CN1328390C - 苎麻微卫星dna标记 - Google Patents
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Abstract
本发明涉及一种苎麻的DNA分子遗传标记,提供了15个苎麻微卫星位点的DNA序列及扩增上述15个苎麻微卫星位点的引物序列和扩增方法,15个苎麻微卫星位点用于苎麻品种分类、遗传关系分析和标记辅助育种,重复性好,是一种可靠有效的分子标记。
Description
技术领域
本发明涉及分子标记技术,具体涉及一种苎麻的DNA分子遗传标记。
背景技术
微卫星(又称为简单重复序列,SSR)是由1~6个核苷酸组成的短序列串联重复多次而组成的一段DNA,广泛分布于真核基因组中。微卫星序列两侧有保守的DNA序列,根据其两侧的保守序列设计引物,可用PCR扩增出微卫星位点的序列。由于微卫星中重复单元的重复的次数不同,因而扩增出的微卫星序列的长度呈现多态性(即SSRLP或SSR)。由于其具有多态性丰富、重复性高、共显性遗传等优点,被广泛应用于遗传图谱的构建、基因定位、遗传关系分析,品种鉴定等领域。如用微卫星标记分析苎麻品种的亲源关系(Zhou等,2005,Progress in Natural Science,15:137-142)、用微卫星DNA标记进行猪品种分类的专利申请“适于猪品种分类的猪微卫星DNA标记”(公开号CN 1370834A)。
苎麻(又称为“中国草”,种名为Boehmeria nivea(L.)Gaud)属于荨麻科苎麻属,是我国重要的纤维经济作物。苎麻的品种分类、遗传关系分析等一般是根据形态、生理指标进行,准确度低、且受生长季节、发育时期等的限制。苎麻的分子标记的研究起步较晚,有研究者尝试用RAPD(随机扩增多态性DNA)标记进行了苎麻品种亲源关系分析,取得了成功(郭安平.RAPD.湖南农业大学博士学位论文,1999.)。但是,RAPD标记在绝大多数情况下是显性标记,不能区别纯合基因型与杂合基因型。如何解决苎麻品种分类、遗传关系分析中存在的准确度低及受生长季节、发育时期限制的问题,是开展苎麻研究迫切需要解决的技术。
发明内容
本发明旨在分离克隆微卫星DNA标记,建立苎麻的微卫星DNA的技术体系并利用这些分子标记进行品种分类、遗传关系分析和辅助育种。
实现上述发明目的的技术方案包括苎麻(CT/AG)n微卫星富集文库的构建、含微卫星序列的阳性克隆的筛选及测序,得到15个含有(CT/AG)n微卫星克隆,确定了15个多态性丰富的微卫星标记BN19、BN20、BN21、BN22,BN23,BN24、BN25、BN26、BN27、BN28、BN29、BN30、BN31、BN32、BN33;BN19为674个核甘酸、BN20为571个核甘酸、BN21为653个核甘酸、BN22,BN23,BN24均为760个核甘酸、BN25为529个核甘酸、BN26为472个核甘酸、BN27为338个核甘酸、BN28为366个核甘酸、BN29为542个核甘酸、BN30为670个核甘酸、BN31为1068个核甘酸、BN32为649个核甘酸、BN33为326个核甘酸。
附图说明
图1是用BN19位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图2是用BN20位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图3是用BN21位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图4是用BN22位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图5是用BN23位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图6是用BN24位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图7是用BN25位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图8是用BN26位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图9是用BN27位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图10是用BN28位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图11是用BN29位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图12是用BN30位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图13是用BN31位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图14是用BN32位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
图15是用BN33位点的特异性引物扩增25个苎麻品种DNA的PAGE电泳图
具体实施方式
1:苎麻(CT/AG)n微卫星富集文库的构建
(1)按郭安平等方法(参见郭安平等,1997,中国麻作,19:4-10)提取苎麻基因组DNA。用限制性内切酶Mse I酶切苎麻基因组DNA,酶切产物在1.2%琼脂糖凝胶上电泳,紫外光下切下300-1000bp大小的DNA片段并用凝胶回收试剂盒(购自Qiagen公司)纯化回收的酶切产物。
(2)将碱基互补的寡核苷酸PHC-1(5’-AGATGGAATTCGTACACTCGT-3’)和磷酸化的寡核苷酸PHC-2(5’-phos-TAACGAGTGTACGAATTCCATCT-3’)等摩尔混合,95℃变性5分钟,60℃退火1小时,形成具有Mse I粘性末端的接头。
(3)将酶切产物和接头用T4DNA连接酶连接。连接产物用等体积的酚/氯仿/异戊醇(25∶24∶1)抽提一次,离心收集上清液。在上清液中加0.3M醋酸钠(pH5.2)和2倍体积的无水乙醇沉淀,70%乙醇清洗一次,真空抽干DNA后溶于50μl无菌水中。
(4)用生物素标记的寡核苷酸探针(CT)15与连接产物DNA片段杂交。具体操作为:在总体积100μl杂交液(6×SSC,0.1%SDS)中加入500ng连接产物和100pmol生物素标记的寡核苷酸探针,并在95℃下变性10min,60℃杂交1小时。
(5)加入100μl(10μg/μl)磁珠(Dynabeads M-280,购自Dynal Biotech公司),并在室温放置30分钟。
(6)用磁铁吸附磁珠,弃上清。磁珠用400μl洗涤缓冲液(6×SSC,0.1%SDS)在60℃下洗涤15分钟,然后在室温下用分别用2×SSC和1×SSC溶液洗涤两次,并弃上清。
(7)最后加入50μl无菌水,在90℃下保温10分钟,小心吸取上清,上清液即为含有(GA)n重复序列的单链DNA片段。以PHC-1为引物,以单链DNA片段为模板进行PCR扩增获得双链目的片段。
(8)将扩增产物纯化后连接到pMD18-T载体(购自大连宝生物公司)上,然后将连接产物转化大肠杆菌DH5α感受态细胞,转化菌液涂抹在含有IPTG和X-gal的LB固体培养基平板上进行蓝白斑筛选。
2.含微卫星序列的阳性克隆的筛选、测序及微卫星引物的设计
(1)挑取白色菌落并接种到3ml LB培养液中,于37℃培养过夜,然后提取质粒。
(2)质粒DNA为模板,采取PIMA方法(参见Lunt等,1999,Molecular Ecology,8:891-894)筛选含有(CT/AG)n微卫星序列的阳性克隆。
(3)将阳性克隆测序,分析是否含有(CT/AG)n重复序列。通过测序,共得到了16个含(CT/AG)n微卫星序列的阳性克隆。
(4)根据微卫星重复序列两侧的保守序列设计引物。
3.利用苎麻微卫星DNA标记分析苎麻品种间的遗传差异
将上述微卫星标记用于25个苎麻品种的分类,确定了15个多态性丰富、适于苎麻品种分类的微卫星标记BN19、BN20、BN21、BN22,BN23,BN24、BN25、BN26、BN27、BN28、BN29、BN30、BN31、BN32、BN33(表1)。具体操作过程如下:
(1)按郭安平等方法(参见郭安平等,1997,中国麻作,19:4-10)提取各个苎麻品种的DNA作为模板,进行PCR。
(2)分别用BN19、BN20、BN21、BN22,BN23,BN24、BN25、BN26、BN27、BN28、BN29、BN30、BN31、BN32、BN33。15个位点的15对引物(引物序列见表1)扩增这25个品种苎麻的DNA。
(3)将PCR产物在6%变性聚丙烯凝胶上电泳,然后进行银染并拍照,得到图1-18。
(4)结果分析:每个样品的扩增条带按有或无记录,扩增条带存在时赋值为1,否则赋值为0。采用SPSS10.0 for windows统计分析软件中的系统聚类分析程序,距离测量选用相关系数距离法,用类间平均连锁法(Between-groups linkage)进行聚类分析并建立系统树。
具体分析方法参见《系统与进化植物学中的分子标记》(邹喻苹、葛颂、王晓东编,科学出版社,2001年)中的方法进行。
由附图1-15可看出,本发明的15个微卫星点的序列长度在供试的25个品种中都出现多态性,由此可见,本发明的这15个微卫星标记能用于苎麻的品种分类和遗传关系分析。
表1 引物特性表:引物序列,退火温度,片段大小
位点 | 基序 | 引物序列(5’→3’) | 产物大小(bp) | 退火温度(℃) |
BN19BN20BN21BN22BN23BN24BN25BN26BN27BN28BN29BN30BN31BN32BN33 | (AG)40(GA)26(CT)28(AC)9(GAC)3C(CCGACG)3(ACG)2(CT)2(TCT)7(TCG)2(GA)29(AG)2(CT)21(GA)7T(TG)2(GAGAAA)2(GA)7(AG)8(GAA)5(AC)2(AG)18(GA)6(CT)22(GA)16(GTT)2(GA)8GG(GA)14(TC)9 | F:GCT AAC GCC CAT TTG TAT CTT GR:GGC CCA CCT GAC TTT GAG CF:TGG CAA AGG AAG GTG AAG AAC AAGR:TGA GGC ACG TGA CTG GGT ATC CF:AGA TGC TTT TGG CTA TGCR:TCA AAC GAT ATC ACA GAC AACF:GCC CAG GAA GCC ATT GAA GCR:GAC GAC GGC AAT GGC TCT CAF:TGC CGT CGT CTC TGC CAT TAT CGR:GCC CCA CCC AGC CCT ATC TTC AF:CTT CTT CGT CGG ATT CTT GTT CR:GCT TTT CTC TTC TCC TTC TTG TTTF:CTC TCT CAA CTC ATA CCA ATA ATR:AGA GAA CAG CCA ACA TCA AF:CGT GCC TAT GAC TTC GTT TCT CR:TTT CCA ATA ATT CAG CCT TCA CTTF:GGA CAT AAG AAC CCC CAA CAC TR:GGC CAC CCT ATA AAT ATC CTT CACF:TTC CGA TGA CAA ACC ACT GCTR:ACA CTC ACC AAC CCA TTA CAT CACF:CTT GCC GCT TCT TCA GTA GGT CTGR:GGC ATT TTC CAA GGG CAC AAF:AAT TAG CAG AGA AAA GGG CAG TTR:AAA GCG TGC GTC AAC ATT CAF:AAG GTA CGC CGC GAT TCT CR:TGG GCA AAG CAA CCT ACA CCF:CGC CAC AAA CAA TTA CCA CCA AR:AAT AGG ACA ACC CCA TGA GAG CACF:AAG GTA AAG TTT CTC TCT CTC TCR:AGA ATC TCC AAT CAC TAC CTT | 194bp218bp290bp216bp187bp269bp273bp201bp240bp282bp274bp316bp277bp267bp217bp | 60℃64℃51℃64℃69℃58℃51℃60℃61℃60℃64℃60℃61℃63℃51℃ |
表1中F表示5’端引物,R表示3’端引物。
本发明提供了15个苎麻微卫星位点的DNA序列及扩增上述15个苎麻微卫星位点的引物序列和扩增方法,15个苎麻微卫星位点用于苎麻品种分类、遗传关系分析和标记辅助育种,重复性好,是一种可靠有效的分子标记。
序列表
<110>中国农业科学院麻类研究所
<120>苎麻微卫星DNA标记
<160>13
<170>PatentIn version 3.3
<210>1
<211>674
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(193)..(272)
<220>
<221>重复单元
<222>(441)..(456)
<400>1
taaattgtga aggccatttt gggccttcac tttgttgaat gcccaaccca tttatgggcc 60
taacagcaag aaagctgtga aggctatttt cgaccttgtt ctagcgcctc ataagtggct 120
aacgcccatt tgtatcttgg ccaaatgtgt aagaaggcct tgaacctagt agtgggccac 180
ttaattactc aaagagagag agagagagag agagagagag agagagagag agagagagag 240
agagagagag agagagagag agagagagag aggggttttc tgagatgggc tagctcaaag 300
tcaggtgggc ctggcgttgg cccgaaatga ccaaactcga cccgacccgg aagtgaccaa 360
cctcgggtca agaagtgaca cgtggacaga agtacctaac tgtcagcctc aggtaatttc 420
atggaagtgg ataaagaaac agagagagag agagagcgcg agattttagc gccttttaac 480
cagacacaac cgttccaagt tttgaaaagc gctataaggg tgaatctgct ccacgacaga 540
ttcgatttca tcattacttt cgaacccaaa agacttcaat tgacgaaagc cttctttatc 600
taattgataa acaatatttt tgacacccgc aaatccatat aagtgcaata cttcagattt 660
tttcacgatc attt 674
<210>2
<211>571
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(340)..(411)
<400>2
taaggcttgt tggacctcca agtgagtccc taatacttat tataatatat tatatgttca 60
aatactcagt tgggatcaaa acaactttgt tagagtcgct tcggagccaa aaaactattt 120
tgaaggctat atttcgacaa aactagttat aacgatttac tttttgcgcg aaataaattt 180
ttaagcttgg agtagtcaag agtggttgaa gctctaacca ctagagattt tgagaagcgc 240
acagtatgag cgaaaatggg cttgtgggtt ttggtgtaga tggcaaagga aggtgaagaa 300
caagcgtaga atgtcgaagc agagaacgcc atcgaagggg agagagagag agagagagag 360
agagagagag agagagagag agagagagag aattctagaa aatctcttac ttgcttttta 420
cttgtaatct ccagattagc aaagtcgaca cgtggcgcaa cggacatccc gagttgggat 480
acccagtcac gtgcctcacg tgaagttggg acaaaatttt gtcccaacat ttacatttag 540
cataagatag tgttactgtg atatcccgat t 571
<210>3
<211>653
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(32)..(36)
<220>
<221>重复单元
<222>(309)..(328)
<220>
<221>重复单元
<222>(328)..(363)
<400>3
caagtgaata tcaagcatat agcttcttct ttctctctct cgtttccctt tttcccttct 60
tttcaggaca tttcagatac tttctggttt tatccaatgt tgcacaaaag aaaatatgat 120
tcactaacat gtcaatacgg gaaagagaac tcaatcaaat caaatcaaat caaacgatat 180
cacagacaac aatcacgtat catgaattac tggtcacaca tattcttgtg atagtcaaga 240
ctagatcaag aaatcatgca ttttcctttt atacatagca gagaaaacct tagacttgta 300
tatatgtctg tgtgtgtgtg tgtgtgtgag agagagagag agagagagag agagagagag 360
agagagagag agagagagag agaattttgc acgtaactgg caataattgg ccgtgtattc 420
caaaaaactt ggaaaatgaa ggcatagcca aaagcatctt caatcttcat ctccggggtc 480
gattggtaat ttacacaatc ttcttcttat gatatgttat tgtttgatca aagaaatttt 540
atgttgaacg atttttcatt gctgtttata acttcaggga aattacagaa atggacgctt 600
catgtcctga ttatgattat catctttacc ctttgaccag cttgcagatt ggg 653
<210>4
<211>760
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(601)..(658)
<400>4
taataataaa caataataat aataataata atcacctagc actggggact cttctgggta 60
gctggggctc gagctcagtg ggcaagccca ggaagccatt gaagcggtcg aaggtcacgt 120
gcgacacgtg cctgacctct gtgggccagc tgatctccaa cgccgacgac gaccccgacg 180
ccgacgccga cgacgacgtc gacgacacgt cgtctctctc gacgctgcat gtgactatgg 240
acttcctcag agccgtcacc agcacatcca cgatcgcgaa ctgatgagag ccattgccgt 300
cgtctctgcc attatcgccg ccgtggtaaa gccgagcctt gctgctaaac ggcgtcgtca 360
cggggttgta caacgcctcg tcttcgccgt cttcgtacaa ttcgttctcc gactcttctt 420
cttcttcttc ttcttcttcg tcggattctt gttcgtctat gaagataggg ctgggtgggg 480
ccgtgttgaa ctcggcgagc ccgcaagatt ttgaccggaa aagccgagtc atggcgagag 540
gtcaagatca gagaaaccaa aacagagtag tgtttgtgtg tttgatttgt tttgagagtt 600
gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagagaag 660
agccggcctt ggaagaaaca agaaggagaa gagaaaagct tgatttttcc aagctaaaac 720
gtaaggtaac ttagccagct tttcttatag tttgagtttt 760
<210>5
<211>529
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(306)..(347)
<400>5
taacaactca ccactgtgat ggaccaattg actcatcgtc aacagaatga ttgatcggct 60
cactttacaa cgatcggtcg gctccttgca atgaccgatt ggtccagttg gctcgtcacc 120
gacatgatga cgagttggtt ctccttctca acgttagatt ggctcgcttt ctggccacca 180
acataatgat taaataaatt ttattctctc tcaactcata ccaataatta cttcacaaat 240
atcatttttc tacttctttt atcttttctc tctaggctct ctctttcttc cgatcttctt 300
tcttttctct ctctctctct ctctctctct ctctctctct ctctctctgg aattctcgct 360
ccctctctgt ttcatctctc acactctccc tctctctcat atctgctctg atcactatac 420
aacggggtta gactttggag tttgaatatg ttattacgct tgatgttggc tgttctctct 480
tctaagctca aaactgaaat actttttttt ttactggtat tgatagaat 529
<210>6
<211>472
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(305)..(318)
<220>
<221>重复单元
<222>(335)..(348)
<400>6
taacccaact caagctgaag aaacttattc aatggtcacc gctaaccgct tttggtccca 60
aatctttggg gttgcttttt ccaataaacg ttggttacat ttctttatgt tatttgtacc 120
agtcactggt ttatggatga gtgctcttgg agtagtcggt ctggctttga atctacgtgc 180
ctatgacttc gtttctcaag aaatccgtgc agcggaagat cctgaatttg agactttcta 240
cacaaaaaat atcctcttaa tttgttgttt gtgtgtcagt gtttgtggtg atttcagggg 300
agacagagag agagagagat tgtgagaaag agaaagagag agagagagat gaaagtgaag 360
gctgaattat tggaaagata gatacagaga catgtcgttt gtgggttctg agttggaatt 420
ggttgagata taatcatgcc ggccaatggc aatcacggaa agtttagttt tt 472
<210>7
<211>338
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(220)..(235)
<400>7
taagatgtac caaagaaatt ggggggccag agaagcacaa gagagaggct aatctggaca 60
taagaacccc caacactttg gaagtggaat caatatatgc ctctagaaca aagccatcaa 120
gaaatcgatc atcagatctc aaacccttga agccgaaagg agaggcattt atactcaacc 180
cacgcacaca gagccaaaat gaaaattgct ttacaccaaa gagagagaga gagagggagt 240
aatgattcga ggcttagtga agagtagagt tgtgaaggat atttataggg tggcctaaaa 300
ccctaggttg tgaacgatgc tgagaagata tcagctgc 338
<210>8
<211>366
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(264)..(273)
<220>
<221>重复单元
<222>(291)..(300)
<400>8
acaaaacctc cccaatcaat ttcaagtttg gaggccgacg atggtgaggt ggatggctgc 60
ggcggctgtg cgattttcgg catagtccga cagtttccga tgacaaacca ctgctcaaaa 120
tattcctctc gtcgagagct ttccaacggt accaagtatg catcaatcgg acttaggatg 180
agaaagttat gagtgttcga attttgaaga atttttgaaa aaaataattg aggagagaaa 240
gagaattcga atttagagaa gaaagagaga gagggtgaca gaaatagggg gaagaagaag 300
aagaaaagga aaagaaaagg ggaaaaagaa aaagaaaaag tgatgtaatg ggttggtgag 360
tgtatt 366
<210>9
<211>542
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(194)..(229)
<220>
<221>重复单元
<222>(230)..(241)
<400>9
taatcatcaa tccatgtcgt tttcagcctt gccgcttctt cagtaggtct cacttgttct 60
ctaattgtcg ttttcacgtc tccttcgaca acaaaaacga cacgtcgttt acatcccccg 120
ctctcgcagc ctcaaaaacc cccaaaaacc ccaacaaatc tccttcggcg tttagcttct 180
cagattgaga cacagagaga gagagagaga gagagagaga gagagagagg agagagagag 240
aggggtgggt cagtacatgt ggcagattcg cacgtgcctc tttgtgccct tggaaaatgc 300
ccaaaattag cacttggcct ctgcaaaaac tagtactttg agttgagcaa catatatgaa 360
ttcacatttc cacgctaata cactatcttt cctattatct cttaaaagaa ataaaaaaaa 420
tttccaaaat acgaaggaac aattttcatt gaaactacga taaatcaatg ttcctagaca 480
tttgttgttg caaataccaa acataatcac tttcttcacc tcttgcatct tgaatttcct 540
tt 542
<210>10
<211>670
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(380)..(423)
<400>10
taagaaagac tttttttgac caaaatgatc atatcataca ttagtcaact tgattttgat 60
aaaatcaagg agcttggcat ttgaagaata gttctatatg tactactttt tttttagaaa 120
aaaatgtgat tgactttgat ataattaaac atttataaac cttttgaatt agcagagaaa 180
agggcagttg gttattattg agctagctca atacagtttg gtcaggctga gaaaattggc 240
gatataagta gatatctaat tttacctata ataagtcata ggcaaatgat gcactaaacg 300
caaatttcca agatgattta agggtccaaa atccttttat acttaggtga gtttttagag 360
agagtttcac ttgtgtcttt ctctctctct ctctctctct ctctctctct ctctctctct 420
ctctatctct atatccattt acagctccat tttctttatg aatgaatgtt gacgcacgct 480
ttcacctaac tctgctttct aatctaaaat caagattgga agagttttat gttttctttt 540
tcgacttcta gagataacat tttttttggg tattgcgtat gatgattgga tataaatgtt 600
gactttttaa agggcttttt ttggcaccga aatgactatg gcgaagttgt tttgatccca 660
aatgagtgtt 670
<210>11
<211>1068
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(683)..(714)
<400>11
taaaaaatgg agacaccatt atcatctaaa attcgaagaa aattcaaaag cagtgacagg 60
agttgtagac ttacacccat acgtttgccc ttgcctccag ccagaagaat gaccgaaacg 120
cttttttcct ttactaaacc aaccttttca gcagaacaac aaaccaacaa acttggcttt 180
gaaccaactc ttccacgtct cactgcacgc ccaacaattg caagaccatg taaagattca 240
gaatctattt aggaaaacag accaaaaaaa aaaaaaaggg gactacttgc ctgggaatgg 300
aagattgaaa tgcggaagtg tgtgagaaag agggatagtc ctcacatcgg aagaaaaagc 360
agaaaaggtg aggctcagac ggagagtggg agtgggtatg gccatatctg ttttagtgtt 420
ttcggattgg aagtggcatt cactgccact gctgactttg atacgcagag cacaacgcaa 480
cacagaatga agttcgggcg gcgatggatg gcttggggtt tctcgagacg atgggcggtg 540
gtcggagggg aaggtacgcc gcgattctca gcggcagagg cgacggcgtt cggcagtgat 600
tttgggtggt tcaattttgg gtttctctcg gaaaaagatg gcggtgaccg actttgttgc 660
cggcagttgg ataacaaaga tcgagagaga gagagagaga gagagagaga gagagtgagt 720
acgctgttcc tggaagtagt agtttccttg gaggcgacag ccgaggcgat cgatggcttg 780
gccggtctcg cggatccaga atccgacggt gtaggttgct ttgcccaaag tcccatctta 840
gcgagctcgg agtcgtcgtc ttcagtgggg atttaggttt ctgagtcaaa cagaccaaga 900
ttttgatttc gggagaagag aaatgatacc cgagaaagcg ggggattggg gtataaaacg 960
acgactcacc ggtcgtggat gttttttttt gacaattacg tgattcccat tttaccaggg 1020
gacgaattca tctatcctaa agattccgta ccgactcaat tcaacttg 1068
<210>12
<211>649
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(331)..(346)
<220>
<221>重复单元
<222>(349)..(376)
<400>12
taatcgcctt actagttgct gtactgatat acttgggagc gaagaaaaga aaaatgatca 60
agctcaagga gaaattcttt gtagaaaatg gaggattgat gttacaacaa caacttgcta 120
gtagaagacc agtcgagacg acaaaaatat tttctgagga cgaactgaga agcgccacaa 180
acaattacca ccaaaagaga gttctaggag aaggaggatt tggaatagtg tataaaggaa 240
tatttccaga tcaaagggtt gttgcaatta ataacacctc aaaaaagctc agaaaaaaaa 300
aaaaaccaga aaccaaaatc atgtttgttt gagagagaga gagagaggga gagagagaga 360
gagagagaga gagagagtcc aagtctcttt gtccttctac gccagcctct gacacgtgct 420
ctcatggggt tgtcctattc tcatctcatc tcatcatgct tggccttggc ctttgccttc 480
ccaatttttc ctctgtaaca ctcttctatt caaaataatc tcatattgtt gatattcaat 540
caaaagaaga ggcgctattg tttatttgtt ttcatactgc aaattctacg cgtatatgta 600
cgcacatgaa aatgtattag agttattatt gcgttattta taagtgaga 649
<210>13
<211>326
<212>DNA
<213>苎麻(Boehmeria nivea(L.)Gaud)
<220>
<221>重复单元
<222>(26)..(43)
<400>13
attctctctc ttcaaaggta aagtttctct ctctctctct ctcctttttt ttttctgtga 60
atgtatagta atggaatgaa tgtcaagaaa ccatgctcat gcaagttctg cgatgatagt 120
tttggtgacg ggtttcgctt ctgttctctt agtttgtgcc gaatttagca ttttcgtagg 180
catttcttag ggtcggaagg tagtgattgg agattctagt ttcggtaatg gcggatcttt 240
aggttctact ctctgattgt ggtttttttc tgtatggttg tgttcgttgc gtgttggatc 300
tgtgggaaat tttggatttt ttttgg 326
Claims (2)
1.一种苎麻微卫星标记,其特征在于,所述微卫星标记的编号分别为BN19、BN20、BN21、BN22、BN23、BN24、BN25、BN26、BN27、BN28、BN29、BN30、BN31、BN32、BN33,其核苷酸序列是:
BN19:
taaattgtga aggccatttt gggccttcac tttgttgaat gcccaaccca tttatgggcc 60
taacagcaag aaagctgtga aggctatttt cgaccttgtt ctagcgcctc ataagtggct 120
aacgcccatt tgtatcttgg ccaaatgtgt aagaaggcct tgaacctagt agtgggccac 180
ttaattactc aaagagagag agagagagag agagagagag agagagagag agagagagag 240
agagagagag agagagagag agagagagag aggggttttc tgagatgggc tagctcaaag 300
tcaggtgggc ctggcgttgg cccgaaatga ccaaactcga cccgacccgg aagtgaccaa 360
cctcgggtca agaagtgaca cgtggacaga agtacctaac tgtcagcctc aggtaatttc 420
atggaagtgg ataaagaaac agagagagag agagagcgcg agattttagc gccttttaac 480
cagacacaac cgttccaagt tttgaaaagc gctataaggg tgaatctgct ccacgacaga 540
ttcgatttca tcattacttt cgaacccaaa agacttcaat tgacgaaagc cttctttatc 600
taattgataa acaatatttt tgacacccgc aaatccatat aagtgcaata cttcagattt 660
tttcacgatc attt 674
BN20:
taaggcttgt tggacctcca agtgagtccc taatacttat tataatatat tatatgttca 60
aatactcagt tgggatcaaa acaactttgt tagagtcgct tcggagccaa aaaactattt 120
tgaaggctat atttcgacaa aactagttat aacgatttac tttttgcgcg aaataaattt 180
ttaagcttgg agtagtcaag agtggttgaa gctctaacca ctagagattt tgagaagcgc 240
acagtatgag cgaaaatggg cttgtgggtt ttggtgtaga tggcaaagga aggtgaagaa 300
caagcgtaga atgtcgaagc agagaacgcc atcgaagggg agagagagag agagagagag 360
agagagagag agagagagag agagagagag aattctagaa aatctcttac ttgcttttta 420
cttgtaatct ccagattagc aaagtcgaca cgtggcgcaa cggacatccc gagttgggat 480
acccagtcac gtgcctcacg tgaagttggg acaaaatttt gtcccaacat ttacatttag 540
cataagatag tgttactgtg atatcccgat t 571
BN21:
caagtgaata tcaagcatat agcttcttct ttctctctct cgtttccctt tttcccttct 60
tttcaggaca tttcagatac tttctggttt tatccaatgt tgcacaaaag aaaatatgat 120
tcactaacat gtcaatacgg gaaagagaac tcaatcaaat caaatcaaat caaacgatat 180
cacagacaac aatcacgtat catgaattac tggtcacaca tattcttgtg atagtcaaga 240
ctagatcaag aaatcatgca ttttcctttt atacatagca gagaaaacct tagacttgta 300
tatatgtctg tgtgtgtgtg tgtgtgtgag agagagagag agagagagag agagagagag 360
agagagagag agagagagag agaattttgc acgtaactgg caataattgg ccgtgtattc 420
caaaaaactt ggaaaatgaa ggcatagcca aaagcatctt caatcttcat ctccggggtc 480
gattggtaat ttacacaatc ttcttcttat gatatgttat tgtttgatca aagaaatttt 540
atgttgaacg atttttcatt gctgtttata acttcaggga aattacagaa atggacgctt 600
catgtcctga ttatgattat catctttacc ctttgaccag cttgcagatt ggg 653
BN22,BN23,BN24:
taataataaa caataataat aataataata atcacctagc actggggact cttctgggta 60
gctggggctc gagctcagtg ggcaagccca ggaagccatt gaagcggtcg aaggtcacgt 120
gcgacacgtg cctgacctct gtgggccagc tgatctccaa cgccgacgac gaccccgacg 180
ccgacgccga cgacgacgtc gacgacacgt cgtctctctc gacgctgcat gtgactatgg 240
acttcctcag agccgtcacc agcacatcca cgatcgcgaa ctgatgagag ccattgccgt 300
cgtctctgcc attatcgccg ccgtggtaaa gccgagcctt gctgctaaac ggcgtcgtca 360
cggggttgta caacgcctcg tcttcgccgt cttcgtacaa ttcgttctcc gactcttctt 420
cttcttcttc ttcttcttcg tcggattctt gttcgtctat gaagataggg ctgggtgggg 480
ccgtgttgaa ctcggcgagc ccgcaagatt ttgaccggaa aagccgagtc atggcgagag 540
gtcaagatca gagaaaccaa aacagagtag tgtttgtgtg tttgatttgt tttgagagtt 600
gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagagaag 660
agccggcctt ggaagaaaca agaaggagaa gagaaaagct tgatttttcc aagctaaaac 720
gtaaggtaac ttagccagct tttcttatag tttgagtttt 760
BN25:
taacaactca ccactgtgat ggaccaattg actcatcgtc aacagaatga ttgatcggct 60
cactttacaa cgatcggtcg gctccttgca atgaccgatt ggtccagttg gctcgtcacc 120
gacatgatga cgagttggtt ctccttctca acgttagatt ggctcgcttt ctggccacca 180
acataatgat taaataaatt ttattctctc tcaactcata ccaataatta cttcacaaat 240
atcatttttc tacttctttt atcttttctc tctaggctct ctctttcttc cgatcttctt 300
tcttttctct ctctctctct ctctctctct ctctctctct ctctctctgg aattctcgct 360
ccctctctgt ttcatctctc acactctccc tctctctcat atctgctctg atcactatac 420
aacggggtta gactttggag tttgaatatg ttattacgct tgatgttggc tgttctctct 480
tctaagctca aaactgaaat actttttttt ttactggtat tgatagaat 529
BN26:
taacccaact caagctgaag aaacttattc aatggtcacc gctaaccgct tttggtccca 60
aatctttggg gttgcttttt ccaataaacg ttggttacat ttctttatgt tatttgtacc 120
agtcactggt ttatggatga gtgctcttgg agtagtcggt ctggctttga atctacgtgc 180
ctatgacttc gtttctcaag aaatccgtgc agcggaagat cctgaatttg agactttcta 240
cacaaaaaat atcctcttaa tttgttgttt gtgtgtcagt gtttgtggtg atttcagggg 300
agacagagag agagagagat tgtgagaaag agaaagagag agagagagat gaaagtgaag 360
gctgaattat tggaaagata gatacagaga catgtcgttt gtgggttctg agttggaatt 420
ggttgagata taatcatgcc ggccaatggc aatcacggaa agtttagttt tt 472
BN27:
taagatgtac caaagaaatt ggggggccag agaagcacaa gagagaggct aatctggaca 60
taagaacccc caacactttg gaagtggaat caatatatgc ctctagaaca aagccatcaa 120
gaaatcgatc atcagatctc aaacccttga agccgaaagg agaggcattt atactcaacc 180
cacgcacaca gagccaaaat gaaaattgct ttacaccaaa gagagagaga gagagggagt 240
aatgattcga ggcttagtga agagtagagt tgtgaaggat atttataggg tggcctaaaa 300
ccctaggttg tgaacgatgc tgagaagata tcagctgc 338
BN28:
acaaaacctc cccaatcaat ttcaagtttg gaggccgacg atggtgaggt ggatggctgc 60
ggcggctgtg cgattttcgg catagtccga cagtttccga tgacaaacca ctgctcaaaa 120
tattcctctc gtcgagagct ttccaacggt accaagtatg catcaatcgg acttaggatg 180
agaaagttat gagtgttcga attttgaaga atttttgaaa aaaataattg aggagagaaa 240
gagaattcga atttagagaa gaaagagaga gagggtgaca gaaatagggg gaagaagaag 300
aagaaaagga aaagaaaagg ggaaaaagaa aaagaaaaag tgatgtaatg ggttggtgag 360
tgtatt 366
BN29:
taatcatcaa tccatgtcgt tttcagcctt gccgcttctt cagtaggtct cacttgttct 60
ctaattgtcg ttttcacgtc tccttcgaca acaaaaacga cacgtcgttt acatcccccg 120
ctctcgcagc ctcaaaaacc cccaaaaacc ccaacaaatc tccttcggcg tttagcttct 180
cagattgaga cacagagaga gagagagaga gagagagaga gagagagagg agagagagag 240
aggggtgggt cagtacatgt ggcagattcg cacgtgcctc tttgtgccct tggaaaatgc 300
ccaaaattag cacttggcct ctgcaaaaac tagtactttg agttgagcaa catatatgaa 360
ttcacatttc cacgctaata cactatcttt cctattatct cttaaaagaa ataaaaaaaa 420
tttccaaaat acgaaggaac aattttcatt gaaactacga taaatcaatg ttcctagaca 480
tttgttgttg caaataccaa acataatcac tttcttcacc tcttgcatct tgaatttcct 540
tt 542
BN30:
taagaaagac tttttttgac caaaatgatc atatcataca ttagtcaact tgattttgat 60
aaaatcaagg agcttggcat ttgaagaata gttctatatg tactactttt tttttagaaa 120
aaaatgtgat tgactttgat ataattaaac atttataaac cttttgaatt agcagagaaa 180
agggcagttg gttattattg agctagctca atacagtttg gtcaggctga gaaaattggc 240
gatataagta gatatctaat tttacctata ataagtcata ggcaaatgat gcactaaacg 300
caaatttcca agatgattta agggtccaaa atccttttat acttaggtga gtttttagag 360
agagtttcac ttgtgtcttt ctctctctct ctctctctct ctctctctct ctctctctct 420
ctctatctct atatccattt acagctccat tttctttatg aatgaatgtt gacgcacgct 480
ttcacctaac tctgctttct aatctaaaat caagattgga agagttttat gttttctttt 540
tcgacttcta gagataacat tttttttggg tattgcgtat gatgattgga tataaatgtt 600
gactttttaa agggcttttt ttggcaccga aatgactatg gcgaagttgt tttgatccca 660
aatgagtgtt 670
BN31:
taaaaaatgg agacaccatt atcatctaaa attcgaagaa aattcaaaag cagtgacagg 60
agttgtagac ttacacccat acgtttgccc ttgcctccag ccagaagaat gaccgaaacg 120
cttttttcct ttactaaacc aaccttttca gcagaacaac aaaccaacaa acttggcttt 180
gaaccaactc ttccacgtct cactgcacgc ccaacaattg caagaccatg taaagattca 240
gaatctattt aggaaaacag accaaaaaaa aaaaaaaggg gactacttgc ctgggaatgg 300
aagattgaaa tgcggaagtg tgtgagaaag agggatagtc ctcacatcgg aagaaaaagc 360
agaaaaggtg aggctcagac ggagagtggg agtgggtatg gccatatctg ttttagtgtt 420
ttcggattgg aagtggcatt cactgccact gctgactttg atacgcagag cacaacgcaa 480
cacagaatga agttcgggcg gcgatggatg gcttggggtt tctcgagacg atgggcggtg 540
gtcggagggg aaggtacgcc gcgattctca gcggcagagg cgacggcgtt cggcagtgat 600
tttgggtggt tcaattttgg gtttctctcg gaaaaagatg gcggtgaccg actttgttgc 660
cggcagttgg ataacaaaga tcgagagaga gagagagaga gagagagaga gagagtgagt 720
acgctgttcc tggaagtagt agtttccttg gaggcgacag ccgaggcgat cgatggcttg 780
gccggtctcg cggatccaga atccgacggt gtaggttgct ttgcccaaag tcccatctta 840
gcgagctcgg agtcgtcgtc ttcagtgggg atttaggttt ctgagtcaaa cagaccaaga 900
ttttgatttc gggagaagag aaatgatacc cgagaaagcg ggggattggg gtataaaacg 960
acgactcacc ggtcgtggat gttttttttt gacaattacg tgattcccat tttaccaggg 1020
gacgaattca tctatcctaa agattccgta ccgactcaat tcaacttg 1068
BN32:
taatcgcctt actagttgct gtactgatat acttgggagc gaagaaaaga aaaatgatca 60
agctcaagga gaaattcttt gtagaaaatg gaggattgat gttacaacaa caacttgcta 120
gtagaagacc agtcgagacg acaaaaatat tttctgagga cgaactgaga agcgccacaa 180
acaattacca ccaaaagaga gttctaggag aaggaggatt tggaatagtg tataaaggaa 240
tatttccaga tcaaagggtt gttgcaatta ataacacctc aaaaaagctc agaaaaaaaa 300
aaaaaccaga aaccaaaatc atgtttgttt gagagagaga gagagaggga gagagagaga 360
gagagagaga gagagagtcc aagtctcttt gtccttctac gccagcctct gacacgtgct 420
ctcatggggt tgtcctattc tcatctcatc tcatcatgct tggccttggc ctttgccttc 480
ccaatttttc ctctgtaaca ctcttctatt caaaataatc tcatattgtt gatattcaat 540
caaaagaaga ggcgctattg tttatttgtt ttcatactgc aaattctacg cgtatatgta 600
cgcacatgaa aatgtattag agttattatt gcgttattta taagtgaga 649
BN33:
attctctctc ttcaaaggta aagtttctct ctctctctct ctcctttttt ttttctgtga 60
atgtatagta atggaatgaa tgtcaagaaa ccatgctcat gcaagttctg cgatgatagt 120
tttggtgacg ggtttcgctt ctgttctctt agtttgtgcc gaatttagca ttttcgtagg 180
catttcttag ggtcggaagg tagtgattgg agattctagt ttcggtaatg gcggatcttt 240
aggttctact ctctgattgt ggtttttttc tgtatggttg tgttcgttgc gtgttggatc 300
tgtgggaaat tttggatttt ttttgg 326。
2.根据权利要求1所述的苎麻微卫星标记,其特征在于所述BN19、BN20、BN21、BN22-23-24、BN25、BN26、BN27、BN28、BN29、BN30、BN31、BN32、BN33十五个位点的引物序列为:
BN19-F:GCT AAC GCC CAT TTG TAT CTT G
BN19-R:GGC CCA CCT GAC TTT GAG C
BN20-F:TGG CAA AGG AAG GTG AAG AAC AAG
BN20-R:TGA GGC ACG TGA CTG GGT ATC C
BN21-F:AGA TGC TTT TGG CTA TGC
BN21-R:TCA AAC GAT ATC ACA GAC AAC
BN22-F:GCC CAG GAA GCC ATT GAA GC
BN22-R:GAC GAC GGC AAT GGC TCT CA
BN23-F:TGC CGT CGT CTC TGC CAT TAT CG
BN23-R:GCC CCA CCC AGC CCT ATC TTC A
BN24-F:CTT CTT CGTCGG ATT CTT GTT C
BN24-R:GCT TTT CTC TTC TCC TTC TTG TTT
BN25-F:CTC TCT CAA CTC ATA CCA ATA AT
BN25-R:AGA GAA CAG CCA ACA TCA A
BN26-F:CGT GCC TAT GAC TTC GTT TCT C
BN26-R:TTT CCA ATA ATT CAG CCT TCA CTT
BN27-F:GGA CAT AAG AAC CCC CAA CAC T
BN27-R:GGC CAC CCT ATA AAT ATC CTT CAC
BN28-F:TTC CGA TGA CAA ACC ACT GCT
BN28-R:ACA CTC ACC AAC CCA TTA CAT CAC
BN29-F:CTT GCC GCT TCT TCA GTA GGT CTG
BN29-R:GGC ATT TTC CAA GGG CAC AA
BN30-F:AAT TAG CAG AGA AAA GGG CAG TT
BN30-R:AAA GCG TGC GTC AAC ATT CA
BN31-F:AAG GTA CGC CGC GAT TCT C
BN31-R:TGG GCA AAG CAA CCT ACA CC
BN32-F:CGC CAC AAACAA TTA CCA CCA A
BN32-R:AAT AGG ACA ACC CCA TGA GAG CAC
BN33-F:AAG GTA AAG TTT CTC TCT CTC TC
BN33-R:AGA ATC TCC AAAT CAC TAC CTT。
Priority Applications (1)
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CNB2005100324228A CN1328390C (zh) | 2005-11-24 | 2005-11-24 | 苎麻微卫星dna标记 |
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CNB2005100324228A CN1328390C (zh) | 2005-11-24 | 2005-11-24 | 苎麻微卫星dna标记 |
Publications (2)
Publication Number | Publication Date |
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CN1814787A CN1814787A (zh) | 2006-08-09 |
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CN101805734B (zh) * | 2008-10-23 | 2012-02-01 | 浙江师范大学 | 一种棘胸蛙微卫星dna标记ⅱ及其用途 |
CN101805733B (zh) * | 2008-10-23 | 2012-02-01 | 浙江师范大学 | 一种棘胸蛙微卫星dna标记ⅲ及其用途 |
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CN102634512B (zh) * | 2009-12-31 | 2013-07-03 | 北京林业大学 | 一种适用于尾叶紫薇遗传多样性评价的微卫星分子标记 |
CN102634513B (zh) * | 2009-12-31 | 2013-07-03 | 北京林业大学 | 尾叶紫薇i2h微卫星分子标记的开发和应用 |
CN105274103B (zh) * | 2015-11-22 | 2018-05-25 | 保琦蓓 | 一种鉴别汉麻、苎麻、亚麻原麻纤维种类的pcr引物组以及pcr鉴别方法 |
CN109706264A (zh) * | 2018-12-04 | 2019-05-03 | 中国农业科学院麻类研究所 | 苎麻叶背毡毛主效qtl及其紧密连锁的分子标记和应用 |
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用微卫星DNA标记分析苎麻品种的亲缘关系 周建林 等,作物学报,第30卷第3期 2004 * |
用微卫星DNA标记分析苎麻品种的亲缘关系 周建林 等,作物学报,第30卷第3期 2004;苎麻栽培品种亲缘关系的RAPD分析 揭雨成 等,作物学报,第28卷第2期 2002 * |
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