【作者】 袁录霞；

【导师】 罗正荣；

【作者基本信息】 华中农业大学 ， 果树学， 2012， 博士

【摘要】 柿属(Diospyros Linn.)是柿科中种类最多、分布最广、经济价值最大的属。我国是柿属植物的分布中心和原产中心之一,拥有丰富的种质资源。柿(Diospyros kaki Thunb.)是柿属植物作为果树栽培的代表种,分为完全甜柿、不完全甜柿、不完全涩柿和完全涩柿四种类型。我国大多数柿品种为完全涩柿。中国甜柿和完全雄性柿种质仅分布在湖北、河南和安徽三省交界的大别山区,因其甜涩性状为显性遗传等特点而在世界柿基因库和甜柿遗传改良中具有重要的地位,但其遗传多样性和起源与进化至今鲜见报道。另外,柿属植物的系统发育关系至今仍不清楚。本研究首次利用ISSR、IRAP和cpDNAPCR-RFLP分子标记对大别山区特产柿种质的遗传多样性和系统发育学进行系统分析,探索其起源与传播途径,并对柿属植物DNA条形码进行初探,以期为柿属植物的快速鉴定、系统发育学、起源与进化和遗传育种等研究提供方法和思路。主要研究结果如下：1.利用ISSR和IRAP对43份‘罗田甜柿’单株间的遗传差异进行分析,两种分子标记共扩增280条谱带,长度范围在250～2000bp,均具有较高的多态性比例(PiISSR=100%和PiiRAP=97.93%)和多态性信息含量(PICISSR=0.20和PICIRAP=0.26),是有效的分析方法(标记指数MIISSR=1.78和MIIRAP=1.14); ISSR和IRAP分析试材间的SM相似系数分别为0.104-0.985(平均0.795)和0.566-0.903(平均0.734),前者鉴定的遗传变异范围较后者更广,说明ISSI适用于筛选中国甜柿新种质而IRAP适宜研究其居群遗传多样性和起源与进化；UPGMA聚类图中,ISSR将试材分为两大类,IRAP将其分为三大类,整合两种方法的结果与ISSF类似,这说明‘罗田甜柿’单株间存在遗传差异,并且少数遗传差异较大的试材可能为隐存新种质,如11、36、40、41等,‘罗田甜柿’是遗传背景复杂的品种群而非遗传背景一致的单一品种。2.利用IRAP对大别山区甜柿的遗传多样性进行分析,共扩增101条谱带,长度范围在250-2000bp,IRAP具有高的多态性比例(Pi=100%)和多态性信息含量(PIC=0.35),标记指数(MI)为1.92,是有效的分析方法；大别山区甜柿在物种水平上的Nei’s基因多样度指数(H)为0.2992,Shannon信息指数(I)为0.4630,在居群水平上分别为0.2615和0.4156,说明大别山区甜柿存在较高的遗传多样性水平,并且物种水平略高于居群水平,存在丰富的遗传变异；七个居群中,八迪河居群的Nei’s基因多样度指数(H=0.3194)和Shannon信息指数(I=0.4864)均最高,推测其为大别山区甜柿的起源中心；甜柿居群总基因多样度(Ht)为0.2967,居群内基因多样度(Hs)为0.2615,占总基因多样度的比率为88.14%,说明大别山区甜柿的遗传多样性主要是由居群内的基因多样性引起的,遗传变异主要发生在居群内单株间；居群间存在强的基因流(Nm=3.7205),可能发生过突变、杂交或人为引种等事件；大别山区甜柿的传播途径是从罗田县的八迪河村起始,向西南方传播至罗田县的唐家山村、錾字石村和东冲畈村等地,向东北方传播至安徽省的马石村等地进而传播至河南省的大埠河村等地,向西方传播至麻城市的凌家塝村等地,最终在湖北、安徽和河南三省交界的大别山区进行广泛栽植和遗传进化。3.利用cpDNA PCR-RFLP和IRAP对完全雄性柿种质的分类学地位和系统发育学进行分析,前者多态性比例为75.0%,多态性信息含量为0.13,标记指数为0.26,后者多态性比例为97.61%,多态性信息含量为0.32,标记指数为2.49,说明cpDNA PCR-RFLP的鉴定力较低,适合种间或者利以上柿属植物的鉴定、系统发育学和遗传进化研究,而IRAP的鉴定力和灵敏度高,适合鉴定系统发育关系近的种内试材；cpDNA PCR-RFLP分析试材间的Jaccard相似系数范围为0.318-1.0(平均值为0.836),而IRAP分析的SM相似系数范围为0.394～0.912(平均值为0.671)；两利,方法获得的UPGMA聚类图表明完全雄性柿种质属于柿种,并且与中国柿品种系统发育关系紧密------雄株1号、雄株2号、雄株3号和雄株9号与‘罗田甜柿’和‘宝盖甜柿’等中国完全甜柿的系统发育关系较近,雄株8号与‘磨盘柿’和‘铜盆柿’等中国完全涩柿的关系很近,雄株10号则和日本柿品种关系近,这说明完全雄性柿种质间具有不同的遗传背景,其起源与进化途径不同；另外,君迁子与柿种内材料的遗传关系较浙江柿、老鸦柿和美洲柿近。4.利用matK和rbcL序列分析柿属植物DNA条形码及其适用性,通过比较分析筛选X+3.2和a f+724r引物组合分别对柿属植物natK和rbcL序列进行扩增,扩增效率均为84.47%,测序成功率分别为82.35%和100%；由于X+3.2扩增产物(1031bp)过长,推荐2.1a+5和a f+724r分别作为柿属植物DNA条形码matK和rbcL序列的通用PCR扩增引物;matK和rbcL序列长度分别为1031bp和744bp,相应序列已提交GenBank数据库(登录号GU471696～GU471727);功能分析可知前者为成熟激酶(matK)超家族蛋白的部分编码序列(编码约343个氨基酸),后者为核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)超家族蛋白的部分编码序列(编码约248个氨基酸),多数碱基位点高度保守；matK序列在所有试材间存在238个变异位点(23.92%)和114个简约信息位点(11.46%),约为rbcL)序列变异位点(12.84%)和简约信息位点(6.64%)的两倍,matK+rbcL序列组合居二者之间；matK检测最大的平均种内遗传距离(0.0009),因此当种间遗传距离大于0.001时确定为物种鉴定成功：当对110种柿属植物进行DNA条形码分析时,鉴定力大小依次为matK+rbcL (92.73%)>matK(91.82%)>rbcL(79.09%),因此建议matKK+rbcL片段组合作为柿属植物核心DNA条形码；利用matK+rbcL对110种164份试材(其中2份假乌木属试材作为外类群)构建NJ树、进行系统发育学分析,大多数柿属植物的分类学地位和系统发育关系得到鉴定,支持野毛柿和金枣柿分别作为新种,并且野毛柿与油柿、D. glandulosa聚类在一起,浙江柿和君迁子聚类在一起,说明他们之间的系统发育关系近,可能具有共同的祖先,但是一些未知试材[如Diospyros sp. SD-2009voucher Lowry et al.5783(MO)、Diospyros sp. K20616和Diospyros sp. K20613等]无法鉴定,柿属植物DNA条形码仍需深入研究。更多还原

【Abstract】 Diospyros Linn. is the biggest genus including the most species distributed most widely and of the largest economic value in Ebenaceae. China is one of the originating and distributing centers of Diospyros Linn. with abundant genetic resources. D. kaki Thunb. is the representative species cultivated as fruit and classified into PCNA, PVNA, PVA and PCA types. Most of the cultivars in China are PCA types. Chinese non-atringent persimmon and staminate germplasm are only distributed in Dabieshan Mountain around the junction of three provinces, Hubei, Henan and Anhui in central China. They play important roles in the research and industry of the oriental persimmon in the world because that their trait of natural astringency-loss of fruit is dominant to the non-PCNA types whereas Japanese PCNA cultivars’ is recessive. The genetic diversity and phylogenetics of the persimmon germplasm native to Dabieshan region are still unclear now. Here they were analyzed by ISSR, IRAP and cpDNA PCR-RFLP markers, meanwhile DNA barcoding for Diospyros Linn, was researched in the present study, and the main results were shown as follows:1. ISSR and IRAP markers were used to predict the genetic differences among43’Luotian-tianshi’collected from three major producing areas in China. A total number of280amplicons were observed ranging between250～2000bp. Both of them were effective methods because of the high Pi (ISSR=100%; IRAP=97.93%), PIC (ISSR=0.20; IRAP=0.26) and MI (ISSR=1.78; IRAP=1.14). The SM coefficient between ’Luotian-tianshi’were from ranged from0.104to0.985(average0.795) analyzed by ISSR and from0.566to0.903(average0.734) by IRAP, indicating that ISSR is suitable for selecting new germplasm while IRAP for studying the population genetic diversity, origin and evolution of Chinese non-atringent persimmon. On the basis of cluster analysis in the UPGMA dendrogram, two and three clusters were observed by ISSR/ISSR+IRAP and ISSR, indicating that ’Luotian-tianshi’ existed the genetic differences and some could be potential germplasms because of the significant genetic differences, such as Accll, Acc36, Acc40and Acc41. So ’Luotian-tianshi’ was a complex group including different cultivars rather than a single cultivar with the same genetic background. 2. IRAP was used to study the genetic diversity, origin and evolution of non-atringent persimmons native to Dabieshan Mountain. A total number of101amplicons were observed ranging between250-2000bp. IRAP was the effective method because of the high Pi (100%), PIC (0.35) and MI (1.92). The values of H and I of Chinese non-atringent persimmon were0.2992and0.4630in the level of individuals while0.2615and0.4156in the level of population, indicating that the high genetic diversity existed among non-atringent persimmons native to Dabieshan Mountain. Among the seven populations, Badihe population had the highest H (0.3194) and I (0.4864) so that it was supposed to be the origin center of non-atringent persimmons native to Dabieshan Mountain. The values of Hs was88.14%of Ht (0.2967), illustrating that the genetic diversity of non-atringent persimmons native to Dabieshan Mountain is mainly contributed by individuals in the population. The gene flow from Gst was strong (Nm=3.7205) among the populations, suggesting that mutation, crossover or artificial introduction might have happened in the evolution of non-atringent persimmons native to Dabieshan Mountain. Basing on the Nei’s similarity, UPGMA and NJ dendrograms, the probable spreading pathway of non-atringent persimmons native to Dabieshan Mountain was as follows:they origined from Badihe village, then migrated to Tangjiashan village, Zanzishi village, Dongchongfan village and so on in the southwest, to Mashicun village, Dabuhe village and so on in the northeast, to Lingjiabang village and so on in the west, and finally were cultivated widely and evolved in Dabieshan Mountain around the junction of three provinces, Hubei, Henan and Anhui in central China.3. The taxonomy and phylogenetic relationships of Chinese staminate germplasm and the other genotypes in Diospyros Linn. were analyzed by cpDNA PCR-RFLP and IRAP. cpDNA PCR-RFLP was suitable for the identification, phylogenetics and evolution of inter-species or above-species level because of the low values of Pi (75%), PIC (0.13) and MI (0.26), meanwhile IRAP was appropriate for that of intra-species level because of high values (Pi=97.61%; PIC=0.32; MI=2.49). The Jaccard similarity of samples analyzed by cpDNA PCR-RFLP ranged from0.318to1.0(average0.836) and the SM coefficient analyzed by IRAP was from0.394to0.912(average0.671). The UPGMA dendrograms constructed via the combination of the two methods indicated that Chinese staminate germplasm belonged to D. kaki Thunb. indeed and had different genetic background. Male strain No.l, Male strain No.2, Male strain No.3and Male strain No.9were closely related with Chinese PCNA persimmons, such as ’Luotian-tianshi’ and ’Baogai-tianshi’. Interestingly, Male strain No.8was closely related with Chinese PCA persimmons, such as ’Mopanshi’ and ’Tongpanshi’, and Male strain No.10was closely related with Japanese persimmon. In addition, the phylogenetic relationship between D. kaki Thunb. and D. lotus L. was closer than that between D. kaki Thunb. and D. glaucifolia Mete., D. rhombifolia Hemsl., D.virginiana L.4. matK and rbcL sequence fragments were selected to analyze DNA barcode for Diospyros Linn. and its applicability. X+3.2and a_f+724r primers combinations were screened to amplify mat K. and rbcL sequences of17persimmon samples used in the study. The amplification efficiency of mat K and rbcL were the same (84.47%) and the sequencing success rates were82.35%and100%, respectively. Due to the long X+3.2amplicons (1031bp), we recommended2.1a+5and a_f+724r as the universal PCR amplification primers for matK and rbcL sequence in Diospyros Linn, respectively. The length of matK and rbcL sequences, which were deposited in GenBank database (Accession nunbers were from GU471696to GU471727), were1031bp and744bp, respectively. The sequence analysis showed that matK sequence includes gene fragment encoding about343amino acids of mature kinase (matK) superfamily protein, and rbcL sequence is the partial gene fragment encoding about248amino acids of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) superfamily protein, most nucleotide sites in which were highly conserved. matK sequences in all samples had238variable sites (23.92%) and114parsimony informative sites (11.46%), which were approximately twice of those of rbcL sequences, and matK+rbcL sequences between them. The identification of Diospyros Linn, was regarded as successful when the inter-specific genetic distance is greater than0.001because that the largest average intra-specific genetic distance (0.0009) were detected by aligning the matK sequences. When110species in Diospyros Linn.were analyzed by DNA barcoding, the percent of discrimination success were matK+rbcL (92.73%)> matK (91.82%)> rbcL (79.09%), so that matK+rbcL was recommended to be the core DNA barcode for Diospyros Linn.. On the basis of the NJ tree of164samples from110species constructed by matK+rbcL, the taxonomic status and phylogenetic relationships of most species were identified. It was supported that D. kaki Thunb. var. sylvestris Makino and Jinzaoshi would be new species. In addition, D. kaki Thunb. var. sylvestris Makino, D. oleifera Cheng, and D. glandulosa, D. glaucifolia Metc. and D. lotus L. had the close phylogenetic relationships, suggesting that they might have the same ancient. But some unknown species, for example, Diospyros sp. SD-2009voucher Lowry et al.5783(MO), D. sp. K20616and D. sp. K20613, couldn’t be identified so far and DNA barcode for D. spp. needed to be further researched.更多还原