,’Make’,’Kexi’ and’267’carried out self-cross, the number of style growing pollen tubes were less, the rate was low. Combination of self-cross of’Shuangguo’ had growed pollen tubes from three style incision, which was 1/3 location,1/2 location and base of style, respectively, which indicated some self-compatibility; Two combination of self-cross of’Zhipi’and’Kexi’had nothing pollen tubes growed from any style incision, another combination of self-cross of’Make’ and’267’had growed pollen tubes only from 1/3 location of style incision, which showed typical self-incompatibility.When the five varieties outcrossed each other, the number of style growing pollen tubes of the most crosses were higher than self-cross, which meaned configuration of major cultivars and pollination tree in the scientific manner could increased effectively the setting rate of almond. When the same male parent and female parent is not the same affinity performance between the different species varied greatly, which showed pollinated varieties had highly selective when the almond in outcrossing. Therefore, we should choose appropriate pollinated varieties in the production, meanwhile, noting that the cultivars and pollination species had a reasonable collocation. When selecting pollinated trees and cultivars, we also should consider economic value of pollinated trees. Three varieties of ’Zhipi’,’Shuangguo’,’Make’could use as pollinated trees each other, their outcrossing affinity were high, they acted as pollinated trees also had higher economic value.3 The test of almond genomic DNA extraction using four methods showed that the DNA quality extracted by improved CTAB method was the best, OD260/OD280 was close to 1.8, and there were no smearing phenomenon in the picture of electrophoresis, which indicated purity of DNA was good, RNA was less, pollution of polysaccharide, protein, phenol and other impurities was less. DNA precipitation was transparent jelly, and without pollution caused by phenolic oxidation or other pigments. The next was CTAB method and improved SDS method; The purity of DNA extracted by using SDS method was very poor, OD260/OD280 was less than 1.8, and there existed pollution of protein or phenol.4 Amplification coefficient and amplification successful rates of different primer combinations to tested almond cultivars vary widely, amplification effect of primer combination of AmyC5R+AS1Ⅱwas the best.5 The amplification results of eight primer combinations showed if we wanted to carry out comprehensive and accurate identification of the S-genotypes from Xinjiang almond varieties, in addition to using different primer combinations, but also need to further develop and design new specific primers according to the 5-gene sequence of Xinjiang almond.6 Using primer combination AmyC5R+AS1Ⅱcarried out S-gene specific PCR to 18 genomic DNA of Xinjiang almond cultivars,32 clear fragments were amplified. After cloning and sequencing, the software of DNAMAN analysis showed the bands located at identical position of different cultivars, their DNA sequence similarity was above 99%. So we could think they were the same gene fragments, that meaned 32 gene fragments total included 11 nucleotide sequence, whose size involved 1688 bp (Snt),1404 bp (S,9),1330 bp (S61),1222 bp (S24),1141 bp (S25),1087 bp (S17),979 bp (S,o),785 bp (S63),777 bp (S6), 690 bp (S50),602 bp (5/5), respectively.7 According to the identification results of S-gene and cross pollination test, we determined S-genotype of 10 almond cultivars, which was ’Amaiti’ (S15S17S63),’Hongbaoshi’(S15S17),’Shazhou’ (S15S17),’Yingzui’(S10S24),’Aifeng’(Sn1S25),’Bianshitou’(Sn1S25),’Taobiantao’(S6Sn1),’Kaixinguo’(S6S61), ’Shuangguo’(S63Sn1) and ’Zhipi’(S50S61).更多还原