龙须菜杂种优势株的筛选及AFLP分析
摘要
龙须菜(Gracilaria lameneiformis)是重要的产琼胶海藻,目前已形成我国继海带和紫菜之后的第三大海藻栽培业,有力地促进了琼胶制造业的发展,同时也是进行遗传学研究的良好材料。本论文通过不同产地野生型龙须菜间的杂交来筛选杂种优势株,并进行重要数量性状和AFLP分析,探索龙须菜杂种优势的遗传机理,以期为龙须菜栽培良种选育提供依据。
以中国山东青岛、石岛和龙须岛三个不同产地野生型龙须菜四分孢子体为亲本,在获得配子体的基础上,通过自交和杂交,建立起了包括亲代四分孢子体、亲代雌雄配子体、自交F1四分孢子体、杂交F1四分孢子体和杂交F1配子体在内的材料体系,并进行了经济性状分析。筛选到一株石岛雌配子体与青岛雄配子体杂交的F1四分孢子体(F1sq),与对应亲代四分孢子体相比,其在生长速度方面具有明显优势,线生长速度和生物量累积速度比母本与父本均值高60%以上,α-半乳糖苷酶活性及光合色素含量分析则未表现出明显差异。此外根据正反交F1四分孢子体在性状上的分析结果,推测龙须菜线生长速度可能与细胞质遗传无关。
成功建立起龙须菜AFLP反应实验体系,通过比较选择出适宜的限制性内切酶组合EcoRⅠ-TaqⅠ,并对AFLP分析中各实验步骤进行了优化固定。首次利用优化的AFLP反应条件对青岛野生型龙须菜与养殖品系981进行了比较分析,64种引物组合共扩增出1794条可区分的条带,其中69条呈多态性,多态性比例仅为3.8%,两者在具有极其相近的亲缘关系的同时,又在生长速度、耐高温性能和琼胶含量与质量等方面存在显著的差异,本研究中通过AFLP技术检测到的这些差异位点很可能包含着与以上的优良性状有关的遗传信息。
对包含中国青岛、石岛、龙须岛、南非、委内瑞拉产地的5株龙须菜、养殖品系981以及细基江蓠繁枝变型在内的7种不同材料数量性状与AFLP分析结果显示,生长速度与色素含量在种间和种内均显示出较明显的差异。6种龙须菜中线生长速度和生物量累积速率最高的分别是最低的2.56倍和5.46倍;三种藻胆蛋白中,别藻蓝蛋白含量最为保守,藻红蛋白差异程度最大,藻蓝蛋白介于两者之间。AFLP分析中,16对引物组合共扩增出762条可区分的条带,多态性比例为91.6 %。青岛野生型龙须菜与养殖品系981拥有最小的遗传距离0.0422,委内瑞拉龙须菜与养殖品系981间的遗传距离最大,为0.8881。UPGMA聚类分析结果表明中国4株龙须菜与南非龙须菜在遗传距离为0.2618处聚合成一大个群,委内瑞拉龙须菜与作为种外对照的细基江蓠繁枝变型(G. teniustipitata var. liui)聚合成另一大群,为把委内瑞拉材料划分到龙须菜以外的种提供了更加有力的证据。
Gracilaria lemaneiformis is one of the important agarophytic species of Gracilaria , and the cultivation of G. lemaneiformis has ranked the third place of algal mariculture following Laminaria and Porphyra in China, which greatly promoted the industry of agar production and exportation of China. Besides, G. lemaneiformis is also an ideal material for studies of genetics and molecular biology. The aim of this study is to conduct the selection of crosses with heterosis developed through sexual crossing between G. lemaneiformis from different locations, and the analyses of relative quantitative traits and AFLP, so as to investigate the heterosis mechanism of G. lemaneiformis and to provide practical data for its genetic breeding.
In this study, three wild type tetrasporophytes of G. lemaneiformis from Qingdao(QD), Shidao (SD) and Longxudao (LXD) of China were used as parents, and their male and female gametophytes were obtained by laboratory incubation. A material system including parent tetrasporophytes, parent gametophytes, self-fertilization tetrasporophytes, hybrid tetrasporophytes and hybrid gametophytes was constructed. Comparison of relative economic traits indicates that, both tip elongation rate and biomass increase rate of hybrid F1 tetrasporophyte (F1sq), which is generated by Shidao female gametophyte and Qingdao male gametophyte, is 60% higher than the average value of its corresponding parent tetrasporophytes. While, no significant differences on contents ofα- galactosidase and photosynthetic pigment were detected. Besides, the comparison result between crosses and reciprocal crosses shows that cytoplast genetic contribution may not be involved in tip elongation rate of G. lemaneiformis.
The AFLP analysis system for G. lemaneiformis was established, and the key steps were optimized. EcoRⅠ-TaqⅠrestriction enzymes combination was selected for digestion reaction through comparison. Then the optimized reaction system was applied to investigate the genetic background of QD and 981. A total of 1794 loci were detected by 64 pairs of EcoRⅠ/TaqⅠprimer combinations, and 69 polymorphic AFLP markers were revealed, the ratio of polymorphic marker was only 3.8%. AFLP analysis showed that G. lemaneiformis from Qingdao and its selected strain 981 displayed the highest genetic similarity, on the other hand, 981 differs greatly from QD in high growth rate, high temperature tolerance, and the yield and quality of agar, the different loci of AFLP might include genetic information relative to these fine traits.
Quantitative traits of growth rate and photosynthetic pigment content as well as amplified fragment length polymorphism (AFLP) were detected for investigating the genetic diversity of six strains of Gracilaria lemaneiformis, three of which were collected in Qingdao (QD), Shidao (SD) and Longxudao (LXD) of China, the forth named 981 is high-temperature selected strain from QD , the other two strains came from South Africa (Sa) and Venezuela (Lv) respectively, and Gracilaria tenuistipitata var. liui (Ten) was used as outgroup control. The results of quantitative traits examination showed that obvious difference of growth rate and pigment content occurred in G. lemaneiformis from different locations. The largest value of tip elongation rate and biomass increase rate is 2.56 and 5.46 times of the smallest value respectively. Among three phycobiliproteins, Allo-phycocyanin (APC) is relatively conservative, phycoerithrin (PE) is more flexible, and phycocyanin(PC) is in the middle. In AFLP analysis, a total of 762 loci were obtained by 16 pairs of EcoRⅠ/TaqⅠprimer combinations, and the ratio of polymorphic marker was 91.3%. The genetic distance between pairs of genotypes ranged from 0.0422 (981 and QD) to 0.8881(981 and Lv). Cluster tree obtained with UPGMA analysis demonstrates that all the materials involved were divided into 2 groups. One group includes 4 strains G. lemaneiformis from China and Sa, the other consists of Lv and Ten. In addition, both quantitative traits examination and AFLP analysis indicate that Lv should not to be divided into the species of G. lemaneiformis.
以中国山东青岛、石岛和龙须岛三个不同产地野生型龙须菜四分孢子体为亲本,在获得配子体的基础上,通过自交和杂交,建立起了包括亲代四分孢子体、亲代雌雄配子体、自交F1四分孢子体、杂交F1四分孢子体和杂交F1配子体在内的材料体系,并进行了经济性状分析。筛选到一株石岛雌配子体与青岛雄配子体杂交的F1四分孢子体(F1sq),与对应亲代四分孢子体相比,其在生长速度方面具有明显优势,线生长速度和生物量累积速度比母本与父本均值高60%以上,α-半乳糖苷酶活性及光合色素含量分析则未表现出明显差异。此外根据正反交F1四分孢子体在性状上的分析结果,推测龙须菜线生长速度可能与细胞质遗传无关。
成功建立起龙须菜AFLP反应实验体系,通过比较选择出适宜的限制性内切酶组合EcoRⅠ-TaqⅠ,并对AFLP分析中各实验步骤进行了优化固定。首次利用优化的AFLP反应条件对青岛野生型龙须菜与养殖品系981进行了比较分析,64种引物组合共扩增出1794条可区分的条带,其中69条呈多态性,多态性比例仅为3.8%,两者在具有极其相近的亲缘关系的同时,又在生长速度、耐高温性能和琼胶含量与质量等方面存在显著的差异,本研究中通过AFLP技术检测到的这些差异位点很可能包含着与以上的优良性状有关的遗传信息。
对包含中国青岛、石岛、龙须岛、南非、委内瑞拉产地的5株龙须菜、养殖品系981以及细基江蓠繁枝变型在内的7种不同材料数量性状与AFLP分析结果显示,生长速度与色素含量在种间和种内均显示出较明显的差异。6种龙须菜中线生长速度和生物量累积速率最高的分别是最低的2.56倍和5.46倍;三种藻胆蛋白中,别藻蓝蛋白含量最为保守,藻红蛋白差异程度最大,藻蓝蛋白介于两者之间。AFLP分析中,16对引物组合共扩增出762条可区分的条带,多态性比例为91.6 %。青岛野生型龙须菜与养殖品系981拥有最小的遗传距离0.0422,委内瑞拉龙须菜与养殖品系981间的遗传距离最大,为0.8881。UPGMA聚类分析结果表明中国4株龙须菜与南非龙须菜在遗传距离为0.2618处聚合成一大个群,委内瑞拉龙须菜与作为种外对照的细基江蓠繁枝变型(G. teniustipitata var. liui)聚合成另一大群,为把委内瑞拉材料划分到龙须菜以外的种提供了更加有力的证据。
Gracilaria lemaneiformis is one of the important agarophytic species of Gracilaria , and the cultivation of G. lemaneiformis has ranked the third place of algal mariculture following Laminaria and Porphyra in China, which greatly promoted the industry of agar production and exportation of China. Besides, G. lemaneiformis is also an ideal material for studies of genetics and molecular biology. The aim of this study is to conduct the selection of crosses with heterosis developed through sexual crossing between G. lemaneiformis from different locations, and the analyses of relative quantitative traits and AFLP, so as to investigate the heterosis mechanism of G. lemaneiformis and to provide practical data for its genetic breeding.
In this study, three wild type tetrasporophytes of G. lemaneiformis from Qingdao(QD), Shidao (SD) and Longxudao (LXD) of China were used as parents, and their male and female gametophytes were obtained by laboratory incubation. A material system including parent tetrasporophytes, parent gametophytes, self-fertilization tetrasporophytes, hybrid tetrasporophytes and hybrid gametophytes was constructed. Comparison of relative economic traits indicates that, both tip elongation rate and biomass increase rate of hybrid F1 tetrasporophyte (F1sq), which is generated by Shidao female gametophyte and Qingdao male gametophyte, is 60% higher than the average value of its corresponding parent tetrasporophytes. While, no significant differences on contents ofα- galactosidase and photosynthetic pigment were detected. Besides, the comparison result between crosses and reciprocal crosses shows that cytoplast genetic contribution may not be involved in tip elongation rate of G. lemaneiformis.
The AFLP analysis system for G. lemaneiformis was established, and the key steps were optimized. EcoRⅠ-TaqⅠrestriction enzymes combination was selected for digestion reaction through comparison. Then the optimized reaction system was applied to investigate the genetic background of QD and 981. A total of 1794 loci were detected by 64 pairs of EcoRⅠ/TaqⅠprimer combinations, and 69 polymorphic AFLP markers were revealed, the ratio of polymorphic marker was only 3.8%. AFLP analysis showed that G. lemaneiformis from Qingdao and its selected strain 981 displayed the highest genetic similarity, on the other hand, 981 differs greatly from QD in high growth rate, high temperature tolerance, and the yield and quality of agar, the different loci of AFLP might include genetic information relative to these fine traits.
Quantitative traits of growth rate and photosynthetic pigment content as well as amplified fragment length polymorphism (AFLP) were detected for investigating the genetic diversity of six strains of Gracilaria lemaneiformis, three of which were collected in Qingdao (QD), Shidao (SD) and Longxudao (LXD) of China, the forth named 981 is high-temperature selected strain from QD , the other two strains came from South Africa (Sa) and Venezuela (Lv) respectively, and Gracilaria tenuistipitata var. liui (Ten) was used as outgroup control. The results of quantitative traits examination showed that obvious difference of growth rate and pigment content occurred in G. lemaneiformis from different locations. The largest value of tip elongation rate and biomass increase rate is 2.56 and 5.46 times of the smallest value respectively. Among three phycobiliproteins, Allo-phycocyanin (APC) is relatively conservative, phycoerithrin (PE) is more flexible, and phycocyanin(PC) is in the middle. In AFLP analysis, a total of 762 loci were obtained by 16 pairs of EcoRⅠ/TaqⅠprimer combinations, and the ratio of polymorphic marker was 91.3%. The genetic distance between pairs of genotypes ranged from 0.0422 (981 and QD) to 0.8881(981 and Lv). Cluster tree obtained with UPGMA analysis demonstrates that all the materials involved were divided into 2 groups. One group includes 4 strains G. lemaneiformis from China and Sa, the other consists of Lv and Ten. In addition, both quantitative traits examination and AFLP analysis indicate that Lv should not to be divided into the species of G. lemaneiformis.
引文
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