CN105624282B - 一种濒危羊踯躅多态性ssr分子标记开发与应用的方法 - Google Patents
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Abstract
本发明公开了一种濒危羊踯躅多态性SSR分子标记开发与应用的方法,利用简化基因组测序技术开发得到11,687对羊踯躅SSR分子标记,从中鉴定获得了16对多态性SSR分子标记,它们能够检测到羊踯躅基因组的SSR位点信息,其有效等位基因数范围为3‑15个,杂合度(He)范围为0.489‑0.908。本发明提供一批可用于群体遗传学、系统发生学及保护生物学等研究的SSR引物,应用这些多态性SSR分子标记能评估分析濒危羊踯躅遗传多样性和遗传结构,为该物种的保护提供科学依据和参考。
Description
技术领域
本发明涉及一种濒危羊踯躅多态性SSR分子标记开发与应用的方法。
背景技术
羊踯躅(Rhododendron molle G.Don),又名黄杜鹃、闹羊花和八厘麻等,是中国羊踯躅亚属中仅有的一个原生种,具有较高的观赏价值和药用价值。羊踯躅全株器官均含有多种活性成分,其提取物可治疗类风湿、温疟、慢性肾小球肾炎和高血压等,对血吸虫和多种害虫也具有非常好的灭杀效果。
上世纪80年代以前,羊踯躅遍及华中、华南,西南地区也有少量分布。但由于人们的滥挖乱采及生境的破坏,羊踯躅的生殖环节受到严重影响,羊踯躅野外分布的地区和种群数量急剧减少,栖息地已呈“岛屿化”,有些省份野外已难见其踪影,羊踯躅濒于灭绝的风险日益加剧,亟待加以保护。
遗传多样性分析是评价和保护稀有濒危物种的重要指标,能够反映一个物种适应环境的能力和对环境变迁持续进化的潜力和效应,它能为确定濒危植物优先保护种群和制定有效的取样策略提供重要依据。简单重复序列(simple sequence repeat,SSR),广泛存在于真核基因组中,具有共显性、高度重复性、高度丰富性等优点,是研究群体遗传多样性的理想工具,但羊踯躅的多态性SSR分子标记还很缺乏。为此本发明利用我们此前对羊踯躅简化基因组测序(RAD-seq)的数据,合成SSR分子标记引物,鉴定筛选一批多态性SSR分子标记,应用这些多态性SSR分子标记评估分析濒危羊踯躅遗传多样性和遗传结构,为该物种的保护提供科学依据和参考。
发明内容
本发明的目的在于提供一种濒危羊踯躅多态性SSR分子标记开发与应用的方法,提供一批可用于群体基因组学、系统发生学及比较基因组学等研究的SSR引物,应用这些多态性SSR分子标记评估分析濒危羊踯躅遗传多样性和遗传结构,为该物种的保护提供科学依据和参考。
本发明通过以下技术方案来实现的:
1、羊踯躅SSR分子标记的获得:利用简化基因组测序技术对濒危羊踯躅(Rhododendron molle G.Don)基因组进行测序,经序列数据过滤、聚类和组装等步骤,得到171.534Mb的基因组序列,包含498,252contigs,测序质量合格,GC分布正常,采用SSR搜索软件对contig序列进行SSR检测搜索,最后从11,961微卫星位点中开发得到11,687SSR分子标记;
2、SSR分子标记的筛选与合成:从11,687SSR分子标记中选取94对SSR进行遗传多样性的筛选鉴定,SSR分子标记初选的条件是2个碱基重复单元的SSR需要有10~15个拷贝的重复,3个碱基重复单元的SSR需要有6~12个拷贝的重复,开发设计好的SSR分子标记引物序列合成;
3、羊踯躅多态性SSR的鉴定:采用不同地理分布的羊踯躅DNA对合成94对SSR引物进行PCR扩增及聚丙烯酰胺凝胶电泳检测,最后从94对SSR引物中获得16对的多态性SSR分子标记,见表1;
4、羊踯躅多态性SSR分子标记的有效性评估:将筛选获得16对多态性SSR标记在2个羊踯躅自然居群,每个居群含21个个体,进行PCR扩增,评估分析了这16对多态性SSR标记能够用于羊踯躅的遗传多样性分析和保护生物学研究,见表1;
5、多态性SSR分子标记应用:运用本研究获得的多态性SSR分子标记在8个羊踯躅自然居群,含193个个体,进行了遗传多样性分析,获得了一些保护羊踯躅的科学依据,即羊踯躅Shannon多样性指数(I)为1.768,基因多样性指数H为0.777,江西省金溪县居群的遗传变异最丰富,而福建政和的遗传多样性水平最低,见表2,PCA分析和UPGMA分析都表明8个自然群体被分为两组,江西永修居群独立为一组,羊踯躅最好以就地保护为主,应优先保护金溪、永修居群,同时增加对政和和京山居群的保护权重。
本发明的技术效果是:本发明提供一批可用于群体基因组学、系统发生学及比较基因组学等研究的SSR引物,应用这些多态性SSR分子标记评估分析濒危羊踯躅遗传多样性和遗传结构,为该物种的保护提供科学依据和参考。
附图说明
图1为引物H88在京山自然居群中的PCR扩增的聚丙烯酰胺凝胶电泳结果,该居群共22个个体,每个个体扩增效果良好。
图2为基于12对SSR分子标记对8个羊踯躅自然居群扩增电泳后,经条带统计,利用Genalex 6.1和PowerMarker V3.25软件进行分析得到的羊踯躅群体间聚类图,永修居群为独立居群。
在图中,JZ代表安徽金寨居群,PA为浙江磐安,YX为江西永修,JS为湖北京山,JX为江西金溪,SD为湖南邵东,PY为江西鄱阳,ZH为福建政和。
具体实施方式
下面将结合附图1、2来详细说明本发明所具有的有益效果,旨在帮助阅读者更好地理解本发明的实质,但不能对本发明的实施和保护范围构成任何限定。
1、羊踯躅SSR分子标记的获得:采用改良的2×CTAB法,提取羊踯躅幼嫩叶片中的基因组DNA,检测合格的DNA样品交于北京诺禾致源生物信息科技有限公司,采用IlluminaHiSeq/MiSeq(Illumina HiSeqTM2500/MiseqTM测序平台进行简化基因组技术(RAD-seq),测序完成后的Raw Data或Raw Reads,经序列数据质控、过滤,用cd-hit-est聚类软件进行聚类,用VelvetOptimiser组装软件对筛选过后的每类测序数据进行组装,得到171.534Mb的基因组序列,包含498,252 contigs,采用SSR搜索软件对contig序列进行SSR检测搜索,最后从11,961微卫星位点中开发得到11,687SSR分子标记;
2、SSR分子标记的筛选与合成:从11,687SSR分子标记中选取94对SSR进行羊踯躅的遗传多样性的筛选鉴定,SSR分子标记初选的条件是2个碱基重复单元的SSR需要有10~15个拷贝的重复,3个碱基重复单元的SSR需要有6-12个拷贝的重复,开发设计好的SSR分子标记引物序列合成;
3、羊踯躅多态性SSR的PCR扩增与鉴定:2014年和2015年的4月~5月,采集8个羊踯躅自然居群,合计193份羊踯躅的幼叶,包括江西省的金溪县,鄱阳县和永修县,福建省的政和县,安徽省的金寨县,湖南省的邵东县,湖北省的京山县,浙江省的磐安县,采用改良的2×CTAB法,提取羊踯躅幼嫩叶片中的基因组DNA,从不同居群中选取8个羊踯躅个体的DNA对合成94对SSR引物进行PCR扩增及聚丙烯酰胺凝胶电泳检测,PCR扩增体系(15μl)如:7.5μl2×Taq PCR green mix(北京鼎国昌盛生物技术有限公司提供),2.0μl上、下引物,4μlddH2O,1.5μl基因组DNA,PCR扩增程序为:94℃预变性3min,94℃变性45s,55℃(部分引物56℃)退火45s,72℃复性1min,35个循环,72℃延伸7min,最后4℃保存,扩增产物用8%非变性聚丙烯酰胺凝胶电泳检测,以150V为恒定电压,电泳90分钟,银染染色,最后获得16对具有多态性的SSR分子标记(HD24*,HD25,HD26*,HD34*,HD35*,HD37,HD39,HD43*,HD55*,HD62*,HD63*,HD64,HD74,HD80,HD83*,HD88*),它们的序列信息见表1;
4、16对羊踯躅多态性SSR分子标记的评估:将筛选获得16对多态性SSR标记在安徽金寨和浙江磐安两个羊踯躅自然居群(每个居群含21个个体)中进行PCR扩增与电泳检测,参照20bp DNA ladder,根据分子量大小对扩增结果读带,记录碱基数,无条带记为“0”,得到SSR表型数据,利用Genalex 6.1软件各SSR位点在不同自然居群中的等位基因数,观测杂合度、期望杂合度以及是否符
Hardy-Weinberg定律等遗传参数(见表1),结果发现每对SSR标记的等位基因数为4-16个,杂合度(He)范围为0.489-0.908,其中HD24,HD26,HD34,HD35,HD43,HD55,HD62,HD63,HD83和HD88偏离Hardy-Weinberg定律,其余的7对SSR标记至少是在一个自然居群中不偏离Hardy-Weinberg定律,表明这些SSR分子标记可以用于羊踯躅的遗传多样分析和羊踯躅的保护与利用研究;
5、多态性SSR分子标记的应用:为了研究羊踯躅的遗传多样性、有效地保护该物种,从16多态性SSR分子标记中选取扩增效果最好,条带最清晰的12对SSR分子标记(见表2)在8个羊踯躅自然居群(含193个个体)遗传多样性和遗传结构的评估分析(参照图1所示),开展羊踯躅的保护遗传学的研究与应用,PCR扩增、扩增产物的检测及SSR条带统计的方法同上述步骤,统带结束后,利用Genalex 6.1软件计算遗传多样性指数,遗传距离(D)和遗传一致度(I);利用POPGENE计算Nei’s多样性指数(H),遗传分化系数(Fst),基因流(Nm)等。在此基础上利用Genalex 6.1软件进行主成分分析(Principal coordinate analysis,PCA),并分析羊踯躅居群间,居群内的遗传分化水平等,另外,采用PowerMarker V3.25软件对各位点进行分析,得多态信息量(PIC)指数,并进行非加权算术平均聚类(Unweighted pair-group method with arithmetic mean,UPGMA),结果发现羊踯躅Shannon多样性指数(I)为1.768,基因多样性指数(H)为0.777,江西省金溪县居群的遗传变异最丰富,而福建政和的遗传多样性水平最低(见表2);PCA分析和UPGMA分析都表明8个自然群体被分为两组,江西永修居群独立为一组(图2),建议羊踯躅最好以就地保护为主,应优先保护金溪、永修居群,同时增加对政和和京山居群的保护权重。
表1羊踯躅16对多态性引物序列特征及其在个自然居群中的遗传特性
*表示显著偏离哈德温伯定律(p<0.05);F:正向引物;R:反向引物;A:等位基因数;Ho:观测杂合度;He:期望杂合度.
表2羊踯躅8个自然居群的遗传多样性
以上所述的实施例仅仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案作出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (1)
1.一种濒危羊踯躅多态性SSR分子标记开发与应用的方法,其特征在于,
(1)濒危羊踯躅SSR分子标记的获得:利用简化基因组测序技术对濒危羊踯躅基因组进行测序,经序列数据过滤、聚类和组装步骤,得到171.534 Mb的基因组序列,包含498,252contigs,测序质量合格,GC分布正常,采用SSR搜索软件对contig序列进行SSR检测搜索,最后从11,961微卫星位点中开发得到11,687 SSR分子标记;
(2)SSR分子标记的筛选与合成:从11,687 SSR分子标记中选取94对SSR进行遗传多样性的筛选鉴定,SSR分子标记初选的条件是2个碱基重复单元的SSR需要有10~15个拷贝的重复,3个碱基重复单元的SSR需要有6~12个拷贝的重复,开发设计好的SSR分子标记引物序列合成;
(3)濒危羊踯躅多态性SSR的鉴定:采用不同地理分布的羊踯躅DNA对合成94对SSR引物进行PCR扩增及聚丙烯酰胺凝胶电泳检测,最后从94对SSR引物中获得16对的多态性SSR分子标记,见表1;
(4)濒危羊踯躅多态性SSR分子标记的有效性评估:将筛选获得16对多态性SSR标记在2个羊踯躅自然居群,每个居群含21个个体,进行PCR扩增,评估分析了这16对多态性SSR标记能够用于羊踯躅的遗传多样性分析和保护生物学研究,见表1;
(5)多态性SSR分子标记应用:运用本研究获得的多态性SSR分子标记在8个濒危羊踯躅自然居群,含193个个体,进行了遗传多样性分析,获得了一些保护濒危羊踯躅的科学依据,即濒危羊踯躅Shannon多样性指数I为1.768,基因多样性指数H为0.777,江西省金溪县居群的遗传变异最丰富,而福建政和的遗传多样性水平最低,见表2,PCA分析和UPGMA分析都表明8个自然群体被分为两组,江西永修居群独立为一组,羊踯躅以就地保护为主,应优先保护金溪、永修居群,同时增加对政和和京山居群的保护权重;
表1 濒危羊踯躅16对多态性引物序列特征及其在个自然居群中的遗传特性
* 表示显著偏离哈德温伯定律; F: 正向引物; R: 反向引物; A: 等位基因数; H o :观测杂合度; H e : 期望杂合度;
表2 濒危羊踯躅8个自然居群的遗传多样性
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