CN106967805A - 基于高通量测序筛选的乌龟微卫星dna标记 - Google Patents

基于高通量测序筛选的乌龟微卫星dna标记 Download PDF

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CN106967805A
CN106967805A CN201710211233.XA CN201710211233A CN106967805A CN 106967805 A CN106967805 A CN 106967805A CN 201710211233 A CN201710211233 A CN 201710211233A CN 106967805 A CN106967805 A CN 106967805A
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聂刘旺
李军
石琼
汪玉芹
刘谏君
王梦
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Abstract

本发明公开了基于高通量测序方法筛选的乌龟微卫星DNA标记，包括乌龟简化基因组测序文库的构建，测序及利用MISA软件筛选微卫星序列，在得到987个候选的微卫星的序列的基础上，确定了15个多态性丰富的微卫星标记SLN01、SLN02、SLN03、SLN04、SLN05、SLN06、SLN07、SLN08、SLN09、SLN10、SLN11、SLN12、SLN13、SLN14、SLN15；本发明提供了15个乌龟的新的微卫星位点及扩增这15个微卫星位点的引物序列和扩增方法，可应用于乌龟不同种群的遗传多样性研究以及系统进化的研究，是一种可靠有效的分子标记。

Description

基于高通量测序筛选的乌龟微卫星DNA标记
技术领域
本发明涉及分子标记技术，具体为基于高通量测序筛选新的乌龟微卫星DNA标记。
背景技术
在过去的几十年中，由于人为因素的影响和栖息地环境的改变，导致了野生乌龟种群数量急剧下降。目前该物种已经被列入了2004年的《中国濒危动物红皮书》和2006年IUCN濒危动物物种红色名录中。可见，对乌龟的保护工作已经刻不容缓，想要制定出对濒危动物行之有效的保护措施，就必须先要了解该物种的遗传多样性等信息。
微卫星(Microsatellite,MS)又称短串联重复序列或简单重复序列。是指以少数几个核苷酸(多数为2～4个)为单位的多次串联重复序列，重复单位以二核苷酸较为常见，重复次数多为15～60次，总长度一般在400bp以下。由于在不同的生物个体内重复基序的重复次数不同而显示出多态性。研究表明，DNA复制过程中的“链滑”(strand slippage)现象造成微卫星DNA多态性的主要原因。目前，几乎所有的基因组上都能发现微卫星的存在。由于其具有较高的多态性，且为小分子共显性标记，很容易扩增与检验，微卫星检测技术已被广泛应用于遗传变异分析、遗传图谱构建、物种起源、动植物遗传育种、医学等领域的研究。
传统的微卫星标记开发过程是先构建目标物种的基因组文库，利用滤膜富集法或磁珠富集法富集特异性的DNA片段。通过测序，设计引物获得微卫星标记。这一过程流程比较简单，但工作繁琐且效率低下。
发明内容
本发明要解决的技术问题在于：分离和鉴定乌龟微卫星DNA标记，建立基于高通量测序方法的乌龟多态性微卫星DNA标记的筛选技术体系并利用这些分子标记进行乌龟的亲权鉴定及遗传多样性的分析。
本发明解决技术问题的技术方案：基于高通量测序筛选乌龟多态性微卫星DNA标记，包括a)获得含有微卫星的序列；b)微卫星引物的设计：将a步骤获得的序列用MISA软件筛选，得到996个含有较高重复数的微卫星序列，再通过oligo7.0软件设计46对引物；c)乌龟多态性微卫星位点引物的筛选及分析，确定了15个多态性丰富的微卫星标记SLN01、SLN02、SLN03、SLN04、SLN05、SLN06、SLN07、SLN08、SLN09、SLN10、SLN11、SLN12、SLN13、SLN14、SLN15；SLN01为229个核苷酸、SLN02为348个核苷酸、SLN03为359个核苷酸、SLN04为324个核苷酸、SLN05为381个核苷酸、SLN06为314个核苷酸、SLN07为377个核苷酸、SLN08为246个核苷酸、SLN09为396个核苷酸、SLN10为376个核苷酸、SLN11为381个核苷酸、SLN12为324个核苷酸、SLN13为374个核苷酸、SLN14为494个核苷酸、SLN15为500个核苷酸。
附图说明
图1：SLN01位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图2：SLN02位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图3：SLN03位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图4：SLN04位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图5：SLN05位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图6：SLN06位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图7：SLN07位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图8：SLN08位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图9：SLN09位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图10：SLN10位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图11：SLN11位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图12：SLN12位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图13：SLN13位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图14：SLN14位点的引物扩增20个乌龟个体基因组DNA的STR分型图。
图15：SLN15位点的引物扩增20个乌龟个体基因组DNA的STR分型图
在图1-15中，1-20分别代表1-20个乌龟个体。
具体实施方式
下面结合附图进一步详述本发明。
a)获得含有微卫星的序列
参照sambrook等人(2002,Molecular Cloning,3nd463-471)的方法提取乌龟基因组DNA，并用1％的琼脂糖电泳检测其完整性。DNA样本送到生物公司进行RAD(简化基因组)测序。
参照Bonatelli等人(2015，Using Next Generation RAD Sequencing toIsolate Multispecies Microsatellites for Pilosocereus(Cactaceae).10(11):e0142602)的方法获得微卫星序列。
b)微卫星引物的设计
将a步骤获得的乌龟微卫星序列用MISA软件筛选，得到996个含有重复数较高的微卫星序列，利用oligo7软件对含有微卫星位点的序列设计引物，共设计引物46对。引物长度为22～26bp，GC含量在40％～60％之间，引物Tm值小于68℃，且上下游引物Tm值差别不能大于4℃，扩增引物长度在150～500bp。对这46对引物进行PCR扩增检测。PCR反应程序如下：95℃预变性5分钟，94℃变性40秒，退火45秒，72℃延伸1分钟，变性至退火三个步骤重复34次，72℃充分延伸10分钟，反应结束后用1％琼脂糖凝胶电泳检测。检测结果显示有26对引物能稳定扩增出目的条带，对这26对引物的上游引物5＇端分别用FAM,HEX,TAMRA进行荧光标记。
c)乌龟多态性微卫星位点引物的筛选
使用a所示方法提取20只乌龟的基因组DNA；
用b工序设计所得的26对引物扩增基因组,PCR程序如b所示。PCR产物经ABI PRISM3730遗传分析仪(Applied Biosystem公司)进行基因分型(Genotyping)，使用Genemarker1.6软件(Applied Biosystem公司)判读等位基因片段的具体数值。共得到15个多态性丰富的微卫星标记SLN01、SLN02、SLN03、SLN04、SLN05、SLN06、SLN07、SLN08、SLN09、SLN10、SLN11、SLN12、SLN13、SLN14、SLN15；SLN01为229个核苷酸、SLN02为348个核苷酸、SLN03为359个核苷酸、SLN04为324个核苷酸、SLN05为381个核苷酸、SLN06为314个核苷酸、SLN07为377个核苷酸、SLN08为246个核苷酸、SLN09为396个核苷酸、SLN10为376个核苷酸、SLN11为381个核苷酸、SLN12为324个核苷酸、SLN13为374个核苷酸、SLN14为494个核苷酸、SLN15为500个核苷酸。
结果分析：
使用Genepop4.5软件完成期望杂合度和观察杂合度、哈迪一温伯格平衡的计算，以此来描述20个乌龟微卫星DNA多态位点的特征。
由图1-15可看出，本发明的15个微卫星标记在供试的20只乌龟中都出现的多态性，由此可见，本发明的这15个微卫星标记能用于研究乌龟的遗传关系分析。
本发明提供了15个乌龟的新的微卫星位点及扩增这15个微卫星位点的引物序列和扩增方法，可应用于乌龟不同地理种群的遗传多样性研究，重复性好，是一种可靠有效的分子标记。
表1 15个多态性微卫星位点的相关信息
SEQUENCE LISTING
<110> 安徽师范大学
<120> 基于高通量测序筛选的乌龟微卫星DNA标记
<130> 1
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 229
<212> DNA
<213> Chinemys reevesii
<400> 1
tgagtgtatg agcatgttgt tttattgtag gagtgtgcct gcatgtcgtg tgcatctgtg 60
tatgctatgt gtgtatgtat taatgtgtga ctcttcattt ttgtgtgtgc aggagcacag 120
gtgtattcag gaggcgtgtg tgtgtcattg taacagtgtg tgtgtgtgtg tgtgtgccta 180
ctttttgacc cgtgcgtgtt attctgtgtg ttctcccttc agcctgggg 229
<210> 2
<211> 348
<212> DNA
<213> Chinemys reevesii
<400> 2
ttagcattgg tgagaattac accttttcac ttcatttcta tagcttgatt tttcttgcag 60
ctcagaacaa ttctctctct ctctctctca ctaattctca cacacacaca cacacacaca 120
cacaaaagta agggatgaca aaatgtctct gcaggacaag ttaacctcat catattgatg 180
catctttatt catactcccc aagacaatga cattgtgaca cttgaggtga aaccttggca 240
ctattgaaat caatggcaaa actcccaatg ccttcagtag gaacaggatt tcaccctggg 300
ggaacatgtt gccatagcac agtatcccag ttgcctcatc ttttctgt 348
<210> 3
<211> 359
<212> DNA
<213> Chinemys reevesii
<400> 3
aaaggtttgt aagttagcat tagctgcagt acgtgcacat ttctcgggag aatggacaga 60
aggagcacca ctgttatacc tcagagcctg catatttttt gtgtgtgtgt gtgtgtgtgt 120
ggtcaccctt gacatatgtg agtggaaaga acctgtcatt tatcattaga tgaaatagtc 180
aagatttcca cttacaagag atgtactcaa ggcattcagc aattataccc agcaggaaat 240
atcaccaatg acaaactcgt aatggtacta attgctgaac attacattag aagaatgact 300
acacaatggt gcctaggaca tttacttttt attgtaaaaa aattctaaat gttcacaat 359
<210> 4
<211> 324
<212> DNA
<213> Chinemys reevesii
<400> 4
agcaaaaaaa aggaaactgc agcagcaaga aggatgcaga tgccagggcg ggatcacaga 60
gcaccacgga gtttgcttca gaggaagatg agcagactca gagtggacaa aacagtgagg 120
ttcaagagac cctcccaaat tctgcaggag gaggaggagt ttgttttaaa gcccagttcg 180
atcacctcca aaggcagccg gggaggagga acagccctgc tgcggcagcg tgtgtgtgtg 240
tgtgtgtgtg tggccacgga attgtgacac tggcgtgtgt gtgatacaca atccctggga 300
gaagcggctg ccccctgggt gggg 324
<210> 5
<211> 381
<212> DNA
<213> Chinemys reevesii
<400> 5
tgtgtgtgtg tgcgcacgcg cacggaaaca gtgtcccatg tcctcccagc caaacaggaa 60
ggctgctgtg gcagggacaa taggttactg tctgtgaagc agaggagctg acaccttacc 120
cagcagcctg gaaatgctgg gagaactggg ctgcagggga catgcagccg tgtgtgtgtg 180
tgtgtgtgtg tgtgtgtgtg tgtggatttg catccatgtc ccccaaatcc ctgcccagat 240
accacgctct gagtgccccc agcctggatg gacacaggct cacagctctg ctggaggagg 300
agaatgggcc agtcattaag ggggctggac tgggagtcag gactcctggg gtctcacggg 360
attaggaacc aggattgcca g 381
<210> 6
<211> 314
<212> DNA
<213> Chinemys reevesii
<400> 6
attggaacgt caaactcctg aagtaggtgg cctgtcggca gggcttgggc tggctgggcc 60
ccagtacctg gagcgtgtgt gtagggagag aggaggtggg agctagtgac tagagcagga 120
ggtgacgctt ggcagacctg ggctgtgctc cagactctgc cactgagact cgcatgccct 180
gcccagtcta tgcctcggtt tcccctatgc ttcgagggag ctgtggagct atttgctgtt 240
tctggagctt tggcatgggt gcacacacac acacacacac acacatcccc cttccaccct 300
ccagatgcag ctta 314
<210> 7
<211> 377
<212> DNA
<213> Chinemys reevesii
<400> 7
ctcccccccg gttggtgcgc gagcagcggg ggcagccctc tgcgcaaaaa tgggtcctgg 60
gtcggcgctc agggggggcc ccgggctccc tgaggtttcc ttcgggcagg gggtgcgcag 120
ggctgcccgc cggccggctg caggctccga gaggaggagg aggaggaaga ggcagctcgc 180
cgcagcactt cccacgctgc catgctgacc ccggctgact ctgcctcagc cctggccgcc 240
gacttccgca atcaaaaggc aatttcctcg cgtccctctc cagaggcaca agggggtggt 300
tcctcgcgtc cctctccaga ggcaggcagg gcgcccaggc cgggctgccc cttgtgcccc 360
cctcagcctg ctccaag 377
<210> 8
<211> 246
<212> DNA
<213> Chinemys reevesii
<400> 8
cccgctcagc tcattgtcac cgtatgtctc ctgggtgctg ccagacgaca taccacggca 60
agtctacaca gcagcatccc cttgccttgc cttgccttgc cttgccttgc cttgccttgc 120
cttgcggaca gcagacggtg caatacgact gctaaccatc gttgtcccat cgttgtcccg 180
tgggtgcctc ggttaggttg gtcgggggca cctgggcaga catgggtgct cctggccagc 240
ctcggt 246
<210> 9
<211> 396
<212> DNA
<213> Chinemys reevesii
<400> 9
agaagggcca gacaagcagc caggaagcag gtcagagctg ggagcagagt cacagaagca 60
gcccacagag cagacctgtc ctggggcaga gctgtagcaa ccagagccag aggggccaga 120
gaagcagccc agggagctga aggcagagca gcagcagcag ctgtgctgag gcagagtgga 180
gccggagctg gagctggggc tggagcagtc cggagccggg tgcggtgagc agctggggag 240
aggagaggga ccctgggcag tgggcccagc gcagggagac gcctcagcca agaggctctg 300
caggccagac ttggggggga tcgtaaccct gacagggcgg gggcaacgct gggaagaagg 360
gtcctgccac ctagagcctg agagcgtgtg gccacc 396
<210> 10
<211> 376
<212> DNA
<213> Chinemys reevesii
<400> 10
ttgaatgtga gattatacag cataatatag tcctctctga ccagaagatt cagcaccttt 60
tttcccctct cttaacccag ttaaacttga atatgtgtac attttagtac tgtatgtata 120
tcttgtgctg tatgatgtga tttcctgtat tggtaggggt tgtttttttg ttgttgttgt 180
tgggtttttt tttaacatat aaaatggatc cattccagag tataccgaaa ggggtgacaa 240
tgtattgcag aacttagata ttgtaggatt ctgcatcgct ttacttagaa ataacactaa 300
ttggttaatt ctggtcattc tgatttatgc catatattta cttacgcaag ctgttctttt 360
tttcactgaa tttatt 376
<210> 11
<211> 381
<212> DNA
<213> Chinemys reevesii
<400> 11
agaggcaggg actactcctc ccactccggt ttccatggct agtagtggct gctgagaccg 60
tttctcctgt tctagctgca gcctcagcat ctccactagc tgctgcttct gtttcagcat 120
tcgggtgagt tcctcaatct gcctgtcctt ctcctgcaac atctgatctt tatcctggac 180
ctccatatcc ttgctgctgc tgctgctgca ccgctccaac ctggatgctg gatttaggct 240
gcaggaggta gccttagagc tctcttcctt catcaggaac tgcactggag atgcctgcag 300
ggtgagctgg gtcagaggcg aagtcaccgt ctctccaaag gtatctccag tggcagagtt 360
ctcgtcccca gtactcatct g 381
<210> 12
<211> 324
<212> DNA
<213> Chinemys reevesii
<400> 12
tcaacctttt gacatttatt tgtagcagtt gaattgaggt accggtgcat tattagcatt 60
taaacagtaa agtgttccag ctctaccata ataactgagt ttaagttgag tctttagcct 120
gaagtactct gtggaatgta aaaattaccc agacaaacaa acaaacaaac aaataaaaac 180
tgtaatggaa aagaaaatag taatccgatt tctatcttca caatgtgata aaacagaact 240
ccaaaagtgt gtaaagcatt aaggggcttg tacaaaccat cctgttgagt ttaaaacaaa 300
agaaaggtaa aggtggttca agaa 324
<210> 13
<211> 374
<212> DNA
<213> Chinemys reevesii
<400> 13
cggccatctt tttttttgct tcgggcggca aaagcctaga gccggccctg gcagcagcag 60
cgcgtcgccc tggggccact gcggttcgcg ctgtggggga gcagcacctg caccttgtgg 120
ctgggccggg caggctccga gtaggggaca ctcgggctgg gggccacccc gcggggcaca 180
cggagccgcc tgcaggtgct gcagggcggc cgccccccag caccgcagct ggggctgggc 240
gaagcggccc gagccaccca gggactgcgg cagggcggcc agaagcagca gcagcagcgg 300
ggccatagag gggccgtgtg gggcccgcgt cccgctctcc agggatccgc tccctctggg 360
gctaccctgc cccg 374
<210> 14
<211> 494
<212> DNA
<213> Chinemys reevesii
<400> 14
ataaaattgg gtcccctccc ccttttctcg ttttgccttt ttttttaaaa tttcctcctc 60
ccgagaagaa ggatttggag ccagcgtgaa ggataaaaag cctggttgct ccctaggagg 120
cgcgccgaag gagcaggagg gagaaggaag gaaggaagga aggaagttag agagaagctc 180
tggcctcagg ccaaggagtg cgcatgaacc gcacagttaa ggaagcgctc cggaaggtag 240
caaagcacgc ggggaaagat cggccggatt gtctctgcca tccgcagcag tagcagccta 300
agaacaatca tgtaaccctt tcgcgtgctg tttggacatc tcatgcgcca aacgactgag 360
cctagaaccg gaaatgggtc cccgtttcgc aaactaaacg gctgacggaa ataacgacgt 420
ttggattctc tgcagaacag gacgtcccgg aacccgggcg ccaagcgctg cttcaccacc 480
gccattttca cccg 494
<210> 15
<211> 500
<212> DNA
<213> Chinemys reevesii
<400> 15
cagcttgtac tgcacctttg gaacaatgaa ggggtcacac agggagtccg cacttttgct 60
ttctgcttcg ggctcttctt ggggtttggg caggaggggg gacacctcac ggggtgcatt 120
tttctgctct tcttgtttgg gggattcttt ggcagggtct ttgtctgggg tagcagcccc 180
cagggggact ggggtttggc ttcctttggg ctgttgctgc tgctgcactt tctgctgctg 240
ctgctgcact tgcctctgct gctgctgctg ctgctgctgc tgctgcaacg cctcctgcag 300
actctgctga tattgctggt actgctgcag caaggagctg ggggacaggc ctagcagagc 360
ctgggacaga gcagggctgt aagggaacag gccttccatg ccgtacatgg gctgcaggta 420
cccgctctgc aaggccccgg gaatctgagg ggcgtaatag ggtgaaaagc ccgggacgaa 480
gtagggaagg aattggcttg 500

Claims (2)

1.基于高通量测序筛选的乌龟微卫星DNA标记，其特征在于：所述的微卫星标记编号分别为：SLN01、SLN02、SLN03、SLN04、SLN05、SLN06、SLN07、SLN08、SLN09、SLN10、SLN11、SLN12、SLN13、SLN14、SLN15；
其核苷酸序列为：
SLN01
SLN02
SLN03
SLN04
SLN05
SLN06
SLN07
SLN08
SLN09
SLN10
SLN11
SLN12
SLN13
SLN14
SLN15
2.根据权利要求1所述的基于高通量测序筛选的乌龟微卫星DNA标记，其特征在于：所述的SLN01、SLN02、SLN03、SLN04、SLN05、SLN06、SLN07、SLN08、SLN09、SLN10、SLN11、SLN12、SLN13、SLN14、SLN15；扩增15个微卫星位点的引物序列为：
SLN01F:CATGTCGTGTGCATCTGT
R:CAGAATAACACGCACGGGTC。
SLN02F:GCTTGATTTTTCTTGCAGCTCAG
R:TCCCCCAGGGTGAAATCCTGTTC
SLN03F:GCACCACTGTTATACCTCA
R:TAAATGTCCTAGGCACCA
SLN04F:GAGGAGCTGACACCTTACCCA
R:CTGTGAGCCTGTGTCCATCC
SLN05F:GCACGGAAACAGTGTCCCAT
R:CCTGACTCCCAGTCCAGCCCCCT
SLN06F:ATTGGAACGTCAAACTCCTGA
R:GTGTGTGTGCACCCATGCCAA
SLN07F:CAAAAATGGGTCCTGGGTCG
R:TCTGGAGAGGGACGCGAGGAA
SLN08F:CAGACGACATACCACGGCAAG
R:CACCCACGGGACAACGAT
SLN09F:AAGCAGCCCACAGAGCAGACCT
R:AGGACCCTTCTTCCCAGCGTT
SLN10F:TTTTCCCCTCTCTTAACCC
R:GGTCTCATATGGCTTTCCC
SLN11F:AGACCGTTTCTCCTGTTCTAGC
R:GTGACTTCGCCTCTGACCCA
SLN12F:AAGTGTTCCAGCTCTACC
R:GCCCCTTAATGCTTTACAC
SLN13F:CTTCGGGCGGCAAAAGCCT
R:CCCCTCTATGGCCCCGCTGCT
SLN14F:CCCCTTTTCTCGTTTTGCCTT
R:GTCGTTATTTCCGTCAGCCGTT
SLN15F:CTTTTGCTTTCTGCTTCGGGCTC
R:CCTATTACGCCCCTCAGATTCCC。
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