应用AFLP技术对大连沿海裙带菜养殖和野生群体的遗传多样性分析
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摘要
裙带菜是一种广温性海产大型经济藻类,含有大量的维生素A及其它营养成分,具有高蛋白、低脂肪、高纤维等特点,不仅味道鲜美,还具有很高的营养价值和药用价值,而且可作综合利用提取褐藻胶的原料。我国自然生长的裙带菜主要分布在浙江省的舟山群岛和嵊泗岛,大连和青岛地区作为我国主要的裙带菜养殖基地,所用种菜绝大多数是通过早年的引种及野生种苗,这就势必导致遗传多样性水平不断下降。近年来,裙带菜国内外市场需求不断扩大,应用范围不断扩展,显示出巨大的市场潜力,但裙带菜选育工作基础还十分薄弱。因此,对裙带菜遗传背景的深入研究势在必行。本文利用AFLP分子标记技术对大连沿海的付家庄野生、星海公园野生、獐子岛野生和凌水养殖裙带菜群体进行遗传多样性的研究,以期对裙带菜的遗传育种和种质资源保护工作提供一定的理论依据。
本实验共筛选了5对AFLP引物,对四个群体共计120个裙带菜个体(每个群体30个个体)进行了研究。利用Popgen32软件分析了群体内的遗传变异和群体间的遗传分化,计算不同群体的遗传距离,并利用UPGMA法构建了四个群体的系统发生树。所得结果如下:
利用5对AFLP引物共扩增出277个位点,其中有232个多态性位点,平均每对引物组合能检测到46.4个多态位点。四个群体中獐子岛野生群体(ZZD)多态位点比例最高,为73.28%,最低的是凌水养殖群体(YZ),多态位点比例为64.25%。Shannon指数(I)的变化在0.3594~0.3971之间,杂合度(H)的变化范围在0.2408~0.2711之间,两个指数变化规律基本一致,并且与多态位点比例分析结果相吻合。由此可以推断:獐子岛野生群体(ZZD)的遗传变异水平最高,凌水养殖群体(YZ)的遗传变异水平最低。
AFLP扩增获得的四个裙带菜群体总的遗传分化系数Gst为0.1651,即表明在裙带菜总的遗传变异中有16.51%的变异存在于群体间,83.49%的变异存在于群体内。利用成对比较的方法分析各群体间的Gst的值分布在0.0917~0.1416之间,证明群体间没有明显的遗传分化,大部分的变异来自群体内部。所有群体间的基因流Nm的值分布在4.9520~3.0303之间,表现了一定的基因流动性。
四个裙带菜群体遗传距离在0.0532~0.0996之间,可见各群体间遗传变异比较小。基于遗传距离用UPGMA法构建的系统发生树表明,四个群体共分成了三个组,星海公园野生群体(XH)和凌水养殖群体(YZ)首先聚为一支,然后和獐子岛野生群体(ZZD)聚为一支,付家庄野生群体(FJZ)和这些群体最后聚在一起。
通过对裙带菜遗传变异的分析,发现四个裙带菜群体都具有较高的遗传多样性水平,但是与野生群体相比,养殖群体的多样性参数有所降低。为了保护养殖裙带菜种质资源保持在比较高的水平,应该采取有效保护措施防止同地区间的种苗交流。本文的研究结果为进一步深入研究裙带菜遗传背景,实现裙带菜良种化奠定基础。另外实验结果还表明,AFLP分子标记技术相较于RAPD、ISSR等其他常用的分子标记技术来说,具有很高的多态性检出效率,是研究裙带菜群体遗传多样性中高效、可靠的工具。
Undaria pinnatifida is a wide temperature algae of large scale,rich in vitamin A and other nutrients,with high protein,low fat,high fiber,etc.Not only delicious,but also have high nutritional value and medicinal value and can be used for the comprehensive utilization of raw materials extracted alginate.In China,natural growth Undaria pinnatifida are mainly distributed in Zhoushan Islands and Shengsi Island,Zhejiang Province.Dalian and Qingdao as a major breeding base,the majority is used to grow algae are through the introduction or wild seed,which is bound to lead to genetic diversity declining.In recent years,Undaria pinnatifida market demand growing both at home and abroad,expanding the scope of application,showing that the huge market potential.But breeding basis work is very weak.Therefore,depth study of the genetic background of Undaria pinnatifida is imperative.AFLP technique was used to estimate genetic diversity of Undaria pinnatifida which belong to four populations,Fujiazhuang(FJZ) wild,Xinghai(XH) wild,Zhangzi Island(ZZD) wild and Lingshui(LS) cultured.The purpose is to provide theoretical basis for breeding and protection of idioplasm.
120 individual from four Undaria pinnatifida popolations (30 individuals per populations) were analyzed using 5 AFLP primer combinations.By using Popgen32 program , the genetic diversity was estimated , genetic distance was calculated and phylogenetic tree was constructed by UPGMA.The results are as follows:
Total 277 loci were amplified using 5 primer combinations,and 232 were polumorphic loci.On average,46.4 polumorphic loci were amplified by each combination.Among the four populations,ZZD has the highest percentage of polumorphic loci which is 73.28%,YZ has the lowest one which is 64.25%.The range of alteration of index Shannon is from 0.3594 to 0.3971,and the heterozygosity is from 0.2408 to 0.2711.The alteration law of two indexes was identical,and the result was also identical with the analysis of the percentage of polumorphic loci.It can be inferred the intra-populations genetic variation:ZZD is highest,YZ is lowest.
The total Gst was 0.1651.It means 16.51% genetic variation found in groups,83.49% of variation exists in the group.The Gst ranged from 0.0917 to 0.1416,that demonstrate no significant genetic differentiation,most of the variation from the internal populations.Gene flow was from 4.9520~3.0303,which means there was a certain amount of gene flow among the four populations.
Genetic distance ranged from 0.0532 to 0.0996,shows that genetic variation between populations is relatively small.Based on genetic distances,the phylogenetic tree using UPGMA shows that XH was closer to YZ,and they were clustered together as the first grope.ZZD was clustered into the second grope, the last one is FJZ.
By analysis of genetic variation of the four populations of Undaria pinnatifida,we found that they all have a high level of genetic diversity.But compared with the wild populations,the diversity parameters of the cultured population has decreased.In order to protect the cultured population maintained at a relatively high level,effective measures should be taken to prevent the seedlings mixed with other populations.The results of this article lay the foundation for further study on the genetic background and improve idioplasm of Undaria pinnatifida.The result also indicated that AFLP has a much higher polymorphic banding patterns than RAPD、ISSR . AFLP is a high-performance、reliable tool for the analysis of the genetic diversity of Undaria pinnatifida.
本实验共筛选了5对AFLP引物,对四个群体共计120个裙带菜个体(每个群体30个个体)进行了研究。利用Popgen32软件分析了群体内的遗传变异和群体间的遗传分化,计算不同群体的遗传距离,并利用UPGMA法构建了四个群体的系统发生树。所得结果如下:
利用5对AFLP引物共扩增出277个位点,其中有232个多态性位点,平均每对引物组合能检测到46.4个多态位点。四个群体中獐子岛野生群体(ZZD)多态位点比例最高,为73.28%,最低的是凌水养殖群体(YZ),多态位点比例为64.25%。Shannon指数(I)的变化在0.3594~0.3971之间,杂合度(H)的变化范围在0.2408~0.2711之间,两个指数变化规律基本一致,并且与多态位点比例分析结果相吻合。由此可以推断:獐子岛野生群体(ZZD)的遗传变异水平最高,凌水养殖群体(YZ)的遗传变异水平最低。
AFLP扩增获得的四个裙带菜群体总的遗传分化系数Gst为0.1651,即表明在裙带菜总的遗传变异中有16.51%的变异存在于群体间,83.49%的变异存在于群体内。利用成对比较的方法分析各群体间的Gst的值分布在0.0917~0.1416之间,证明群体间没有明显的遗传分化,大部分的变异来自群体内部。所有群体间的基因流Nm的值分布在4.9520~3.0303之间,表现了一定的基因流动性。
四个裙带菜群体遗传距离在0.0532~0.0996之间,可见各群体间遗传变异比较小。基于遗传距离用UPGMA法构建的系统发生树表明,四个群体共分成了三个组,星海公园野生群体(XH)和凌水养殖群体(YZ)首先聚为一支,然后和獐子岛野生群体(ZZD)聚为一支,付家庄野生群体(FJZ)和这些群体最后聚在一起。
通过对裙带菜遗传变异的分析,发现四个裙带菜群体都具有较高的遗传多样性水平,但是与野生群体相比,养殖群体的多样性参数有所降低。为了保护养殖裙带菜种质资源保持在比较高的水平,应该采取有效保护措施防止同地区间的种苗交流。本文的研究结果为进一步深入研究裙带菜遗传背景,实现裙带菜良种化奠定基础。另外实验结果还表明,AFLP分子标记技术相较于RAPD、ISSR等其他常用的分子标记技术来说,具有很高的多态性检出效率,是研究裙带菜群体遗传多样性中高效、可靠的工具。
Undaria pinnatifida is a wide temperature algae of large scale,rich in vitamin A and other nutrients,with high protein,low fat,high fiber,etc.Not only delicious,but also have high nutritional value and medicinal value and can be used for the comprehensive utilization of raw materials extracted alginate.In China,natural growth Undaria pinnatifida are mainly distributed in Zhoushan Islands and Shengsi Island,Zhejiang Province.Dalian and Qingdao as a major breeding base,the majority is used to grow algae are through the introduction or wild seed,which is bound to lead to genetic diversity declining.In recent years,Undaria pinnatifida market demand growing both at home and abroad,expanding the scope of application,showing that the huge market potential.But breeding basis work is very weak.Therefore,depth study of the genetic background of Undaria pinnatifida is imperative.AFLP technique was used to estimate genetic diversity of Undaria pinnatifida which belong to four populations,Fujiazhuang(FJZ) wild,Xinghai(XH) wild,Zhangzi Island(ZZD) wild and Lingshui(LS) cultured.The purpose is to provide theoretical basis for breeding and protection of idioplasm.
120 individual from four Undaria pinnatifida popolations (30 individuals per populations) were analyzed using 5 AFLP primer combinations.By using Popgen32 program , the genetic diversity was estimated , genetic distance was calculated and phylogenetic tree was constructed by UPGMA.The results are as follows:
Total 277 loci were amplified using 5 primer combinations,and 232 were polumorphic loci.On average,46.4 polumorphic loci were amplified by each combination.Among the four populations,ZZD has the highest percentage of polumorphic loci which is 73.28%,YZ has the lowest one which is 64.25%.The range of alteration of index Shannon is from 0.3594 to 0.3971,and the heterozygosity is from 0.2408 to 0.2711.The alteration law of two indexes was identical,and the result was also identical with the analysis of the percentage of polumorphic loci.It can be inferred the intra-populations genetic variation:ZZD is highest,YZ is lowest.
The total Gst was 0.1651.It means 16.51% genetic variation found in groups,83.49% of variation exists in the group.The Gst ranged from 0.0917 to 0.1416,that demonstrate no significant genetic differentiation,most of the variation from the internal populations.Gene flow was from 4.9520~3.0303,which means there was a certain amount of gene flow among the four populations.
Genetic distance ranged from 0.0532 to 0.0996,shows that genetic variation between populations is relatively small.Based on genetic distances,the phylogenetic tree using UPGMA shows that XH was closer to YZ,and they were clustered together as the first grope.ZZD was clustered into the second grope, the last one is FJZ.
By analysis of genetic variation of the four populations of Undaria pinnatifida,we found that they all have a high level of genetic diversity.But compared with the wild populations,the diversity parameters of the cultured population has decreased.In order to protect the cultured population maintained at a relatively high level,effective measures should be taken to prevent the seedlings mixed with other populations.The results of this article lay the foundation for further study on the genetic background and improve idioplasm of Undaria pinnatifida.The result also indicated that AFLP has a much higher polymorphic banding patterns than RAPD、ISSR . AFLP is a high-performance、reliable tool for the analysis of the genetic diversity of Undaria pinnatifida.
引文
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