CN106434942A - 铁线莲耐热性基因位点qHR1的分子标记方法 - Google Patents
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Abstract
本发明提供了铁线莲耐热性基因位点qHR1的分子标记方法,属于分子遗传学领域。利用44对SSR和22对SRAP分子标记确定铁线莲属127份材料的基因型结合其耐热指数进行全基因组关联连锁分析,通过GLM程序和MLM程序均检测到铁线莲耐热性主效基因位点qHR1,分别解释8.96%和5.47%表型变异,SSR分子标记CNSSR80与之极显著相关。本发明不但有助于解决我国铁线莲耐热育种进展迟缓的问题,而且有助于解决现有育种技术存在的育种成本高、时间长、稳定性差等技术难题,将加快我国铁线莲耐热新品种培育。
Description
技术领域
本发明提供了铁线莲耐热性基因位点qHR1的分子标记方法,属于分子遗传学领域,专用于定向培育耐热的铁线莲新品种。
背景技术
铁线莲属(Clematis)为毛茛科(Ranunculaceae)植物,多年生木质藤本。铁线莲属观赏品种的花型、花色丰富多样,花朵美丽繁茂,花期从早春至晚秋,具有单年多次开花的特性,观赏价值极高,是非常优良的垂直绿化材料,享有“攀援植物皇后”的美称。铁线莲属植物在欧洲等国的立体园林绿化中已占据十分重要的地位,亦受到我国园林界和爱好者的喜爱。但是铁线莲栽培品种大部分不耐热,夏季高温易导致铁线莲嫩叶萎蔫,花朵畸形,花期缩短,因此培育耐热性强的铁线莲品种已成为我国乃至世界铁线莲育种的目标之一。
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。
前期研究发现铁线莲材料间的耐热性存在显著差异,根据综合耐热指标可以将其分为不耐热、耐热性中等、较耐热和耐热等4个组 (马育珠,李林芳,高露璐等. 利用隶属函数法对铁线莲属苗期耐热性的综合评价. 中国观赏园艺研究进展, 2016,348-354.)。因此,对铁线莲群体开展田间耐热指数鉴定和关联分析,可能检测到铁线莲耐热性主效基因或分子标记。目前还未见相关研究的论文或专利。
发明内容
一、技术问题:
1 铁线莲材料间的耐热性差异显著,其中,大花园艺系品种因花大色艳、花期长等特点受到喜爱,但是夏季高温天气导致花期变短、花朵畸形和易枯,嫩枝叶萎蔫,影响铁线莲的夏季观赏品质。提高铁线莲栽培品种的耐热性,将是铁线莲育种的方向之一。
2 铁线莲花卉耐热性育种存在周期长、费时费力等问题,开展早代选择有利于缩短育种周期、降低育种成本,分子标记是开展早代选择的重要工具之一,因此开发相关分子标记对铁线莲耐热性育种具有重要意义。
二、技术方案:
利用SSR分子标记引物CNSSR80的正向引物序列5’- ACAAGAGAAAACCCGAAA -3’和反向引物序列5’- GCAGTCCCAGCCGAG -3’结合PCR技术扩增铁线莲嫩叶的基因组DNA,如果能扩增出230 bp 的DNA片段,则表明铁线莲耐热主效基因位点qHR1的存在,利用TASSEL2.1软件的GLM程序和MLM程序,测得其对铁线莲耐热性位点的分别解释8.96%和5.47%的表型变异。
三、有益效果:
本发明首次公布了一个铁线莲耐热性的主效基因位点及其分子标记,该标记将有利于缩短铁线莲耐热性的育种周期,降低育种成本,定向培育铁线莲耐热性强的铁线莲品种,促进铁线莲栽培品种的推广,最终为提高铁线莲栽培品种在我国夏季高温天的观赏性奠定基础。
四、具体实施方式
(一) 铁线莲耐热指数的统计分析
2014和2015年6-9月,高温(T≥30℃)持续三天后观察记录江苏省中国科学院植物研究所铁线莲苗圃中的供试材料的热害症状。热害症状分级标准参照文献(康俊根,2002)并略有改动,系统全面地观察铁线莲高温伤害的表现(叶色、新叶、老叶、花)及发生规律,观察项目及级数为:(1)叶色,0级:正常,无热害症状;1级:小部分叶黄;2级:大部分叶黄;3级:枯黄发黑 .(2)新叶萎蔫程度,0级:正常,无热害症状;1级:新叶萎蔫;2级:新叶部分干枯;3级:新叶完全干枯。(3)老叶萎蔫程度,0级:正常,无热害症状;1级:基部叶片部分萎蔫;2级:基部叶片完全萎蔫;3级:整株叶片干枯.(4)花朵萎蔫程度,0级:正常,无热害症状;1级:花瓣部分萎蔫;2级:花瓣完全萎蔫;3级:花朵枯黄。每个品种或野生种选3株进行观测。根据2014年和2015年观察记录的热害性状,分别计算出供试材料的热害指数。热害指数=Σ各株级数/(最高级数×总株数)×100%。
(二) 铁线莲群体遗传结构分析
利用CTAB法提取铁线莲嫩叶基因组DNA,然后用100对SSR引物和106对SRAP引物随机PCR扩增12个铁线莲家系DNA,结果发现44对SSR引物和22对SRAP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44%。44对SSR引物的多态性频率介于19%~83%,平均的多态性频率为52%,22对SRAP引物的多态性频率介于12%~62%,平均的多态性频率为34%,(表1)。SSR和SRAP引物由英潍捷基(上海)贸易有限公司合成。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10 min,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127份铁线莲材料的基因型数据。利用Structure 2.1软件结合基因型数据对铁线莲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 ofindividuals using the software STRUCTURE: a simulation study. Molecularecology, 2005, 14(8): 2611-2620.)通过△K来确定K值。△K在K=2时出现峰值,所以127份铁线莲材料可被分为2个亚群。将K=2代入Structure软件重新运算,得到各个材料的对应Q值,作为下一步耐热指数与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。
表1 44对SSR和SRAP引物的扩增片段多态性
引物 | 多态性条带 | 多态率(%) | 引物 | 多态性条带 | 多态率(%) | 引物 | 组合 | 多态性条带 | 多态率(%) |
SSR1 | 4 | 40% | SSR63 | 3 | 72% | SRAP1 | Me2 Em11 | 3 | 33 % |
SSR2 | 3 | 60% | SSR66 | 3 | 77% | SRAP4 | Me1 Em16 | 3 | 50 % |
SSR5 | 3 | 28% | SSR68 | 3 | 57% | SRAP8 | Me11 Em16 | 4 | 22 % |
SSR8 | 3 | 52% | SSR69 | 3 | 37% | SRAP14 | Me10 Em5 | 3 | 12 % |
SSR15 | 3 | 61% | SSR70 | 3 | 66% | SRAP15 | Me9 Em3 | 4 | 24% |
SSR20 | 3 | 44% | SSR72 | 3 | 53% | SRAP16 | Me9 Em2 | 4 | 12% |
SSR26 | 3 | 38% | SSR74 | 3 | 83% | SRAP19 | Me8 Em15 | 5 | 16% |
SSR31 | 3 | 28% | SSR76 | 3 | 80% | SRAP21 | Me8 Em13 | 6 | 19% |
SSR34 | 3 | 19% | SSR78 | 3 | 72% | SRAP22 | Me8 Em11 | 4 | 33% |
SSR35 | 3 | 28% | SSR80 | 3 | 61% | SRAP23 | Me7 Em7 | 3 | 39% |
SSR36 | 3 | 40% | SSR82 | 3 | 55% | SRAP24 | Me7 Em6 | 3 | 47% |
SSR37 | 3 | 67% | SSR84 | 3 | 49% | SRAP25 | Me7 Em5 | 4 | 55% |
SSR38 | 3 | 83% | SSR85 | 3 | 44% | SRAP26 | Me7 Em4 | 5 | 21% |
SSR39 | 3 | 51% | SSR86 | 3 | 42% | SRAP27 | Me7 Em3 | 3 | 49% |
SSR49 | 3 | 31% | SSR90 | 3 | 45% | SRAP28 | Me7 Em2 | 4 | 32% |
SSR51 | 3 | 34% | SSR91 | 3 | 38% | SRAP29 | Me7 Em1 | 3 | 62% |
SSR53 | 3 | 73% | SSR92 | 3 | 65% | SRAP30 | Me6 Em18 | 4 | 24% |
SSR56 | 3 | 32% | SSR93 | 3 | 69% | SRAP31 | Me6 Em16 | 4 | 51% |
SSR57 | 3 | 54% | SSR95 | 3 | 67% | SRAP32 | Me6 Em15 | 3 | 22% |
SSR58 | 3 | 37% | SSR96 | 3 | 27% | SRAP33 | Me6 Em13 | 3 | 39% |
SSR59 | 3 | 21% | SSR97 | 3 | 71% | SRAP41 | Me4 Em15 | 3 | 24% |
SSR62 | 3 | 55% | SSR98 | 3 | 73% | SRAP42 | Me1 Em15 | 3 | 62% |
(三) 铁线莲耐热性的关联分析
利用TASSEL2.1软件的GLM程序和MLM程序以127份材料对应的Q值为协变量,将44个SSR标记和22个SRAP标记与耐热指数进行线性回归分析,结果检测到1个耐热性的QTL,命名为qHR1。QTL位点qHR1紧密连锁的标记为CNSSR80,解释的表型变异率分别为8.96%和5.47%。CNSSR80的正向引物序列是:5’- ACAAGAGAAAACCCGAAA -3’,反向引物序列为:5’-GCAGTCCCAGCCGAG -3’,扩增片段大小为230 bp。
SEQUENCE LISTING
<110> 江苏省中国科学院植物研究所
<120> 铁线莲耐热性基因位点qHR1的分子标记方法
<130> 说明书
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Clematis apiifolia
<220>
<221> primer_bind
<222> (1)..(18)
<220>
<221> CNSSR80 正向引物
<222> (1)..(18)
<400> 1
acaagagaaa acccgaaa 18
<210> 2
<211> 15
<212> DNA
<213> Clematis apiifolia
<220>
<221> CNSSR80 反向引物
<222> (1)..(15)
<400> 2
gcagtcccag ccgag 15
Claims (1)
1.铁线莲耐热性基因位点qHR1的分子标记方法,其特征在于:利用SSR分子标记引物CNSSR80的正向引物序列5’- ACAAGAGAAAACCCGAAA -3’和反向引物序列5’-GCAGTCCCAGCCGAG -3’结合PCR技术扩增铁线莲嫩叶的基因组DNA,如果能扩增出230 bp 的DNA片段,则表明铁线莲耐热性基因位点qHR1的存在。
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Non-Patent Citations (5)
Title |
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LINFANG LI ET AL: "Association analysis of heat-resistance traits in Clematis", 《EUR J HORTIC SCI》 * |
XIAOYAN SUN ET AL: "Association of SSR markers with functional traits from heat stress in diverse tall fescue accessions", 《BMC PLANT BIOL》 * |
徐玉仙: "EST-SSR标记开发及莲种资源遗传多样性分析", 《中国优秀硕士学位论文全文数据库 农业科技辑 D048-76》 * |
陈永霞 等: "SSR标记与扁穗牛鞭草农艺性状关联分析", 《湖北农业科学》 * |
马育珠 等: "利用隶属函数法对铁线莲属苗期耐热性的综合评价", 《中国观赏园艺研究进展》 * |
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CN110129336A (zh) * | 2019-05-31 | 2019-08-16 | 上海植物园 | 铁线莲clhsp18基因编码序列及其应用 |
CN110129336B (zh) * | 2019-05-31 | 2024-02-27 | 上海植物园 | 铁线莲clhsp18基因编码序列及其应用 |
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