【作者】 黄绍西；

【导师】 张绍铃；

【作者基本信息】 南京农业大学 ， 发育生物学， 2009， 博士

【摘要】 本研究主要以中国李(Prunus salicina)和中国樱桃(P. pseudocerasus)的‘南京垂丝’及其自交后代为材料,首先鉴定了其自交不亲和性或自交亲和性,进而鉴定花粉和雌蕊S特异性识别决定因子,以及部分品种的S基因型,在此基础上开展了其自交不亲和性或自交亲和性机制的研究,取得主要结果如下：一、首先对中国李花粉悬浮剂进行筛选,进而调查了30个品种的花粉量(粒/花朵)和花粉萌发率,最后采用软件SAS8.2对实验数据进行了平均距离法聚类分析和判别分析,并计算花粉量、萌发率分级临界值。结果表明,中国李具有可育的雄配子体(花粉粒),10%葡萄糖溶液是中国李比较合适的花粉悬浮剂。从试验品种间的花朵花粉量来看,最高为’Redgold’(37,169粒/花朵),最低为‘三华李2号’(4,720粒/花朵),可分为4类：Ⅰ类品种占16.7%,花粉量≥32362粒/花朵；Ⅱ类品种占30.0%,花粉量处于32,361～22,556粒/花朵；Ⅲ类品种占46.7%,花粉量处于22,555～9,559粒/花朵；Ⅳ类品种占6.6%,花粉量≤9,558粒/花朵。从品种间的花粉萌发率来看,最高为品系‘82-1-109’(89%),最低为品系‘84-21-62’(9%),可分为4类：Ⅰ类品种占13.3%,花粉萌发率≥76%；Ⅱ类品种占33.3%,花粉萌发率处于75～52%；Ⅲ类品种占36.7%,花粉萌发率处于51～25%；Ⅳ类品种占16.7%,花粉萌发率≤24%。二、对中国李32个品种进行了自然自交授粉,5个品种进行人工自交授粉,16个组合进行人工杂交授粉,采取花柱固定、苯胺蓝荧光染色后用荧光显微镜观察了人工授粉72 h后花柱内花粉管生长情况,并在人工授粉后不同时期进行了坐果率统计。实验结果表明,所有品种经人工自交授粉与自然自交授粉后,在果实采收期的坐果率均为0,表现出自交不亲和性；“盖县大李×芙蓉李”的杂交组合,在果实采收期的坐果率为0,表现为杂交不亲和性；“盖县大李×黄皮李”和“金砂李×油柰”的杂交组合,在果实采收期的坐果率分别为3.15%和4.67%,表现为弱的杂交不亲和性；其余13个组合,在果实采收期的坐果率处于7.76-22.90%,表现为杂交亲和性；授粉72 h后,杂交不亲和组合的花粉管与自交的一样,在花柱中上部生长受到抑制,而杂交亲和组合的花粉管可生长到花柱基部。以上实验结果表明中国李属于典型的配子体型自交不亲和性果树。人工授粉后不同时期统计坐果率,结果表明,中国李由于自交不亲和性引起的落果,可以持续到授粉后40天左右。三、采用李属雌蕊S-RNase基因特异性保守引物进行PCR和RT-PCR,并对扩增产物进行克隆、测序和序列比对,在中国的10个传统李品种中获得了8个符合雌蕊S特异性识别决定因子必要条件的新基因,分别被命名为中国李S-RNase-7～S-RNase-14(基因库登录号分别为AY71290、AY902455、AY996051、DQ003310、DQ512908、DQ512909、DQ512910和EF177345),同时鉴定了中国李25个品种的S-RNases基因型。在中国的传统李品种中鉴定出了更丰富的S-RNase基因多态性。在授粉后,完全亲和性杂交组合的坐果率不一定比半亲和性杂交组合的高。中国李S-RNase-h在本研究样本中表现最高的基因频率,预示着该基因可能与某些优良农艺性状有连锁关系。四、在中国李上鉴定了8个与李属其它物种SFB基因的序列相似性处于76%(中国李的SFB-c与杏的SFB-2)到87%(中国李的SFB-7与梅的SFB-1)之间,并被命名为PsSFB (Prunus salicina S-haplotype-specific F-box gene)(基因库登录号分别是DQ849084、DQ849085、DQ849086、DQ849087、DQ849088、DQ849089和DQ849090)。它们具有与李属其它物种SFB基因相似的结构特征,表现高度的s单元型特异多态性,在花粉中特异性表达。3个包含PsSFBs和S-RNases基因的基因组片段也被成功扩增,它们的物理距离约为410 bp-2,800 bp。这些研究结果表明PsSFBs基因就是中国李GSI的花粉S特异性识别决定因子。五、以表现自交亲和性的中国樱桃(Prunus pseudocerasus)的‘南京垂丝’及其157个自交后代为材料,开展了其S基因型的鉴定,新发现了中国樱桃4个S单元型(Prunus pseudocerasus S-haplotypes, PpsS-haplotypes)。‘南京垂丝’的S基因型被鉴定为S-1/S-3’/S-5/S-7,每个PpsS-haplotype至少含有中国樱桃1个S-RNase基因(P.pseudocerasus S-RNase, PpsS-RNase)和中国樱桃1个SFB基因(P. pseudocerasus S-haplotype-specific F-box, PpsSFB)。这些PpsS-RNases和PpsSFBs分别在花柱和花粉中特异性表达,且与李属植物的S-RNases和SFBs具有相似的结构特征,满足作为S特异性识别决定因子的必要条件,并遵守四体遗传规律。c-DNAs全长序列分析表明,PpsS-haplotype-3’的SFB不能扩增成功,而PpsS-haplotype-3’的S-RNase以及其余PpsS-haplotype都未发现异常。自交授粉后,S基因型为S-7/S-1,S-7/S-5和S-1/S-5的二倍体花粉表现自交亲和性,而S基因型为S-3’/S-7,S-3’/S-1和S-3’/S-5的二倍体花粉则表现自交不亲和性。这些研究结果表明,PpsS-haplotype-1、-5和-7具有正常的S特异性识别,它们之中的任何2个S-haplotype之间的‘竞争性效应’导致了中国樱桃‘南京垂丝’表现自交亲和性。更多还原

【Abstract】 Using Chinese traditional plum cultivar, Chinese cherry cultivar ’Nanjing Chuisi’ and its selfed progenies, we firstly identified their self-(in)compatibility, and then identified their S determinants as well as their S genotypes to studied on their mechanism of self-(in)compatibility. The results are shared as below:1. Experiments had been done to select appropriate method for suspending Chinese plum pollen grains. Pollen grain numbers and pollen germination ratio of thirty cultivars or strains had been investigated. Average distance cluster and discrimination analysis had been made on pollen grain numbers and pollen germination ratio of thirty cultivars or strains using software SAS8.2. The limit value of every level of pollen grain numbers and pollen germination ratio had been calculated. It was turned out that 10% glucose solution was appropriate for suspending Chinese plum pollen grains, and that Chinese plum had fertile male gametes. According to pollen grain numbers, thirty cultivars or strains had been classified into four obvious clusters, i.e.,Ⅰbeing more than 32,361 per flower,Ⅱranging from 22,556 to 32,361 per flower,Ⅲranging from 9,559 to 22,555 per flower,Ⅳbeing less than 9,559 per flower. According to pollen germination ratio, thirty cultivars or strains had been classified into four obvious clusters, i.e.,Ⅰbeing more than 75%,Ⅱranging from 75% to 52%,Ⅲranging from 51% to 25%,Ⅳbeing less than 25%.2. Natural self pollination have been done on 32 Chinese plum cultivars which fruit sets were caculated just before havest, artificial self pollination had been done on 5 cultivars, artificial cross pollination had been conducted on 16 cross combinations, pollen tube growthes in the style at 72 h after artificial self/cross pollination had been observed, fruit set were caculated at different time after artificial self/cross pollination. It was turned out that the fruit sets of all cultivars at havest after natural/artifcial self pollination were O demonstrating Chinese plum exhibiting self-incompatibility, and that fruit set of "Gaixiandali×Furongli" was O at havest after artificial cross pollination showing Chinese plum having cross-incompatible combination, and that "Gaixiandali×Huangpili" and "Jinshali×Younaili" which fruit sets were 3.15% and 4.67% respectifely at havest after artificial cross pollination showing Chinese plum having week-cross-incompatible combinations, and that there were 13 cross combinations which fruit sets were 7.76-22.90% at havest after artificial cross pollination showing Chinese plum having cross-compatible combinations, and that at 72 h after pollination, the pollen tubes could growth at the base of the styles in cross-compatible combinations, but could not in selfing and in cross-incompatible combination. All of above results demonstrated that Chinese plum exhibited classic gametophytic self-incompatibility. Furthermore, fruits had fallen till 40 d after pollination because of self-incompatibility in Chinese plum.3. Eight new genes, which mach necessary conditions for pistil S-determinant and were named as S-RNase-7～S-RNase-14 (Accession No. is AY71290, AY902455, AY996051, DQ003310, DQ512908, DQ512909, DQ512910 and EF177345 respectively), were identified in 10 Chinese traditional plum cultivars by PCR using S-RNase gene specific consensus primers. Furthrumore, S-RNase genotypes of 25 Chinese plum cultivars in all were determined. Abudent S-RNases diversity was identified in Chinese traditional plum cultivars. Fruit sets of fully compatible cross-combination were not always higher than that of semi-compatible cross-combinations. S-RNase-h had the highest gene frequency in sample of the present study implying its lingkage with some excellent agricultural traits.4. We determined eight novel sequences, which showed sequence high similarity to SFB genes of other Prunus species from 76% (between SFB-c from P. salicina and SFB-2 from P. armeniaca) to 87% (between SFB-7 from P. salicina and SFB-1 from P. mume), and named these sequences PsSFBs (P. salicina S-haplotype-specific F-box genes) (Accession No. is DQ849084, DQ849085, DQ849086, DQ849087, DQ849088, DQ849089 and DQ849090 respectively). The gene structure and the characteristic domains in deduced amino acid sequences of the PsSFB genes were similar to those of SFB genes from other Prunus. The eight identified PsSFB sequences showed S-haplotype-specific polymorphism with 74 to 83% amino acid sequence identity among them, and were exclusively expressed in the pollen. Three sequences from 410 bp to 2,800 bp of the intergenic region between the PsSFB sequences and the S-RNase alleles were obtained indicating their physical linkage. These results suggest that the PsSFB alleles are the pollen S-determinants of GSI in Chinese plum.5. Four PpsS-haplotypes (Prunus pseudocerasus S-haplotypes) comprising of at least two genes i.e., PpsS-RNase (P. pseudocerasus S-RNase) and PpsSFB (P. pseudocerasus S-haplotype-specific F-box), have been isolated in self-compatible tetraploid P. pseudocerasus CV. Nanjing Chuisi and its selfed progenies. S-genotype of ’Nanjing Chuisi’ was determined as S-1/S-3’/S-5/S-7. These PpsS-RNases and PpsSFBs, respectively, expressed exclusively in style and in pollen, share the typical structure features with S-RNases and SFBs from other Prunus species, match with the necessary conditions for S-determinants, and comply with tetrosomic inheritence. SFB in PpsS-haplotype-3’was not amplified by PCR, while no mutations leading to dysfunction were found in S-RNase-3 and other S-haplotype based on their full-length c-DNAs. After self pollinantion, diploid pollen grains with S-genotypes S-7/S-1, S-7/S-5 and S-1/S-5 exhibited self-compatibility, while pollen grains with S-genotypes S-3’/S-7, S-3’/S-1 and S-3’/S-5 exhibited self-incompatibility. These results suggest that the PpsS-haplotype-1, PpsS-haplotype-5 and PpsS-haplotype-7 are functional, and that competitive interaction between two of them confer self-compatibility on cultivar ’Nanjing Chuisi’.更多还原