海南荔枝种质资源考察收集、鉴定评价及分析
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摘要
荔枝(Litchi chinensis Sonn.)原产我国,属无患子科,俗有“岭南果王”之称,已有2000多年的栽培历史。自上世纪九十年代初在广东、广西、福建、海南等省迅速发展,成为我国重要的热带南亚热带代表性水果。我国是荔枝的主产国,面积与产量均居世界第一,海南是荔枝的起源地之一,也是我国早熟荔枝的主要产地,拥有丰富的种质资源,但研究相对滞后。本研究主要开展了海南荔枝种质资源的考察、收集和评价,荔枝种质资源遗传多样性分析,指纹图谱的构建,荔枝叶片、果皮、种子功能成分评价等研究,为保护和利用这些宝贵资源提供依据,获得主要结果如下：
1、在对海南省野生、半野生荔枝调查的基础上,摸清了海南野生、半野生荔枝生长、分布现状,绘制了野生、半野生分布图；抢救性收集到了120余份荔枝种质资源。
2、研究制定了荔枝种质资源描述规范、质量控制规范和数据标准,构建了荔枝种质资源评价技术体系,并依据规范对资源进行了评价,获得10000多项科学数据,建立了海南荔枝种质资源网络共享数据库,并筛选出优异种质30多份。
3、利用ISSR和AFLP分子标记技术分别对96份和60份荔枝种质资源进行了遗传多样性分析。13条ISSR引物共计扩增得到180条谱带,平均每条引物13.8条,多态性条带比率为90.56%；3对AFLP引物扩增得到1218条谱带,多态性条带达1125条,比例为92.36%,ISSR和AFLP遗传相似系数分别分布在0.478-0.874和0.616-0.846之间。两种分子标记聚类结果表明：野生荔枝种质覆盖了各个类群,半野生种质存在少数几个类群,而栽培种质几乎集中在同一类群,两种分子标记的聚类结果表现出较高的一致性。
4、研究表明,海南荔枝种质资源之间遗传基础虽然比较宽,但大部分荔枝材料之间亲缘关系比较接近,遗传基础比较狭窄,其相似系数成偏正态分布,遗传差异比较大的主要集中在野生类型之间。栽培荔枝(半野生荔枝)与野生荔枝明显区分开,而且采自同一林区的野生荔枝的亲缘关系较不同林区的近,有很强的生态区域特点,霸王岭的野生荔枝被分成不同的类群,说明霸王岭的野生荔枝遗传多样性较其它林区的野生荔枝丰富,是野生荔枝中的核心地区。
5、利用SSR和AFLP分子标记技术构建了11个荔枝主要栽培品种的SSR指纹图谱。结果表明,两种分子标记技术均可以将11个品种完全区分,并能赋予每个供试材料唯一的条形码,该结果可为品种鉴别、新品种审定及品种保护提供可靠的DNA分子证据。
6、采用响应面法优化了超声辅助技术提取荔枝叶片、果皮和果核中的总酚、总黄酮和多糖类物质,分析了这三类物质的提取工艺条件和提取工艺,建立了数学模型,为荔枝总酚、总黄酮和多糖的提取制备提供理论参考和依据。结果表明利用所建立的新方法,其提取率较传统方法显著提高。
7、进行了紫娘喜、丁香和南岛无核荔枝果皮和果核的酚类物质含量的分析,经测定荔枝核中主要发现6种酚类物质,分别为原花青素A1、A2、根皮苷、柽柳黄素3-芸香糖苷、儿茶素与芦丁,除根皮苷以外,其他均为黄酮类物质。阐明3个品种荔枝叶的40%醇沉多糖包括阿拉伯糖、鼠李糖、半乳糖和葡萄糖,而80%醇沉多糖则仅包括阿拉伯糖、鼠李糖、半乳糖；对荔枝酚与荔枝多糖的DPPH自由基清除活性进行了评价,就酚类物质而言,三个荔枝品种叶的酚类物质活性均高于果皮和种子。就多糖而言,各个品种的活性差异显著,三个品种不同器官活性有明显差异,紫娘喜品种果皮的活性高于果核与叶子,而丁香品种叶子多糖的活性最高。
Litchi (Litchi chinensis Sonn., Sapindaceae), a tropical and subtropical fruit originated from China, has become one of the most important fruit crops in china and developed rapidly in Guangdong, Guangxi Fujian and Hainan provinces from1990. China is the main country of litchi production, which area and yield are both the first in the world. In this study, investigation, collection、evaluation、genetic diversity analysis、fingerprint construction and functional composition analysis of Hainan Litchi germplasm resources was carried out. The main results were as follows:
1. The growth and distribution status of wild, semi-wild litchi in Hainan was cleared and distribution map was drawed based on wild, semi-wild litchi survey. More than120copies of Litchi germplasm resources were collected and a series of norms, rules and standards were made.
2. The descriptors-standard、data quality control of descriptors and the standar of data for Litchi were made.30of excellent germplasms were selected on the basis of evaluation, classification of litchi germplasm resources according to the norms.
3. ISSR and AFLP molecular marker technique were used respectively for genetic diversity analysis of96and60litchi germplasm resources.180polymorphism bands were obtained using13ISSR primers. The average number of band was13.8per primer and the polymorphic ratio was90.56%.1218straps were amplified using3pairs of AFLP primers and1125straps were polymorphic(92.36%). Genetic similarity coefficients of ISSR and AFLP were0.478～0.874and0.616～0.846, respectively. Results from ISSR and AFLP markers showed that wild litchi germplasm resources covered all the groups, semi-wild germplasm resources had a few groups while cultivated germplasm almost belonged to the same group. The clustering results of the two molecular markers showed high consistency.
4. According to the results of genetic similarity coefficient, the genetic basis of Hainan litchi germplasm was relatively wide while the majority of the litchi had closely phylogenetic relationships and the genetic differences was relatively narrow. The similarity coefficient showed partial normal distribution and wide genetic differences mainly existed in wild type. Cultivar litchi (semi-wild) was obviously different from wild litchi. Moreover, the wild litchi from the same forest had closely genetic relationships and strong eco-regional characteristics. Wild litchi from Bawangling was divided into different groups, indicating that genetic diversity of wild litchi from Bawangling is abundant than other forest and Bawangling is the core area of wild litchi.
5. SSR finger printing of litchi germplasm was constructed using SSR and AFLP molecular marker technology.11germplasm resources could be fully separated by the two markers methods and each tested material had the only bar code. These results might provide useful reliable DNA molecular evidence for identification of species, registration of new varieties, and protection of cultivar.
6. Total phenols, flavonoids and polysaccharides were extracted from litchi leaves, peel and stone by using the response surface method to optimize the ultrasonic aided technology. Three types of substance's extraction condition and extraction process is analyzed and established a mathematical model which provide theoretical reference and basis for total phenolics, flavonoids and polysaccharide extraction of Litchi. Compared to traditional methods, the new method could significantly increased extraction rate.
7. Phenols contents in pericarp and stones were analyzed from Ziniangxi, Dingxiang and Nandaowuhe litchi. Six types of phenolic compounds i.e procyanidine A1and A2, phloridzin, tamarixetin-3-rutinoside, catechin and rutin, were detected in stones. All of them are flavonoids except phloridzin. Polysaccharide in litchi leaves precipitated by40%alcohol included arabinose, rhamnose, galactose and glucose, while polysaccharide precipitated by80%alcohol did not contain glucose. DPPH radical scavenging activities of phenols and polysaccharides were evaluated. Higher DPPH radical scavenging activities of phenols were detected in litchi leaves. The DPPH radical scavenging activities of polysaccharides were significantly different in various species. Activities of polysaccharides in pericarp was higher than that of in stone and leaves of Ziniangxi, and the highest polysaccharides DPPH radical scavenging activities was observed in leaves of Dingxiang.
1、在对海南省野生、半野生荔枝调查的基础上,摸清了海南野生、半野生荔枝生长、分布现状,绘制了野生、半野生分布图；抢救性收集到了120余份荔枝种质资源。
2、研究制定了荔枝种质资源描述规范、质量控制规范和数据标准,构建了荔枝种质资源评价技术体系,并依据规范对资源进行了评价,获得10000多项科学数据,建立了海南荔枝种质资源网络共享数据库,并筛选出优异种质30多份。
3、利用ISSR和AFLP分子标记技术分别对96份和60份荔枝种质资源进行了遗传多样性分析。13条ISSR引物共计扩增得到180条谱带,平均每条引物13.8条,多态性条带比率为90.56%；3对AFLP引物扩增得到1218条谱带,多态性条带达1125条,比例为92.36%,ISSR和AFLP遗传相似系数分别分布在0.478-0.874和0.616-0.846之间。两种分子标记聚类结果表明：野生荔枝种质覆盖了各个类群,半野生种质存在少数几个类群,而栽培种质几乎集中在同一类群,两种分子标记的聚类结果表现出较高的一致性。
4、研究表明,海南荔枝种质资源之间遗传基础虽然比较宽,但大部分荔枝材料之间亲缘关系比较接近,遗传基础比较狭窄,其相似系数成偏正态分布,遗传差异比较大的主要集中在野生类型之间。栽培荔枝(半野生荔枝)与野生荔枝明显区分开,而且采自同一林区的野生荔枝的亲缘关系较不同林区的近,有很强的生态区域特点,霸王岭的野生荔枝被分成不同的类群,说明霸王岭的野生荔枝遗传多样性较其它林区的野生荔枝丰富,是野生荔枝中的核心地区。
5、利用SSR和AFLP分子标记技术构建了11个荔枝主要栽培品种的SSR指纹图谱。结果表明,两种分子标记技术均可以将11个品种完全区分,并能赋予每个供试材料唯一的条形码,该结果可为品种鉴别、新品种审定及品种保护提供可靠的DNA分子证据。
6、采用响应面法优化了超声辅助技术提取荔枝叶片、果皮和果核中的总酚、总黄酮和多糖类物质,分析了这三类物质的提取工艺条件和提取工艺,建立了数学模型,为荔枝总酚、总黄酮和多糖的提取制备提供理论参考和依据。结果表明利用所建立的新方法,其提取率较传统方法显著提高。
7、进行了紫娘喜、丁香和南岛无核荔枝果皮和果核的酚类物质含量的分析,经测定荔枝核中主要发现6种酚类物质,分别为原花青素A1、A2、根皮苷、柽柳黄素3-芸香糖苷、儿茶素与芦丁,除根皮苷以外,其他均为黄酮类物质。阐明3个品种荔枝叶的40%醇沉多糖包括阿拉伯糖、鼠李糖、半乳糖和葡萄糖,而80%醇沉多糖则仅包括阿拉伯糖、鼠李糖、半乳糖；对荔枝酚与荔枝多糖的DPPH自由基清除活性进行了评价,就酚类物质而言,三个荔枝品种叶的酚类物质活性均高于果皮和种子。就多糖而言,各个品种的活性差异显著,三个品种不同器官活性有明显差异,紫娘喜品种果皮的活性高于果核与叶子,而丁香品种叶子多糖的活性最高。
Litchi (Litchi chinensis Sonn., Sapindaceae), a tropical and subtropical fruit originated from China, has become one of the most important fruit crops in china and developed rapidly in Guangdong, Guangxi Fujian and Hainan provinces from1990. China is the main country of litchi production, which area and yield are both the first in the world. In this study, investigation, collection、evaluation、genetic diversity analysis、fingerprint construction and functional composition analysis of Hainan Litchi germplasm resources was carried out. The main results were as follows:
1. The growth and distribution status of wild, semi-wild litchi in Hainan was cleared and distribution map was drawed based on wild, semi-wild litchi survey. More than120copies of Litchi germplasm resources were collected and a series of norms, rules and standards were made.
2. The descriptors-standard、data quality control of descriptors and the standar of data for Litchi were made.30of excellent germplasms were selected on the basis of evaluation, classification of litchi germplasm resources according to the norms.
3. ISSR and AFLP molecular marker technique were used respectively for genetic diversity analysis of96and60litchi germplasm resources.180polymorphism bands were obtained using13ISSR primers. The average number of band was13.8per primer and the polymorphic ratio was90.56%.1218straps were amplified using3pairs of AFLP primers and1125straps were polymorphic(92.36%). Genetic similarity coefficients of ISSR and AFLP were0.478～0.874and0.616～0.846, respectively. Results from ISSR and AFLP markers showed that wild litchi germplasm resources covered all the groups, semi-wild germplasm resources had a few groups while cultivated germplasm almost belonged to the same group. The clustering results of the two molecular markers showed high consistency.
4. According to the results of genetic similarity coefficient, the genetic basis of Hainan litchi germplasm was relatively wide while the majority of the litchi had closely phylogenetic relationships and the genetic differences was relatively narrow. The similarity coefficient showed partial normal distribution and wide genetic differences mainly existed in wild type. Cultivar litchi (semi-wild) was obviously different from wild litchi. Moreover, the wild litchi from the same forest had closely genetic relationships and strong eco-regional characteristics. Wild litchi from Bawangling was divided into different groups, indicating that genetic diversity of wild litchi from Bawangling is abundant than other forest and Bawangling is the core area of wild litchi.
5. SSR finger printing of litchi germplasm was constructed using SSR and AFLP molecular marker technology.11germplasm resources could be fully separated by the two markers methods and each tested material had the only bar code. These results might provide useful reliable DNA molecular evidence for identification of species, registration of new varieties, and protection of cultivar.
6. Total phenols, flavonoids and polysaccharides were extracted from litchi leaves, peel and stone by using the response surface method to optimize the ultrasonic aided technology. Three types of substance's extraction condition and extraction process is analyzed and established a mathematical model which provide theoretical reference and basis for total phenolics, flavonoids and polysaccharide extraction of Litchi. Compared to traditional methods, the new method could significantly increased extraction rate.
7. Phenols contents in pericarp and stones were analyzed from Ziniangxi, Dingxiang and Nandaowuhe litchi. Six types of phenolic compounds i.e procyanidine A1and A2, phloridzin, tamarixetin-3-rutinoside, catechin and rutin, were detected in stones. All of them are flavonoids except phloridzin. Polysaccharide in litchi leaves precipitated by40%alcohol included arabinose, rhamnose, galactose and glucose, while polysaccharide precipitated by80%alcohol did not contain glucose. DPPH radical scavenging activities of phenols and polysaccharides were evaluated. Higher DPPH radical scavenging activities of phenols were detected in litchi leaves. The DPPH radical scavenging activities of polysaccharides were significantly different in various species. Activities of polysaccharides in pericarp was higher than that of in stone and leaves of Ziniangxi, and the highest polysaccharides DPPH radical scavenging activities was observed in leaves of Dingxiang.
引文
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