苎麻自交纯合进度评价研究
摘要
苎麻（Boehmeria nivea L.）为异花授粉作物，目前种植品种都是杂合体，还没有纯合体发现的报道,杂合体遗传分析复杂，严重影响了苎麻遗传学的发展。一般来说，在杂种优势的利用中，前提之一就是获得遗传纯合的优良亲本，没有优良的遗传纯合亲本，很难获得整齐一致的强优势F1代，实际上限制了苎麻杂种优势在生产上的广泛应用。
本研究以中苎1号为材料，连续进行多代自交，利用SSR和SRAP两种分子标记技术对自交后代的杂合度进行分析。主要研究结果如下：
(1)建立了适合苎麻DNA分析的SSR优化技术体系：每25μL体系中含有DNA模板为45ng，引物0.15μmol/L，Taq酶用量1.5U，dNTPs为0.15mmol/L。
(2)以中苎1号群体为模板，从100对SSR引物和16×15对SRAP引物组合中筛选出扩增产物稳定并易于区分的引物，得到了31对SSR引物和48对SRAP引物。
(3)为了确定群体的最适抽样量，本试验从中苎1号群体中随机选取100蔸，并从中随机抽取10、20、30、40、50、60、70、80，90个个体组成抽样群体，计算其遗传多样性参数，得出SSR标记在样本量50以上时参数值变化不大，而SRAP标记在样本量60以上时结果较为稳定。
(4) SSR标记显示经过5个世代的连续自交，遗传多样性参数均明显下降，S5代群体的观测杂合度从0.5414降到0.3596，平均等位基因数从5.5667减少到2.6774个。
(5) SRAP标记结果同样显示出遗传多样性参数随自交世代逐年下降，S5代群体的观测杂合度从0.5587降到0.4018，平均等位基因数从9.9832减少到6.0625个。
(6)用两种分子标记方法估测得到的群体观测杂合度与各植物学性状间的关系进行研究，结果显示观测杂合度与自交世代群体的植物学性状的变化趋势相反，进一步说明群体的杂合性在逐年降低。而且两种标记方法所得到群体观测杂合度数值差异性很小，说明两种标记的都能很好地用做估测群体的杂合度。
Ramie（Boehmeria nivea L.）is a cross-pollinated crop, and all of its the currently cultivatedvarieties are heterozygote. Herefore,there is no report of discovering ramie homozygote.Geneticanalysis on ramie heterozygote is so complicated,that it has seriously affected the development of ramiegenetics. Moreover, excellent parents with genetic homozygous is one of the preconditions in theutilization of heterosis.Without excellent parents, we can not obtain consistently superior F1, which hasgreatly limited the application of ramie heterosis in production.
The trial cultivar used in this study was“zhong zhu No.1”. It was self-pollinated for consecutivepoly-generations and five different generations of ramie inbred lines were gained, then two molecularmarkers SSR and SRAP were used in analyzing selfed generations heterozygosity. The research resultsshowed that:
(1) PCR system for SSR in ramie has been found:25μL reaction solution, contained45ng DNAtemplate,0.15μmol/L primers,1.5U Taq polymerase,0.15mmoL dNTPs.
(2) In this thesis,31primers selected from100SSR primes and48primes combinations selectedfrom the combinations of16forward primers and15reverse primers, which were identifiedpolymorphic and clear strip for “Zhong zhu No.1”.
(3) In order to determine the optimal sampling volume of the group, we randomly selected10,20,30,40,50,60,70,80,90individuals composing sample groups from100tufts “Zhong zhu No.1” tocalculate the genetic diversity parameters. The conclusions showed that SSR molecular markerparameter values preserved uniformity when sampling volume of the group was more than50individuals. And that for SRAP molecular marker, the sample number should be more than60individuals.
(4) SSR molecular marker indicated that after five generations of continuous selfing, all geneticdiversity parameters decreased obviously. Heterozygosity of S5generation groups dropped from0.5414to0.3596, and the average number of alleles dropped from5.5667to2.6774.
(5) SRAP molecular marker also showed that genetic diversity parameters decreased as the numberof selfing generations increased. The observed heterozygosity of S5generaion groups dropped from0.5587to0.4018, the average number of alleles dropped from9.9832to6.0625.
(6) This study discussed the relatitonship between observed groups heterozygosity that estimatedby the methods of molecular markers and the botanical characters.The opposite variation tendencybetween heterozygosity and botanical characters indicated that population heterozygosity wasdecreasing year by year. Through the two molecular markers, the deviation of population heterozygosityvalue drived from two molecular markers was extremely small, and the results verified that SSR andSRAP were effective approaches to estimate the heterozygosity of groups.
本研究以中苎1号为材料，连续进行多代自交，利用SSR和SRAP两种分子标记技术对自交后代的杂合度进行分析。主要研究结果如下：
(1)建立了适合苎麻DNA分析的SSR优化技术体系：每25μL体系中含有DNA模板为45ng，引物0.15μmol/L，Taq酶用量1.5U，dNTPs为0.15mmol/L。
(2)以中苎1号群体为模板，从100对SSR引物和16×15对SRAP引物组合中筛选出扩增产物稳定并易于区分的引物，得到了31对SSR引物和48对SRAP引物。
(3)为了确定群体的最适抽样量，本试验从中苎1号群体中随机选取100蔸，并从中随机抽取10、20、30、40、50、60、70、80，90个个体组成抽样群体，计算其遗传多样性参数，得出SSR标记在样本量50以上时参数值变化不大，而SRAP标记在样本量60以上时结果较为稳定。
(4) SSR标记显示经过5个世代的连续自交，遗传多样性参数均明显下降，S5代群体的观测杂合度从0.5414降到0.3596，平均等位基因数从5.5667减少到2.6774个。
(5) SRAP标记结果同样显示出遗传多样性参数随自交世代逐年下降，S5代群体的观测杂合度从0.5587降到0.4018，平均等位基因数从9.9832减少到6.0625个。
(6)用两种分子标记方法估测得到的群体观测杂合度与各植物学性状间的关系进行研究，结果显示观测杂合度与自交世代群体的植物学性状的变化趋势相反，进一步说明群体的杂合性在逐年降低。而且两种标记方法所得到群体观测杂合度数值差异性很小，说明两种标记的都能很好地用做估测群体的杂合度。
Ramie（Boehmeria nivea L.）is a cross-pollinated crop, and all of its the currently cultivatedvarieties are heterozygote. Herefore,there is no report of discovering ramie homozygote.Geneticanalysis on ramie heterozygote is so complicated,that it has seriously affected the development of ramiegenetics. Moreover, excellent parents with genetic homozygous is one of the preconditions in theutilization of heterosis.Without excellent parents, we can not obtain consistently superior F1, which hasgreatly limited the application of ramie heterosis in production.
The trial cultivar used in this study was“zhong zhu No.1”. It was self-pollinated for consecutivepoly-generations and five different generations of ramie inbred lines were gained, then two molecularmarkers SSR and SRAP were used in analyzing selfed generations heterozygosity. The research resultsshowed that:
(1) PCR system for SSR in ramie has been found:25μL reaction solution, contained45ng DNAtemplate,0.15μmol/L primers,1.5U Taq polymerase,0.15mmoL dNTPs.
(2) In this thesis,31primers selected from100SSR primes and48primes combinations selectedfrom the combinations of16forward primers and15reverse primers, which were identifiedpolymorphic and clear strip for “Zhong zhu No.1”.
(3) In order to determine the optimal sampling volume of the group, we randomly selected10,20,30,40,50,60,70,80,90individuals composing sample groups from100tufts “Zhong zhu No.1” tocalculate the genetic diversity parameters. The conclusions showed that SSR molecular markerparameter values preserved uniformity when sampling volume of the group was more than50individuals. And that for SRAP molecular marker, the sample number should be more than60individuals.
(4) SSR molecular marker indicated that after five generations of continuous selfing, all geneticdiversity parameters decreased obviously. Heterozygosity of S5generation groups dropped from0.5414to0.3596, and the average number of alleles dropped from5.5667to2.6774.
(5) SRAP molecular marker also showed that genetic diversity parameters decreased as the numberof selfing generations increased. The observed heterozygosity of S5generaion groups dropped from0.5587to0.4018, the average number of alleles dropped from9.9832to6.0625.
(6) This study discussed the relatitonship between observed groups heterozygosity that estimatedby the methods of molecular markers and the botanical characters.The opposite variation tendencybetween heterozygosity and botanical characters indicated that population heterozygosity wasdecreasing year by year. Through the two molecular markers, the deviation of population heterozygosityvalue drived from two molecular markers was extremely small, and the results verified that SSR andSRAP were effective approaches to estimate the heterozygosity of groups.
引文
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