CN105368971B - 楸树扦插生根率主效QTL位点qRR1的分子标记方法 - Google Patents
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Abstract
本发明提供了楸树扦插生根率主效QTL位点qRR1的分子标记方法,属于分子遗传学领域。利用70对SSR分子标记确定楸树87份无性系材料的基因型结合其扦插生根率进行全基因组关联连锁分析,检测到楸树扦插生根率主效QTL位点qRR1,解释14.58%表型变异,SSR分子标记CB093与之极显著相关。本发明不但有助于解决我国楸树育种进展迟缓的问题,而且有助于解决现有育种技术对提高楸树扦插生根率成本高、时间长、稳定性低等技术难题,将加快我国楸树新品种培育与无性系林业发展。
Description
技术领域
本发明提供了楸树扦插生根率主效QTL位点qRR1的分子标记方法,属于分子遗传学领域,专用于定向培育扦插易生根的楸树新品种。
背景技术
楸树(Catalpa bungei)属于紫葳科(Bignoniaceae)梓属(Catalpa)的优良珍贵用材树种。随着工业的发展和世界人口的增长、木材需求与日剧增,木材短缺已成为世界性问题,我国尤为突出。发展无性系林业成为缓解我国木材短缺问题的有效途径,而扦插生根率是制约无性系林业发展的瓶颈。楸树为扦插生根率低的树种,扦插生根困难限制了楸树无性系林业的发展。因此挖掘与扦插生根率相关的标记位点对培育扦插易生根的楸树新品种具有重要的意义。
SSR分子标记是目前应用最广泛的分子标记技术之一,被广泛的应用于基因定位、QTL定位、标记辅助育种和遗传连锁图谱的构建等方面。陈永霞等(陈永霞, 张新全, 马啸,谢文刚. SSR标记与扁穗牛鞭草农艺性状关联分析. 湖北农业科学, 2011, 50(7):1494-1498.)选用10对SSR引物对44份外部形态差异较大的扁穗牛鞭草进行扫描,进行性状与SSR标记的关联分析,其中找到18个SSR标记与8个农艺性状显著相关,并筛选出优异种质,加快扁穗牛鞭草的育种进程。
前期研究发现不同楸树无性系间扦插生根能力存在显著差异,根据生根率、单株生根数和单株最长根长等3个指标可以将其分为生根能力较差、生根能力中等、生根能力较好和易生根类等4个组 (马玲玲, 王鹏, 张振宇, 李林芳, 杨如同, 李亚. 梓属植物嫩枝扦插生根能力的评价. 北方园艺, 2014, (15): 72-77.)。该研究结果表明利用此群体开展关联分析,可能检测到扦插生根率相关的QTL位点或分子标记。目前还未见开展相关研究的论文或专利。
发明内容
本发明的目的是提供楸树扦插生根率主效QTL位点及其分子标记。
本发明的目的是通过以下技术方案实现的:利用SSR分子标记引物CB093的正向引物序列5’-CAAACGAATGTACGGAGGCT-3’和反向引物序列5’-GAGATTGAGGCAGAGGATGC-3’结合PCR技术扩增楸树嫩叶的基因组DNA,如果能扩增出370bp 的DNA片段,则标志着楸树扦插生根率主效QTL位点qRR1的存在,利用TASSEL2.1软件的GLM程序测得对楸树扦插生根率的贡献率为14.58%。
有益效果:本发明首次公布了一个楸树扦插生根率的主效QTL位点及其分子标记,该标记将有利于缩短楸树的育种周期,降低育种成本,定向培育扦插生根率高的楸树品种,进而加速楸树的推广,最终为在缓解我国的优质木材短缺的问题上发挥重要的作用。
具体实施方式
楸树扦插生根率统计分析:2013年3月上旬和2014年3月上旬,将87个楸树无性系的五年生枝条截成30cm长后均匀地卧置于平整的沙床内,然后覆盖3~5cm的细沙,浇透水。每周用500倍的多菌灵浊液喷洒浇透沙床。将泥炭和珍珠岩按1:1的体积比混匀后装进育苗盆(规格为15×15×20 cm)中并用500倍的多菌灵浊液喷洒浇透。当沙床内新萌发的嫩枝高度达到10~15cm时,去掉嫩枝的顶端,一周后,将嫩枝带踵取下,作为插穗。将插穗上的叶片连同叶柄一起剪掉,只剩顶部的一片叶子,并将其剪掉一半。将处理好的插穗基部浸蘸75%的酒精消毒30s,用清水冲洗干净,然后在3,000 mg/L的IBA中浸蘸1min,将插穗***育苗盘,每个穴杯插1根,深度为插穗长度的1/2~2/3。每个无性系扦插20个插穗,重复3次。扦插完之后用85%遮阴网遮盖防晒,自动喷雾装置喷雾给水。每周用500倍的多菌灵浊液喷洒浇透。扦插后70天统计各无性系生根率,结果发现楸树不同无性系间的生根率差异极大,生根率变幅为10.06%~95.80%。
楸树群体遗传结构分析:利用CTAB法提取楸树嫩叶基因组DNA,然后用277对SSR引物随机PCR扩增12个楸树无性系DNA,结果发现70对引物可以成功的扩增出条带清晰且多态性好的条带,合成引物的可用性为25.26%。70对SSR引物的多态性频率介于40%~100%,平均的多态性频率为87.17%,多态性丰富(表1)。SSR引物由南京思普金生物科技有限公司合成。PCR扩增体系为10 μL包括20 ng基因组DNA,2.5mM的MgCl2,0.5mM的dNTPs,20 ng的引物,0.5U TaqDNA聚合酶。PCR反应程序为:94℃预变性5 分钟, 94℃变性30 秒,57℃退火45秒,72℃延伸60 秒,循环32 次,最后72℃延伸5 分种。PCR反应在伯乐T100PCR仪上完成。PCR扩增产物检测:利用8%浓度的聚丙烯酰胺凝胶电泳,进行PCR产物检测,上样量为1.5 µL,电泳缓冲液为1×TBE,电压设为220 V,电泳至溴酚蓝带跑出胶最低端为止。胶片用银染方法染色:先用固定液(去离子水、10%乙醇、1%乙酸)固定10 min,再1.5%硝酸银溶液浸泡10min,去离子水迅速洗涤2遍后,显色液(去离子水、1.5%氢氧化钠、1%甲醛)显色10 min,最后用去离子水冲洗,放入胶片观察灯上拍照。以二进制记录SSR引物扩增结果,同一位点上具有相同迁移率的条带记为1,无带记为0,获得87个楸树无性系的基因型数据。利用Structure 2.1软件结合基因型数据对梓属群体结构进行分析,结果表明对应似然值lnP(D)随K值的增大而持续增大,因此参照Evanno等(Evanno G, Regnaut S, Goudet J.Detecting the number of clusters of individuals using the software STRUCTURE:a simulation study. Molecular ecology, 2005, 14(8): 2611-2620.)通过△K来确定K值。△K在K=7时出现峰值,所以87份楸树材料可被分为7个亚群。将K=7代入Structure软件重新运算,得到各个材料的对应Q值,作为下一步生根性状与SSR标记关联分析的协变量,可以有效降低群体结构对关联分析的影响。
楸树连锁不平衡分析:通过对70个SSR标记486个位点分析,表明在117,855个成对组合的SSR位点中,存在一定程度的LD。 统计概率(P<0.01)支持的LD成对位点77,106个,占全部位点组合的65.42%, 平均值为0.557, 值在0.4以上的LD位点组合数为42,523,占总LD位点组合数的55.15%,其中位于0.8-1之间的位点数最多,有29,667个,以上均说明位点间整体LD水平较高。
楸树扦插生根率的关联分析:利用TASSEL2.1软件的GLM程序以87个无性系对应的Q值为协变量,将70个SSR标记与生根率进行线性回归分析,结果检测到6个扦插生根率的QTL,分别命名为qRR1~qRR6。QTL位点qRR1紧密连锁的标记为CB093,解释的表型变异率为14.58%。CB093的正向引物序列是:5’-CAAACGAATGTACGGAGGCT-3’,反向引物序列为:5’-GAGATTGAGGCAGAGGATGC-3’,扩增片段大小为370 bp。
表1 70对SSR引物的扩增片段多态性
SEQUENCE LISTING
<110> 江苏省中国科学院植物研究所
<120> 楸树扦插生根率主效QTL位点qRR1的分子标记方法
<130> 说明书
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<220>
<221> CB093正向引物序列
<222> (1)..(20)
<223>
<400> 1
caaacgaatg tacggaggct 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<220>
<221> CB093反向引物序列
<222> (1)..(20)
<223>
<400> 2
gagattgagg cagaggatgc 20
Claims (2)
1.一种用于楸树扦插生根率主效位点检测的分子标记引物CB093,其特征在于:所述分子标记引物CB093的正向引物序列为5’-CAAACGAATGTACGGAGGCT-3’,反向引物序列为5’-GAGATTGAGGCAGAGGATGC-3’。
2.楸树扦插生根率主效QTL位点qRR1的分子标记方法,其特征在于:利用权利要求1所述分子标记引物CB093结合PCR技术扩增楸树嫩叶的基因组DNA,如果能扩增出370bp的DNA片段,则标志着楸树扦插生根率主效位点qRR1的存在。
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