苎麻分子标记方法比较和遗传图谱的初步构建
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摘要
苎麻（Boehmeria nivea(L.)Gaud），又叫“中国草”，起源于中国，属荨麻科苎麻属的多年生宿根性草本植物。中国是世界上种植和使用苎麻历史最悠久的国家。苎麻纤维优良，是纺织工业的重要原料。我国的苎麻栽培面积和总产量均占世界的90%以上，是重要的出口创汇产品之一。
限制性位点扩增多态性（restriction site amplified polymorphism, RSAP）是一种新型的分子标记技术，为了明确该标记技术在苎麻上利用的可行性。首先对限制性位点扩增多态性（RSAP）-PCR反应体系利用正交试验进行优化。结果表明：PCR扩增的前5个循环采用35℃的退火温度，随后的35个循环采用48℃的退火温度；25μL体系中，其最优条件：Taq DNA聚合酶1U，dNTPs0.3mmol/L，引物0.25μmol/L，DNA浓度10ng。扩增产物在8％变性聚丙烯酰胺凝胶分离，银染显色。
分别采用RSAP、SRAP和SSR3种分子标记和田间性状对16份苎麻种质进行亲缘关系聚类，结果表明：每对引物扩增出的多态性位点，SRAP标记最多，RSAP次之，SSR较少；基于SRAP标记和RSAP标记分类的结果都与RSAP+SRAP+SSR联合分类的结果基本一致，相关达到了极显著水平，而与SSR标记分类结果差异较大；分子标记聚类结果与田间性状的聚类结果接近程度，依次为SRAP+RSAP+SSR>SRAP>RSAP>>SSR。在检测苎麻种质亲缘关系中，SRAP标记效果最优，RSAP标记稍逊，SSR标记最差。RSAP标记能较好地显示苎麻种属间的多态性和亲缘关系。以RSAP、SRAP、SSR标记联合分析，能更好地揭示种质之间的亲缘关系。RSAP方法无需酶切，比以往基于限制性位点的标记技术操作简便，并具有中等产率、稳定可靠的特点和较广泛的适用性，在苎麻上利用是可行的。
采用中苎1号x合江青麻F1分离群体的180个单株，利用JoinMap3.0软件对352个SSR、RSAP和SRAP标记进行分析后，得到了一张包含34个连锁群的苎麻遗传图。该图谱包含103个标记，其中RSAP标记55个，SSR标记40个，SRAP标记8个。标记间的最大图距为50.9cM，最小图距为0.8cM，平均图距为10.7cM，图谱覆盖基因组的总长度1089.0cM。标记在各连锁群上的分布数目为2~17个不等，17个标记、8个标记、6个标记、5个标记各有一个连锁群，有2个连锁群有4个标记标定，还有3个连锁群上有3个标记，25个是二联体(doublet)，每个连锁群上平均有3.0个标记。
Ramie [Boehmeria nivea (L.) Gaud.], called "China grass", originated in China. Ramie is a kind ofratoon perennial herbs which belongs to nettle boehmeria. China has a long history to cultivate and useramie. Ramie with high quality bast fiber is a significant raw material for the textile industry. Itplays an indispensable role in national economy for its unique characteristic and usage. Ramieplanting area and total output in China take up more than90%of the world. It is an importantexport product of China.
Restriction site amplified polymorphism (RSAP) is one of new molecular marker techniques. Inorder to identify the feasibility of RSAP for the utilization on ramie, restricted site of amplifiedpolymorphic (RSAP)-PCR reaction system was optimized at first and the result showed as following:Taq DNA polymerase1U, dNTPs0.3mmol/L, primer0.25μ mol/L, DNA concentration10ng.Amplification products were separated on6%denaturing polyacrylamide gel8%, and colored aftersilver staining.
Then, three molecular markers-RSAP, SRAP and SSR were used to cluster16Ramie germplasm.The result showed that each pair of primers could provide polymorphic sites and the site number wasSRAP>RSAP>>SSR. Both results clustered based on SRAP and RSAP showed high consistency withthat clustering based on combination of SRAP, RSAP and SSR, and the highly significant correlationcoefficients were given. However, results of clustering based on SSR marker were quite different fromthe results based on combined markers. The rank order of similarities between the clustering resultsbased on the molecular marker and phenotype was SRAP+RSAP+SSR>SRAP>RSAP>>SSR. For thestudy of genetic relationship among ramie germplasm, the efficiency of SRAP marker was slightlyhigher than that of RSAP marker, but that of SSR was low. Based on RSAP marker could efficientlydisplay the polymorphism and genetic relationship among ramie varieties. Using the combined markersof RSAP&SRAP&SSR could be to reveal the genetic relationship of germplasm. RSAP method withoutrestriction enzyme is easier than markers based on restricted sites, which has the characteristics ofmedium yield, stability and reliability, and wider applicability, and it is feasible for the utilization oframie.
Using JoinMap3.0software to analysis352polymorphism markers based on SSR, RSAP andSRAP marker, a piece of ramie genetic map contains34chain groups was acquired. The map containedaltogether103markers, which had55RSAP markers,40SSR markers and8SRAP markers. The mapcovered the total length of1089.0cM, the average interval distance between markers was10.7cM, thelargest interval distance was50.9cM, the smallest distance was0.8cM. Number of markers in eachlinkage group varied from2to17.4linkage groups had17markers,8markers,6markers and5makersrespectively, two linkage groups had4markers, but also there were3markers in three linkage groups,and25doublets, the average number makers in each linkage group was3.0.
限制性位点扩增多态性（restriction site amplified polymorphism, RSAP）是一种新型的分子标记技术，为了明确该标记技术在苎麻上利用的可行性。首先对限制性位点扩增多态性（RSAP）-PCR反应体系利用正交试验进行优化。结果表明：PCR扩增的前5个循环采用35℃的退火温度，随后的35个循环采用48℃的退火温度；25μL体系中，其最优条件：Taq DNA聚合酶1U，dNTPs0.3mmol/L，引物0.25μmol/L，DNA浓度10ng。扩增产物在8％变性聚丙烯酰胺凝胶分离，银染显色。
分别采用RSAP、SRAP和SSR3种分子标记和田间性状对16份苎麻种质进行亲缘关系聚类，结果表明：每对引物扩增出的多态性位点，SRAP标记最多，RSAP次之，SSR较少；基于SRAP标记和RSAP标记分类的结果都与RSAP+SRAP+SSR联合分类的结果基本一致，相关达到了极显著水平，而与SSR标记分类结果差异较大；分子标记聚类结果与田间性状的聚类结果接近程度，依次为SRAP+RSAP+SSR>SRAP>RSAP>>SSR。在检测苎麻种质亲缘关系中，SRAP标记效果最优，RSAP标记稍逊，SSR标记最差。RSAP标记能较好地显示苎麻种属间的多态性和亲缘关系。以RSAP、SRAP、SSR标记联合分析，能更好地揭示种质之间的亲缘关系。RSAP方法无需酶切，比以往基于限制性位点的标记技术操作简便，并具有中等产率、稳定可靠的特点和较广泛的适用性，在苎麻上利用是可行的。
采用中苎1号x合江青麻F1分离群体的180个单株，利用JoinMap3.0软件对352个SSR、RSAP和SRAP标记进行分析后，得到了一张包含34个连锁群的苎麻遗传图。该图谱包含103个标记，其中RSAP标记55个，SSR标记40个，SRAP标记8个。标记间的最大图距为50.9cM，最小图距为0.8cM，平均图距为10.7cM，图谱覆盖基因组的总长度1089.0cM。标记在各连锁群上的分布数目为2~17个不等，17个标记、8个标记、6个标记、5个标记各有一个连锁群，有2个连锁群有4个标记标定，还有3个连锁群上有3个标记，25个是二联体(doublet)，每个连锁群上平均有3.0个标记。
Ramie [Boehmeria nivea (L.) Gaud.], called "China grass", originated in China. Ramie is a kind ofratoon perennial herbs which belongs to nettle boehmeria. China has a long history to cultivate and useramie. Ramie with high quality bast fiber is a significant raw material for the textile industry. Itplays an indispensable role in national economy for its unique characteristic and usage. Ramieplanting area and total output in China take up more than90%of the world. It is an importantexport product of China.
Restriction site amplified polymorphism (RSAP) is one of new molecular marker techniques. Inorder to identify the feasibility of RSAP for the utilization on ramie, restricted site of amplifiedpolymorphic (RSAP)-PCR reaction system was optimized at first and the result showed as following:Taq DNA polymerase1U, dNTPs0.3mmol/L, primer0.25μ mol/L, DNA concentration10ng.Amplification products were separated on6%denaturing polyacrylamide gel8%, and colored aftersilver staining.
Then, three molecular markers-RSAP, SRAP and SSR were used to cluster16Ramie germplasm.The result showed that each pair of primers could provide polymorphic sites and the site number wasSRAP>RSAP>>SSR. Both results clustered based on SRAP and RSAP showed high consistency withthat clustering based on combination of SRAP, RSAP and SSR, and the highly significant correlationcoefficients were given. However, results of clustering based on SSR marker were quite different fromthe results based on combined markers. The rank order of similarities between the clustering resultsbased on the molecular marker and phenotype was SRAP+RSAP+SSR>SRAP>RSAP>>SSR. For thestudy of genetic relationship among ramie germplasm, the efficiency of SRAP marker was slightlyhigher than that of RSAP marker, but that of SSR was low. Based on RSAP marker could efficientlydisplay the polymorphism and genetic relationship among ramie varieties. Using the combined markersof RSAP&SRAP&SSR could be to reveal the genetic relationship of germplasm. RSAP method withoutrestriction enzyme is easier than markers based on restricted sites, which has the characteristics ofmedium yield, stability and reliability, and wider applicability, and it is feasible for the utilization oframie.
Using JoinMap3.0software to analysis352polymorphism markers based on SSR, RSAP andSRAP marker, a piece of ramie genetic map contains34chain groups was acquired. The map containedaltogether103markers, which had55RSAP markers,40SSR markers and8SRAP markers. The mapcovered the total length of1089.0cM, the average interval distance between markers was10.7cM, thelargest interval distance was50.9cM, the smallest distance was0.8cM. Number of markers in eachlinkage group varied from2to17.4linkage groups had17markers,8markers,6markers and5makersrespectively, two linkage groups had4markers, but also there were3markers in three linkage groups,and25doublets, the average number makers in each linkage group was3.0.
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