CN114574564B - 一种高效的基于y染色体特异序列鉴定山羊性别的方法 - Google Patents
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Abstract
本发明公开了一种高效的基于Y染色体特异序列鉴定山羊性别的方法，通过已知性别个体的测序数据筛选Y染色体特异序列，并利用筛选到的序列与未知样品的重测序数据进行比对，直接通过比对率鉴定山羊的性别，以实现准确、高效、低成本的性别鉴定。
Description
技术领域
本发明属于动物性别鉴定技术领域，具体涉及一种基于Y染色体特异序列鉴定山羊性别的方法。
背景技术
山羊，是人类最早驯化的一种家畜，由于其具有繁殖率高、适应性强、易管理等特点，因而广泛饲养于我国的农牧地区。由于雌性和雄性山羊的适宜配种时期和饲养方式均存在一定的差异，不同性别的山羊通常独立饲养，以期获取品质较高的山羊。因此，性别区分在山羊的饲养过程中是不可或缺的。
在遗传学兴起之前，鉴定动物性别仅能通过雌雄外形来判定。在哺乳动物中，性别决定包括性染色体造成的初级性别决定和激素作用下的次级性别决定。哺乳动物的雌性性染色体组成为XX，雄性为XY，其中Y染色体只能通过雄性传递到子代的雄性个体中去。在早些时期，性别鉴定主要利用胚胎细胞染色体核型分析法，通过细胞在***中期的染色体形态判断是否存在Y染色体，从而确定胚胎性别。此方法准确性高，但实验操作难度大，耗时长，难以应用于生产实际中。另一种生物化学的方法是在胚胎的特定时期检测由X染色体调控的特异性酶活性，对着床前的胚胎进行性别鉴定。但这种方法有较多的限制：实验试剂可能存在毒性而降低胚胎存活力；不同物种早期胚胎特异性酶活性增强的准确时间尚不确定，且不同样品之间存在个体差异，容易产生误判；同时，此方法只能进行胚胎的性别鉴定，无法对出生后的个体进行鉴定。在生产中也使用免疫学方法来鉴定胚胎性别，利用H-Y抗原血清来检测胚胎中雄性特异性H-Y抗体的存在。这类方法相较之前的方法更为简便可行，但仍存在很多的问题：存在损害胚胎的风险；胚胎的生长时期存在个体差异，容易造成估计的不准确等。
随着分子生物学的发展，DNA水平的性别鉴定被广泛应用于生产实际中。目前应用的主要手段为聚合酶链式反应，即PCR技术。PCR技术主要是通过扩增Y染色体上的SRY基因并利用凝胶电泳检测SRY基因是否存在来鉴定性别。此外，在某些物种中，如在人类的Y染色体上还找到了特异性的微卫星标记，用于辅助性验证。近年来二代测序的快速发展，推动了人们对遗传信息的探索并完成了大量动物的基因组测序工作，这极大的促进了全基因组重测序工作的进行。高通量测序技术还可以获取到包含与性别决定相关的序列信息，为性别鉴定提供了可能。因此，需要一种新的思路，利用重测序数据对大规模群体进行准确有效的性别鉴定。目前山羊的Y染色体尚未被完整测序，根据人类和其他动物的Y染色体测序结果估计(Benjamin M Skinne et al,2016)，Y染色体除了5％的拟常染色体区域，其余95％为非重组区域，即NRY(non-recombining region of the Y chromosome)，不发生配对交换现象。NRY区包括异染色质序列和常染色质序列，其中常染色质序列包括X移位区(X-transposed)、X退化区(X-degenerate)和扩增区(Ampliconic)。山羊NRY区域存在大量的雄性特异序列，这些序列长短不一，与雌性山羊基因组仅仅具有极低或者完全没有同源性；因此，理论上通过雌雄序列比较可以筛选精准的Y染色体雄性特异序列集合，再通过生物学手段鉴定全基因组测序的山羊个体的性别。
发明内容
本发明的目的在于有效准确的鉴定已知重测序数据的山羊个体的性别，通过已知性别的个体数据筛选Y染色体特异序列，并利用筛选到的序列与未知样品的重测序数据进行比对，直接通过比对率差异区分公母，以鉴定个体的性别。
为了实现上述目的，本发明采用以下技术方案：
用于鉴别山羊性别的Y染色体特异序列，所述特异序列的核苷酸序列如SEQ IDNO.1～24所示。
上述Y染色体特异序列在山羊性别鉴定中的应用，包括以下步骤：将未知性别的山羊的全基因组重测序数据与上述Y染色体特异序列进行比对，根据比对率鉴定性别。
具体地，若比对率≥0.04％，则鉴定为雄性个体，若比对率为0.00％，则鉴定为雌性个体。
与现有技术相比，本发明具有以下优点及有益效果：
1、本发明采用序列相似性比对的方法，不需要通过具体的生物分子实验操作，不需要特定的鉴定时期，减少了实验成本。
2、本发明不需要生物样本组织，可以解决缺失原始样本组织而无法进行分子水平实验验证的问题。
3、本发明能够一体化运行，只需要提供相应物种的全基因组重测序数据和待测个体的全基因组测序，便可利用软件自动运行，可操作性强，耗时短并且结果准确性高。
4、本发明可解决没有SRY基因的物种的性别鉴定，应用更加广泛。
具体实施方式
实施例1山羊Y染色体特异序列的获得
一、山羊全基因组序列数据的获取
本实施例中所使用的山羊样本是从NCBI上下载的20头山羊的全基因组双端重测序数据(BioProject number：PRJEB3135)，其中雌性山羊13头，雄性山羊7头。具体信息如表1所示。
表1 下载的山羊全基因组重测序数据信息
二、样品质量控制
在ILLUMINA测序过程中需要添加接头序列，而这些序列可能作为原始测序数据的一部分被用于后续的序列比对，因此需要提前将这些接头序列去除以避免影响分析结果。利用Trimmomatic对下载的数据进行质量控制，去除接头并过滤质量低的reads，然后使用FastQC进行质量检测，使测序数据符合分析标准。
三、序列比对
从下载的样品中随机抽取性别分别为一公一母的两个样品，作为已知性别的标准样品。使用BWA分别将两个质量控制后的测序文件与已下载的该物种Y染色体参考序列进行序列比对，生成SAM格式的结果文件。
四、比对结果处理
原始比对结果存在文件占储存空间过大、比对片段次序混乱等问题，也可能存在低质量结果导致的假阳性比对错误，因此需要进行质量过滤和排序。使用Samtools和Picard进行排序和去除假阳性重复，并保存为二进制的BAM文件。
五、获得Y染色体特异序列
利用Samtools的“depth”工具对两个处理后的结果文件分别进行深度计算，生成一公一母两个深度统计文件，利用Linux“awk”和“paste”命令整合两个深度文件，作为Y染色体特异序列识别脚本的输入文件。然后，利用自主构建的Python脚本，设定50bp滑窗，筛选满足雄性深度大于a倍(可选参数，默认10)、雌性深度小于b倍(可选参数，默认1)的基因位点的窗口，并将其拼接成长段序列(可指定长度，默认大于1,000bp)，最后输出Y染色特异序列，共24条长段序列，其核苷酸序列如SEQ ID NO.1-24所示。
实施例2Y染色体特异序列在山羊性别鉴定中的应用
利用BOWTIE2将待测个体的全基因组测序数据与筛选到的Y染色体特异序列进行比对，统计样品比对率。
表2 公山羊比对率结果统计表(单位：％)
表3 母山羊比对率结果统计表(单位：％)
鉴定结果的统计分析
比对率的检验，分类资料某一类别的个体数服从二项分布，因此对不同性别样品比对率进行分类资料的假设检验，在比对率差异显著的情况下进行率的区间估计以确定鉴定性别的阈值。
(1)分类资料的假设检验
利用统计得到的数据，对不同性别比对率的差异进行检验：
设P1是雌性山羊总比对率，P2是雄性山羊总比对率，根据实际情况，此处应做左侧检验，检验假设H0：P1＝P2，不同性别比对率相等；HA：P1<P2，雄性比对率高于雌性。
率的检验中分类资料某一类别的个体数服从二项分布，但由于样本很大，p接近正态分布，计算检验统计量：
取显著性水平α＝0.005时，Z＝-28.8624<-u2α＝-2.5758，由此可以否定原假设，接受备择假设，即雄性比对率显著高于雌性比对率。
(2)确定雌性比对率
分析可知，对于Y染色体的特异性序列，母山羊理论上属于完全无法比对上，比对率应为0，与实验数据观察值一致，因此规定将比对率为0.00％的检测个体鉴定为雌性。
(3)雄性比对率的阈值估计
采用属于二项分布资料的率的区间估计方法。山羊的比对率统计符合二项分布，使用样本比对率p作为总体概率P的点估计，因为样本很大，此时的统计量p接近正态分布。
样本比对率p＝460÷1200000＝3.8333×10-4
取α＝0.01，uα＝2.5758，总体概率P的左侧临界值为p-uα×Sp，则总体比对率的置信度为99％时，总体概率的左侧临界值为3.7301×10-4，即0.0370％。
基于上述计算结果，本实验中取0.04％作为确认为雄性个体的阈值，即当比对率为0.00％时鉴定为雌性个体，当比对率大于等于0.04％时鉴定为雄性个体，当比对率介于两者之间时，作为难以判断性别的特殊个体。
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ttctcttaca ccgtacttct gaagctacag caagcaaata ttttcagtca tgctaagccc 1020
cttctcacaa ctttgtctca actctttctt gggattggaa ggaaattctg tttacctcct 1080
acgaagaagg cagagatatc aactcttcaa tgtgttttgg gactacactc tttactttat 1140
gtatctttca catcagctga aacatagagc ctgccaatga cacatttaag tgcttactga 1200
attatcaata aagaaatgca ataaccaatc aaatgctctg cacattactc accaacattc 1260
tgtaacctgt actactatct taattca 1287
<210> 2
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgcttcatgg ttttgtttac tgtttctgtc agcagtctgt attttaagtt gatgctcaag 60
taaacattag gttgaacatt acccttcaat tgctaggagc cagtgacttt gtcatgtcac 120
aactaacctt cgattgctag cagaccactg attttgtgat gtcacagctt tttgactggg 180
aggacctctg tgattgtcag agagtttctc tctgtaggcc agccaaacat catttgaagt 240
tgcattgtgt aaaaccagac aaatgagaaa attatctgaa gaaagatttc cctgagaggc 300
acatatttct tcagaaattc aagatggacc tccgaaagat gaatccactg gttcagaaaa 360
ctccattaga tcttattggt atgattatac aactgggact cagttttgag atctacgatt 420
gaagaatatg cttttcaggc tttgaggatc tcgtgatcaa aaggccacac tacacatatt 480
ctgtctctga aacagtcatg tgaatgattt tctttccaca aaaactttgg aatagagttg 540
aaagtgatca gtttgaatct gtttggtggg atgagtcagg cacatgtaga gtgacaatga 600
agaactcttt aagaaagaag tcttggaaag aaagcctttc agaatagttg aaaccaagag 660
tatgaaaagt ttaattcgac agcttaatct ttaggattta atcaaaaccg acaaagtgtt 720
caaagatcgg cttcaccacc tgtctttctg gaagaagaaa acaacatctc tcttttgagt 780
aaggtattcc agattttcac ttattggcaa tatgtcagac taaatcatgt gacctagaaa 840
gcatgttcat atatggagct aaaccggagt ttaaacttga gataacaaat gttgaaaaag 900
ttttattttt ttgaagttga gaaccagctg gaggttagaa tattggatgt ttaccaaacc 960
ttcctagcaa cttgaaacca gatacatgtt tgaagctact taaagtggta tttactgtat 1020
acatacaaca tatttttagt tgagcatata cgttctaaca tgttacctgt taaaactacc 1080
aacagaggtt tttatttgat gttatatcat tgcttccatt aaaggaaact tatagtgata 1140
tgaaggaatg aaattagtaa agaatgaaaa atgaatataa tagagaagaa atattgtaac 1200
caaaccaaaa ctttattctc ccatatgact actaggagac tcccagcaga ccaggacatt 1260
taaaaaacac tgtgtaaacg gccaaaaaga aacagtttag gttttgtgaa tatgatcatt 1320
ggcgattaat taagttttca gccatagata acaaccaaag aaactggcaa gtgcctgttc 1380
caactt 1386
<210> 3
<211> 1089
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gagtattacc ttgacatcac tggtaaaacg tgaatcctgg aatgatgatt gtgggtgtgc 60
aggggtgtgg atgatccacc tatgggatcc acacccatcc ctctcttcct gcagggtata 120
ggggacatca ttccactcca gtccattggt tctgggactt tgaacaggga gcaccagctg 180
caggctggac acctggagct taactttctc aactgattgt caggccgtaa ctgcccagaa 240
tcagacagga ttgctgaggt ggtattgttt tggtcctgca tacagttggt ggtcattttg 300
gagttcttgg ctgctcgtgt gagggttcgg attctggtca gcccagggct gagcttgctc 360
agcagcaaag tgttgccagc agattatcag caaagagagt gggatgatcc cctgaaggac 420
tatcccagga gagaagatac ttctatgaca gaaacacatg tggtaaaggg ctctggaggt 480
aggccatgaa cggacgccct gtactagctg ggacactgtc ctttcccaaa tggggacgct 540
cagtctcacg gaagctggct gcagaatctg aatttgggcc atgcatgtag caagggtctg 600
ctgagactgg gggcttgggg attcagtgtg tccgtgagtg aagcaagtcc cctcaaatca 660
gttgtggttg cagagtgaga gtcctaggtg atatactaga ggcagggtga attaagtttc 720
tttgtcagaa tcatccttcc atgggtcctg ccatcccctg gcctcctgtt gctcactgag 780
tttgagattg ccgggcctta ctctcccgcc ccttaccctg ggaaggccta tcattgcaag 840
ggcgtcctct ctggggtccc agtgcacctg tgtgctgact ttgggatctt cgtgaatgtc 900
cacatccacc actcggtggc tcagcaccac cacacatgct gcattggaag tggcttcagg 960
ctaccttggg aaaatatgga agagctcggg aggaggtaga caggaatcac aaggacagga 1020
cgtggtgcct actgactcca tctccgacat tgcagcgacc acacaccctt agactgggga 1080
cgcctcgtg 1089
<210> 4
<211> 1287
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccttctgcag ttcagcagtg accgcccact gttgactttc gagtttgaga aagcactctc 60
cgaatgtgtg gactgacaaa gtctgatctc ccatgcgtga actcactaag gaggagcagg 120
gcttgccttc tcatgtaaac ttctcgtatg agcactggtg tccagagagt ggcagacagc 180
aagagcacta cttagagaat ggagacaaac agacaccagc ccttggtaca cacacagaag 240
gattgcccaa aacagtgata tgccagaagc agagatgaag agacacagag ccagctcgta 300
ccctaaggct ctggtgtccc tgaacagtgg agatcaaccg tagcaaacag tggggttttt 360
cccttctccc ctagcatccg gacaacaaca ggttcttgtc ccatggagaa aagtatgagc 420
cacgtggagc acctgtttat cctttggata tccggagagt tctcagttat tgcggatcct 480
gagggacaca ggagttgctg ttcagactgt gaagcaggaa cccgtccttg gaattttcag 540
agcctggaca atgacagcgc ctgggggatc agcagatgca aaacccatta cctcctggag 600
cagtatatgc catgtgggtg cgctgtagcc caggaaccct ggaagatgcc ctacgtttga 660
ggggcgtgtc cataagggtg cctcccacca cttgactaga tttgggccag cagcctgtgc 720
gtggctcaca gcctagggat ccgcctattc ggctgatttc cccagggccc cttcaccctg 780
tcccgtgctg tcgccacccc tttcttcagc ctcaggccac cggacacaca ccagcacccg 840
cttccctacc atccccctca ctccccgccc ccaaagagct agtgggtccc aaggattcct 900
gaagcctgag ccacatttgc acaccacctg ttgtgctatt atgtcccatc ccttcacatc 960
tgccctagcc tcgggtcatg accagggcca gaggagtgtg aaaggcagtc agaggaatgc 1020
aggagcatcc tgggaccgca gaccttccga gttgtgagcc ccggtttcag ggcgctgggg 1080
ttggtggccg ccatcctcag gctttttgag gctgggatct tgcagccaca agttgtcatg 1140
ccaggcaaag gagccacgct atgcagagtg gaggcgatgg aggaaggtca cccctggtgg 1200
acggttgggt ggttgttcta gctgcttgtg gaagacgtca tggaggaggt ggaggttgtg 1260
gcagatgagg agcagcaata gggcttc 1287
<210> 5
<211> 1089
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cacaaccact gtgtctatga cagtacaccc actctattta tgacatcaca tccactatat 60
atgatataac atccactgta tatgaagtca cacactctaa tttagggcat cacatccact 120
atgtcggccg acacacccac tttatctgtg tcgtcacccc aactgtgtca ataacatcac 180
acccactgaa tgtatgactc acattcagtg tatgaataat atcctaggca gtatatatag 240
cataacatgt accttttatg acatcaaatc actgtattta tgacataact tcattgtttt 300
ttgatgtcac atgcataatt gtatttatga tgtcacatac atccattgta tgacatcacc 360
tccactatac atgtggcatc acatccactg tatctaaaca tcacatccaa tatattgaga 420
acatcatatg cagtatatat gacatgacac ccactatgtt tatgccatca tatcaactat 480
ttatgacatc acaccattct gtttattaca acacagctgt atctatgaca tcacacccac 540
tgtatttgac agcacatcca atatatgagg tcacaccctc ggtatttatg acatacaccc 600
actatctgcc acccaccctc tatctgacat cacatccact ctatctctga attaatatcc 660
accatatcta tgatcacacc cactgtattt gtgcatcacc tgcactatct atgacatcac 720
ggcacttatt tatgacatca ccccaagtat gtttcacatc cactacatct gtgacatcac 780
atgcaccata tcatgatcaa acccattgta tcattgacat cacatcaaat atatatgaca 840
tcaaatcccc tgtatatgat atcgcatcct ttatatatgt gacttcacac caccgtatcc 900
atgatatcat aaccactata tctatggcat catacccact gtttttactg acatcacacc 960
cagtatactt gtgacatctc cccagtcatt ctatgatcac actcactgta tctatgacat 1020
cacacccact gtatttgtga catcacatcc actatgtatg atatcacacc cactgtatga 1080
ggtcacacc 1089
<210> 6
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgaatgtcaa agttcacggg tttatggatg ttcccttcaa gatgtcctga atgagatctc 60
ctgctcaggg ccagcttttc catttgaata tctacatcca gctttgctcc tacagagatg 120
catctacagc tcgagaaggc aaggcttggc cttctctggg tggtttctga acaagcagcc 180
caggagacac tggaaacagt cttcttactt tcttaatctg taaacttcaa agaatcctta 240
gggctctggc aggctggtac ctgtctttct tagtgctagt tcaaaacaag acaaagcgaa 300
acgacggtgt agtaagtcta cagcaggtgt gagatagcat attggggcat ggatctcggg 360
accttagact gggtcattct agggactgca tggcattatc cacattcaga agttttaagc 420
atcgcaaaga taaagacaag aaaagtttag gacagtgtcc ccccaacacc taatatttcc 480
tcccagtgct tcaggccagg ggtggcttta gctctaacct gtcctgcagt gaagtgcaga 540
gatagtcatg gatcagttag taaatctgaa ctgtggatgg ttgactggat ggtcttctct 600
ggaaacttgc catgacttgt gcccagactc tttagggctt tcacttgggc cctaaagtct 660
aggtggtatt agatttgact gcacactcgg atgtgtgtaa ggttttcttc caccgaatat 720
caatgcatgt gagactcggt ggattagtga tgaattcatg gccttctggc tcaatcatcc 780
tgagctgcat ttcttcctgt gccctggatg tgttcatgtt ttcctatgtt tagaatgaag 840
acggggtgtg tccattgtgt tggcgcatga gtgtgagttg atatttcttc cgtgagtctg 900
cctatgcctc ctgtcttctt tcatgttgca cccatgcttg tgccattatg cacagctctg 960
atgctatatc tgtgttgcac gttcctattc acatacatgc gttgtattgc ttgtatttac 1020
tccttttttt aagtttgtgt atctttgttg gactgcatgg gaatttgacc tattctttca 1080
ttcctggata aaggattcca acatcttgaa ttgtgacttt tttaaagact tccatgatat 1140
ttgcgcacat caagaaatgc tttatcacag gaatagttaa cagtgaccac atgtgggaag 1200
aagaataccc aaacacttac cgtaatgcct gatgggcaag aatatcgctc attgaagtga 1260
gaatattaat taggatcaca gagggcaaga tattgcagga atgcacctgt caggaaattt 1320
ttaatctgtc aggaaagatt tggggttgtc ttaagtgttc taaaaacact actgcaggac 1380
tgaaat 1386
<210> 7
<211> 1485
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
agcctcaaca gatacttctg aattgagatc taaggacttt cagtgaccca ggcttggttc 60
aggccttgtg caggacactt acaaacacac ccgagcctcc ttccagtctc tgcacggaac 120
caagctgggt cctgcccttc ttctacccca acaaatactc ctgtggctca tttcggttct 180
tacccagacc aggctgagcc tgcctttcca aggccatgac aaatactcgt gaacacacgt 240
ctctgtctgt gcactgaacc aggctagctt cttcccatgt gcaagcactt acagacaccc 300
tgagcccaaa tgtcgcgtcc tgcacggaag gaggccaagt gctgcccatg ttcatgaccc 360
ctaataacaa gcttgcggca acttcctgag cctggcctga tccattttgt gagctaccac 420
ttttaaaccc caggaaatgc gcctatgccc aaatgaaggg ccctgcgctg atcccaggcg 480
tgggccagag ggggcatgcc tttgtccaag gccccaacac acactcctga gctctctgct 540
tggtttggca ctgaaccatg ctgggccctg cctctgttta ggccccccag aaacctcctg 600
cactggcaca tccgtccctg ctatgaacca ggctgggtcc ggcacgtatt caaggccctg 660
accaacatac ctgagtctgt atctgactct catctagttc ccggccttat ttgagccacc 720
agcaagcaat cctgagtcga ctagcaactc catgctttgg aactatggat tgtgcagcca 780
ttgttcacta cccgggaaac actaacaatc ccatatccca ctaactgcac tcaagaaggc 840
tgggacaggc ctttgcacaa gaagctgaca aacactcctg agccttagaa gctaggtccc 900
tggattgaac caggctgaat cttgcactgg ataaagcctc tggcaatcac tccccagcgc 960
acatctgttg ttccagcttc tgataaacac cctggcccca cctctaggtc catgcactga 1020
atcaggctgg gccctgccct tgtgcatgac accaacaaca ctccttagcc cacatctgtt 1080
gccctgaaag aacccagctg gagtttgcac ttcttcaagg ccctgtaaac gactgctgag 1140
cccacattta ggtttcttcc ctgcctcagg ctggtcctgc gcttgttcga ccccagagaa 1200
atgcgtgagg caacatatgg tccctgcacg gaaccaatct gggccctgtg cttgagcaac 1260
cctcgacaaa tattccagag cctacaatct ccccctcttt tgatcctgtt tggggcctgg 1320
ctttcctcaa gccccagaca atggctcctg ggcccacatc gcggtctctg aactgaattt 1380
tgctggtcct gaactggctc caagtcatga ccagcaactt gagcccatat ctcaatccca 1440
gaactcatgc aggttggacc ctgcctttgg tcatgtcccg agaaa 1485
<210> 8
<211> 1188
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggttcttccc ttctccccta gcatctggac aacaacaggt tctgtgtccc atggagaaaa 60
gtatccagcc acgtggagca cctgtttatc ctttggatat cagggagagt tctcagttat 120
tgcgggtcct gatggacaca ggagtgtgct gttcagactg cgaagcagga acccgtcgtt 180
ggaatttcca gagcctggca atgacagcac ctggggggtc agcagatgca aaacccatta 240
cttccatgga gcagtgtatg ccatgtgggt gcgctgtagc ccaggaaccc tggaagaacc 300
ctacgtttga ggggagtgtc cataagggtg cctcccacca cgctctcggc cattggacta 360
gctttgagcc agcagcctgt gcgtggctca cagcctggga tccgcctatt cggctgattt 420
ccccagggcc ccttcaccat gtcccatgct gtctgcctcc cctttctcag cctcagggga 480
ctggacacac accagccccg cttccctacc atccccctca ctccccgccc ccaaagagct 540
agtgggtccc acagctctga gccgcatttg cgcaccactt gttgtgctat tatgcccatc 600
cctttgcatc tgccccagcc tggggtcacg accagggcca agaggagtgt gaaaggcagt 660
aagaggaatg caggagcatc ctgggaccgc aaacttctta gttgtgagcc ccggtttcaa 720
gcgctggggt tggtggcctg ccatcctcag gctttttgag gtgggacctt gcagccacaa 780
gttgtcatgc cagcagatta gccgcactat gcagggtgga ggcagtggac gacggtgagc 840
ccctggagga cagagacgtg atggggattg ggtggttgtt ctagctgctt gggaagacgt 900
catggaggag gtggaggttg tggcagatga ggagcagcaa tagggcttcc tccaggagct 960
gagaagacag tggaggagca ggtccaggaa ggcccgggag cgctattgag cttccagggc 1020
aagacgcgct gcaggcactg gctgccctgc tggtggaact gagctctgag cataagaaaa 1080
actgcagggc tacttccagt tcatgtgcaa gaaccatcag aggaggaagc ctgacttggc 1140
ttggaggagc gccatcacct ggggcatccc ttgcttctgg gccattgc 1188
<210> 9
<211> 1188
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ttgccagttt ctttggttgt tatctatggc tgaaaactta attaatcacc aatgatcata 60
ttcgcaaaac ctaaaatatt tcttttttgg ccctttacaa gtgtttttta aatgtcctgt 120
tctgctggaa gtctcctagt agtcatatgg gagaataaag ttttggtttg gttacaatat 180
tttcttctct attatattat ttttcattct ttactaattt cattccttca tatcactata 240
agtttccttt attggaagca atgacataac atcaaataaa atatctctgt ttggtagttt 300
aacaggtaac atgttagaac atatatgctt aactaaaaat atgttgtata tatacagtaa 360
ataccacttt aagtagcttc agaatttgta tctggttcaa gttgctagga aggttttgta 420
aacatccaat attcttagct ccagctgttc tcaacttcaa aaaaataaaa ctttttcaat 480
aatttgttat ctcaatttaa tctccagttt agctacatat atgaacatgc tttctcagtc 540
acatgattta atctgacata ttgccaataa gtgaaaattc tggaatacct tactaaaaga 600
gagatgttgt tttcttcttc cagaaagaca ggtggtgaag ccaatctttg aaccgtttgt 660
tggttttgat taaatccata aagattgagc tgggaattaa acttttcata ctcttggttt 720
caactattct gaaaggcttt ctttccaaga cttctttctt aaagagttct tctttgatca 780
ctctacatgt gctgactcat cccaccaagt agattcaaac tgatcacttt caactctatt 840
ccaaagtttt tgtggaaaga aaatcattca catgatctgt ttcagagaca gatatgtgta 900
gtgtggcctt ttgatcacga gatcctcaga caaagcctga aaaacatatt cttcaatcgt 960
agatctgaaa actgagtccc cagttgtata tcataccaat aagatctaat ggaggtttct 1020
gaaccagtgg attcatcttt aggaggtcca tcttgaattt ctgaagaaat atgtgccatc 1080
tcagggaaac tttcttcaga taattttctc atttgtctgg ttttacacaa tgcaacttca 1140
aatgatgttt ggctggccta cagagagaaa ctctctggaa aatcacag 1188
<210> 10
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtgtcagaca cagtaggaaa gcaaattaaa cttcccaaag gaaaaagagc cttgacgtgc 60
actttgattt gtgctggacc catagtttta attgaaaaga tcaaacacag ggaatctgtt 120
cagcttttga attgggctaa attgaggtct cttaccctta agctacaatg gagttgtcca 180
agaatagtaa atacagtgat gaaaatagtc cagggtgatt aaaccctttt tagaagtcgt 240
ccttaccttt tccagttaca tgtgtcttat cctagttgaa catgtgaaaa tttcagtttg 300
tatcttatct tgccttggac acaaatgtag tacagttaac agctaattct ctgcgtgtgt 360
aatttcccat tgtatcaaac taattgagtt tcagtcttct tgacacagtc tgatgtgcaa 420
gaattgtctc atttatttca gtaattttca ggtcatagag ttttccttcc tctaatctat 480
cttaccaact aatcttgatt atgcttgtgg aaaccttttt tcttgcggtc tccaagggct 540
gtttcacaga cctgtggggt tccctcttaa gtattggttc ctgaacatca gcaccttggc 600
tgcctctgta ggttcttgga gttgcgtctc ctcctggaga caaatgttac ccttttccac 660
ttctggcaca gatacccagc aatctctgtg cacattcaag gaagctggga tcctgtctgt 720
agaccttgcc aggagaaatc tctccgtttc ctcttccttt ctcctcctct ttgtctctcc 780
ccactctccc cttcctcaat ttatttaagt cctctaagct ttcacgaaat gctggaaggt 840
gctaatcacc aaggggttag catcgctctc agaactgaag aggaaggact aagatgatga 900
agctgtagtg aatctcttgt ctccatttta ggtaatttcc tgagcgtaaa ttcaaattca 960
aataaattgt atttatttcc tcaaagcttg ttgatttatt tgcactttga atattttact 1020
aggcctttcc agttagttta cttgggccaa agctctttgc tgagtgtcag cttaaaccac 1080
ttttagttga taaagagtgt gtgtgtgttc tggtctcagg tgaaaactct atgcataaat 1140
aaatatgcag actattgaat tcttatctcc tttcttcgat ttatatagaa aaaacacgaa 1200
atgttttcat tatgtgtttt tattgttgct tagtattatc aacactgtta ctgttagttg 1260
ctgtgatgta tgctcctaag gagtacagtt gtgtttattg tcagattaaa aggttccagc 1320
aatatggtcc ctgactgctt cctctcttcc attctctgtc cttctactct actatattcc 1380
ccacta 1386
<210> 11
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tcttcatggg agggcaaaca gaagctcctt ccaatccgaa gaaatgttga gacaaagttg 60
tgagaagggg cttagcatga ttgaaaataa tttgtgtgat gtagcttcag aagcacagta 120
taaaagaaag ataatgaaat tactaaggaa aacagatatc atgtctttca agaactttta 180
accacgctga tgtgctgcgt caccaccagg aatttattct gtctctgtgg cgacagataa 240
gcaacgtcta aaaatgcatt tggaattata gggtggaatc ttggtgtcag gaaataagag 300
ggtccttaag gacagcagag caaaaatgac cctatagaat accacttgta aagtacatat 360
cttgtgctga ccatgggcca gccactgatt atgtcagtta tctttgtgat cacagtgatg 420
gcataattgt gatcaagatt ttggacgtgc tacaatatct atattttcaa ggacagaatg 480
aatttctata atgaagcaac tgctgtctaa ccttttaaga ctctaccagt aactaataga 540
atataatgtg tgaatgcact aaagttcata ctgtgtattt tcatttttct aacttatttg 600
catctcttcc ttcacccata ccatcctaat ttaggcaccc cttcatttca tcagggatga 660
tttcagaaat cactttacat ccattacccc tttccctttg ttctcatttt cttgcttaca 720
aatattatat cagctgtcat ttacctagga tgcataacga atgttttctt gaactgacga 780
gtttataaaa cctgcagtaa aatagccaac cactgaacta aagcctcatt ttactcttcc 840
ttgtttgaaa ataaaaaatg aaactatgaa gatgaacatg gaaatttatt gtcaaataca 900
agaattgtag acatttgttg gcatctccaa acataaattt caatctcctt taggctccat 960
aatctgacac gtgtttgaca gatcagtttt attaggatgg ttttcacata atgatgatta 1020
ttgatgagtt tactcgaaag agaggaggca gatgtatacg caattgttgg cactggaaac 1080
catgagttgc tctctcttgc aggttaagaa aaggactgtc atgggctgac atatcgctat 1140
gcctgtctgg aaatttagaa ggtttcacca tcagtccaga ataactgcct gtaatggaga 1200
ttcgatgtgg ttccgagaag tagatgtagt tgtagtagca aagtcctcaa cgtggccatt 1260
tacttgagtg tagctgctct gtgagtgcaa ttaaaaatgc tcaacttttt taaaacttca 1320
gattgatcta ccacaatctg gtctgtgtgt ctaactgata ttgctgatgg ggtaggtaag 1380
tcacat 1386
<210> 12
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
attcagagtt aagatcatct tttttgacac ttgctttagc atgcttcttg ttaccttgaa 60
ctaatgaaga tattggagac acattgttaa ttccaactct ctttgcattc ttactaaaag 120
ttggggatgg cctcttttga aatttggatt atagtaggtc tgtaactaaa atcaaagaat 180
taaattattt agtttcttat aaaccaaggt aaaactgaca aacaaagcta agttataaaa 240
acgtatccct ttaatggaac caattaaata acatcaaata aaatacctct gttggtattt 300
taacaggtaa catgttagaa agaatatgct caactaaaag tatgttgtat atatacagta 360
aataccactt taagtagctt cagaacttat atctggttca agttgatagg acagtttgtt 420
aacatccaat attttaacct ccagctgttt tcaacttcaa aaaaaataaa actttttcaa 480
caatttgtta tctcagttta aattccgatt tagctacata tatgaacatg cattctaggt 540
cacatgattt aatctgacat attggcaata agtgaaaaat ttggaatacc ttaccaaaag 600
agagatgttg ttttcttctt ccagaaagac aggtagtgaa gccgatcttt gaacagtttg 660
ccggttttta tgaaatccat aaaggttaag ctgcgaatta aagttttcat actcttggtt 720
tcaaatattc tgaaaggatc ctttctttcc aagacttctt tcttgaagag ttcttcattg 780
atcactatac attgcctgtc tcatcccacc aaatagattc aaactgatca ctttcaacta 840
tattccaaag tttttgtgga aatgtgagtg aaagaaaatc attcccatca ttgtttcaga 900
gacagaatat gtgtagtgtg gccttttgat cacaagatcc tcagacaaag cctgaaaagc 960
atattcttca atcatagatc tcaaaactga tccccagttg tataatcata caaagaagat 1020
ctaatggagg tttctgaacc agtggattca tctttaggag gcccatcttg aatttctgaa 1080
gaaatatgtc catctcagag aaacctttct tcagataatt ttctcatttg tgtggtttta 1140
cacactgcaa cttcaaatga tgattgcctg gcctgcagag agaaactcct agaccatcac 1200
agaggtcctc ccagtcaaaa agctgtgaca tcacaaaatc actggtctcc tagcaatcga 1260
aagtttattg tgacatcaca aagtcacggt ctcctagcaa ttgaagggta atgttcaacc 1320
agtgtttact tcagcatcaa cttaaaatac agactgctga cagaaagagt aacaaaaacc 1380
atgaag 1386
<210> 13
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
agttcttcta tgtaatcata ccaccactcc ttaatatctt ccaattcttt taggttcaga 60
catttttggt cctttattgt acacatatgt gcatgaaatc tcctttgata cctcttattt 120
tcctgaagag atctcgagtc tttcccagtc tctcatgccc actcagtagt ttagcaacat 180
ttattgtttc atggatgtac atgcttccag tgcttcttat tccacttgaa ttcctacact 240
cccagtagtg tatcagaatc ccttgtttca ccacagggac ctgctttacc tgcttctctc 300
aaccccagat caaagacact cagtacttta tcaccataga gtattcagtg cattaacatg 360
tgacactttc cagtgtttcc ttgtcgcctg ggactatcac ccagtagtat aagtgccata 420
ctctattttc gtgctgttac acaagtcaag tgcttcattt tctgaagtga aaacacactc 480
atcatccagt gtcttgtgca gaaacgtgct tgaggagctt cttcatccac ccagtcaaac 540
actcccaggg gtttttcacc atccagtgtt caagttctgt gacatgcttc aagtcttctt 600
tgtgtacact gggtcaaaga cacccagtag tttgtcatta tccagtgttt cagttatgca 660
acatgcttct gggtttcttc attccctcag gtctcacgcc aagtagtttc tcactatcca 720
gtgtttcagg gttacagcat gcttccagag cttttctgcc tgggtcaaac ataccaagta 780
gcttgtcccc gtcacgtttg agttctgtga aacggatcct gtgcttcttg tccaccctaa 840
atgaagcaca ctcaaccgtt tgtcaccatc cagtgtttca tgttggtcac aggttcagat 900
acttccccat gccctctggg tcatacacac cctgttgatt atcacctgtt tcagtttttt 960
gacatgcttc aaatgcttct tgcctctcct gatcaaacac accagtagtt tagcaacatc 1020
caatgtttca gtactgtaac atatttcaag aattttgcca cacaccaccg ttcagaagta 1080
cccaacagtt atcagcattc catgcctcag tttgaggact tgcctcaagt gcttcttcgc 1140
aacccgagac aaaacacacc cagtagattg tcaccatcca ttgtttcatc ctgcagcagg 1200
cttcatgtgc ttcttcatcc tctctaggtg aagcacatgc agtagcttat gaccatttag 1260
tgtttccatt ttatggcaga ttccaggctt ttccatcctc accaggccaa acacactcaa 1320
acatttacac catccagtgc tttgctctgt gacattcctc cagtcttcct cattatccca 1380
agtcaa 1386
<210> 14
<211> 1188
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caaaacacac acactcttta tacaactaaa agtggtttaa gctgacactc agcaaagagc 60
tttggcccaa gtaaactaac tggaaaggcc gagtaaaatt tcaaagtgca aataaatcaa 120
tcaagctttg aggaaataaa tacaatttat ttgaatctga atttacactc aggaaattac 180
ctaaaaagga gacagtagtt cactacagct taatcatctg tagtccttcc tcttcagttc 240
tgagagggat gctaacccct tggtgattaa caccttccag cattttgtga aagcttacgg 300
acttaaataa attgaggagg ggggagagtg gggagagaca aagaggagga gaaaggaaga 360
ggaaatggag agatttctcc tggcacaggt ctacagcagg atcccagctt ccttgaatag 420
tgcacagaga ttgctaggta tctgtgccag aagtggaaaa gggtaacatt taactccagg 480
aggagacaca acgccagaac ctacggaggc agccaagtgc gctgatgttc aggaaccaat 540
acttagaagg taccccacag gtctgtgaaa cagcccttgg agaccacaag aaaaaggttt 600
ccacaagtat aatcacagat tagttggtag gatagattag aggaaggaaa actctattac 660
ctgacaatta gtgaaacaca tgagacaatt cttcacatca gactgtgtca agaaagactg 720
aaactcaatt agtttgatac aatgggaaat tacacacgca gagaattagc tgttaactgt 780
actacatttg tgccaaggca agataagata caaaactgaa atcttcacat gttcaactag 840
ggtaagacac atgtaactgg aaaaggtaag gaggagttct aaaaagggtt tattaccctg 900
gactatcttc atccactata tttactattc ttggacaact ccagtgtagc ttaagggtaa 960
gagacctcaa tttagcccaa ttcaaaagct aacagattcc ctgtgtttga tccttttcag 1020
ttaaaactat gggtccagca caaatcaaag tgcacgtcaa ggctcctttt cctttgggaa 1080
ttttaacttc tttgctactg tgtctgacac agatatgcct acctcattgt gactacaggc 1140
cagccaaatg gcaaagggga tggaatgaat caaattcctc tttttatc 1188
<210> 15
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
agtaacacaa tcagatttcc ttctgtggga tcaacaatct tgtctggact tgctaaactt 60
ggaaaatcag tttttgtcag tccattttcc cctaaaggtt tacaggcttg caatgaacac 120
tgtcctttgg aaaaggtgtc ggacaaggtt ctccattctg agcgtggctt agtgaggtat 180
ggacacggtc tggtgtgctt gctgagtggg cactgtatga aaaatacctg aaggggaaaa 240
acctaaagac tgttccccca taatgccatc actgagctta ggccttctgg gatttctcta 300
tttgtttcac aagtggaaag tcgctttgat acatgaggac ttttattcat atctcctgca 360
gaaactgctc tttctactgg atgctgacat gaactttgaa ggtaatcaaa gaagaacata 420
actcctttga aaagcaatta ggtactatct ctggtgaaat atttttataa tcagctttag 480
acggaggttc agtaaaatag gactctctgc tgatcttggt tcagagtcag aaactggcaa 540
gacagtcgtt gcttctgatg taggagtcac atgactaaca ggctgtaatc tgtggcgtta 600
cctttcctga aatgaccata cctttttctc cttgaacaag aacctggggt atctctgttc 660
agtatgtttg aaacgactgg ttttgaagct gagtcatccc aacaaagatc acgtcagcat 720
cttcatttac atcttccact ccaaccaaga tcagttcatc atcatcatca cctacttgtt 780
tggtttctcc ctgcttttcc atagttctgg ttcttgttct tcttcacata acataatggt 840
agttccatat ttttaatacc tttttattct tagaggatgc ggccacaagt ccagtgcttc 900
ctttatacaa actgccacct aaggataaac atactacata ttagttaagt acacaaaaat 960
gggatatttg attatctcca caactctaca tagttataaa cacccttatc ttacagacaa 1020
acacactgag tgtcaatgag acgcagtgac agctgaaaaa cctatgacta caagtctaca 1080
tggctctaga tcatagcttt gtcccacgtt cattttaatc tcttctagtt aaaaaaataa 1140
tgacaaaaaa cccaacacta ctgatcatta ggtgacatca catgtaaaga caagtatttt 1200
tacaaaagca ctgttgtaca gcttttagaa acatggactt tggagtcaaa gaagattaag 1260
ttggaattct aagttaacca cacaccattg catcttcttg ggcaaattta taacttttaa 1320
atgtgcttct gtatcttaaa gacaaacatg cttatttctt agacttaagg ttaacagctc 1380
tttgta 1386
<210> 16
<211> 1287
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
agaaagcttg tacttctgca tatggacagg tttcctctca gcgatgttac tgtgtacacg 60
catatgttgt gccatgtgtg cgtagaaatt tcagtgcttc caaaattcat gtttctggaa 120
aagaaaactt gtacttctgc atatggacaa gtttcctctc agcgttgtta ctgtgtacac 180
atgtatgttt gtgcacattg cctgtagaaa tccctgccag ttcgcttgtg agtatgagct 240
agtgcaactg acctagccag gtgatagacg ttggacagca aaaacatgaa gttcttagga 300
aagatacata gtctctggcc agcagaagca ctgaagccag ggaatcgctc cccgttatct 360
cactgagcaa acatgttcta agtggaatca cagtcggcac ctagaaagcc tggcagaaag 420
gagaggcagg aggaaagagc ctggggagtg tgaagaggaa agtccactac acctgcagtt 480
tatgaaaagt agaaatgttc ctaatgagct gggaggacaa actgtactta attgagttat 540
ctaagaggat ttccatccaa acagccaaaa gtgcttcctg gtttcttctt gctgctgtag 600
taaaacatga agggaatttt tctaaaaaat gtttaagata aagactatta aaacaggaga 660
caaaatttgc tgatttggga attttccagt ctctaggatg acagaagatg ttaaaattaa 720
tgactctcaa gcaagatcat atccagggca ttgagattag gtggtctaaa tataacattt 780
tacaccacct atggccagtt cttcttcatt attttttata aacaaaggtg ttctaaagat 840
gagtgtgact gtaaaattct ttgttaagcc tcagaaagct ggaaggggat gctcatggca 900
ttatttactc aggccaatac cctccaaaga gtttaggggt gtcactcaca gatcctttca 960
gtcacacaat aagacttcta tcaaggttca ggtgttatcc ctcagctatc tcagcagaaa 1020
accaagacag agtcagtctt atcatgaaga catgtgtgat tgtggctttg gagtggggtg 1080
ctttgacaaa caccagtaag attatcagga gacacaccaa ggttttaaga taacatattt 1140
aagttcagct cggactgaaa ggaccagcaa tagtacaaaa taagagtgtg gacttctacc 1200
ttctacaagc aggaagcagg ctagggagac tgtttcatgt aaccatatac tagccacctt 1260
tcatgcaatg gcatcatgtc agggggt 1287
<210> 17
<211> 1287
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ctgagctcat cagcagtggc tgggtaagca gaagtgccca gcatccttga agatacctgt 60
tcccatgtct ttcttccacc ccagactagg gctgatgctc tggctcttct tggcccagca 120
tgtggcatta atggctctgt gacctcaagc agattatgaa ccaccttcaa gtttctgtca 180
tgatcagcga ccaagataag gctttctcag ctacatgatc gacttgaatg tcagtgagag 240
agactgagga caggtgggta tgggacttgg tggggtatgg gaggatcatg ttatccagtt 300
cctacactgt gagagtttgg agaggcttca tggaagctgt taatgcctgc ctgggaggaa 360
agctgaggac cctagaagct tgcacctttt ccctccccct atcctagcag gtacaggtgc 420
gaaccatcca cggtcatgca gcaagctgat cttttccttt caggaccacc cctatttctt 480
gaaaagagtg atcataagga gtattatctt aacatcactg gtaaaacgtg agtccgggaa 540
tgatgattgt gggtgtgcag gggtgtgcat gatccaccta tgggatccac accctctccc 600
ctctcttcct gcagggtata ggggacatcg ttccactcca gtccattggt tctgggactt 660
tgaacaggga gcaccagctg caggctggat accggagcct taactttctt aactggttgt 720
caggccacaa ctgcccagaa tcaaacagga ttgctgaggt ggtattgttt cggtcctgca 780
tacagttgat aacgttggag ttcttggctg ctcgtgtgag ggttcggatc ctggtcagcc 840
cagggctgag cttgctcagc agcaaagtgt tgccagcaga ttatcagcga aacgagtggg 900
atgatcccct gaaggactac cccaggagag aagattcttc catgacagaa acacgtgtgg 960
tgaaggggtc tggaggtggg ccatgaacgt tactagccgg gacactgtcc tttctcaaat 1020
gcagatgctc agtctcatga aagctggctg cgaagtctgc atttgggcca tgcatgtagc 1080
aagtggcctc taaaacttgg gcttagggat tcagtgtgtc tgtgagtgaa gcaagtcccc 1140
tcaaatcagt tgtggttgca gactgagagt cctaggtgat actactaggg cagggtgaat 1200
taagtttctt tgtcagaatc atccttccat gggtcctgcc atcccttggc ctcctgttgc 1260
tcacagagtt tgaaattgcc tgggcct 1287
<210> 18
<211> 1089
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tcagatttcc tatcatgggg tcaacagtct tgctctgact tgcaagagtc aaaaagtcca 60
ttttctctag cccttacaaa ttacatgttc tttctggatc ataagatgga agcactcctg 120
gtcattggca tgagtctgtg aaaatgatgc ctcaccatga acatggctcg gtgaggtcag 180
ggaatcttcc aaggtcatat cgtgtttttt gtaggagaga tacctagagg ggccatgatg 240
gagggatgtc tgcctgggat tccatcaccg agtctgggtc ttttggaatt cccactgtaa 300
atttcacatc tgaagtggga catcgctttg aattaagagg gctttcatcc ttgcctccta 360
atgagagaac agctccagag agacagtgaa gccgcgagag agcaatcctg tattgtgaag 420
gacagtttcc ggagagctca ttttataatc aggtttaggc gaaggcccag aaataataga 480
actgcctttt cctcccatac agactcagaa gctggtgaaa tggcagctgg atctgttgct 540
gaggtcctga gatttgcagg ttgcaacgtg caggagggat ctggattcac atggccttct 600
ttcttcttga agatgagtcc ctggtcactc tgttcaaaac gttagaaatg actggctttg 660
aagttgaagt cactccgaca tagagggttt cagatcctca ctgacatgat ccactccaac 720
aaagatgacc tcagcatcat cattgtcctc atctctggat ttggacacgg tggtcttttt 780
cccaggttgt agggtgcttg tgcttcctca catgggaaga gatgggccat ttgcaatcac 840
ttttggcttc aagtggcccc ttctcgtgtt tttttccacc taaaagcaat ctcagacaag 900
gtcatcccct ctgatctcag atgtcaccct ctgtgtgttc agttaaccct cagttgttcc 960
ccaagaatat taattggaaa actccagaaa atgtaactcg tacattttaa actgcaggta 1020
attctgagga gtgtaattgg aatttcacac caccatgctt cctcctgccc agaatctgaa 1080
tctttcctt 1089
<210> 19
<211> 1089
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gcattggatc acacacaggc ccatcacagt ccctgggggc cgatggaggc tcgggcatct 60
ctttggcctt ttcctgctaa agggagatag cacttggaac acaggtagac aggtgcacat 120
cgaaagctac acagatagaa agaacagggg gcctcattca gaatgttgtg ggccctaagg 180
ggtggaaggc tagcaaaagg acacgaatat gaccaggaat gacctagata ggtgcacacc 240
aagaagacag ttctccaagt cagctgagcc cagacgtcct tgttttctgg tcccctgagg 300
gttgcacaga caatggatca gaatagcctg actggtactg ggaagctgcc caaggtgatg 360
ggagaatgca gaacgcaggt gcctgtcact cagctgccag ggcgagcgtt tccaacctgc 420
cccattctct gcagatcggt ccctgtcagg agcaaatgcc aacattttgt agcctttgag 480
gctaaatcac agagtagaag tgcagtctga catgatcgat gagcatgtga aagaaaataa 540
ccaggacaaa gaaactcggg tggttaccaa aactcatgtt cctggagaag aaagttgtac 600
ttctgtatat ctgcaagttt cctgtcgggg atattagtgt ggacacacgc atgtgtgtgc 660
atatgtgagc caaaatcgcc tgacagtaca ctttgaatat gaagtgttgg aactgaccga 720
gccaggcgat ggaagtcgga tagcgaaaac atgaaagcca tagggaaagg tacatactca 780
ggccatgaga agactggagc cggagtacca atgcctgtta tctcactgag tgagcagttt 840
ctaagtggaa tcacagtggt gcacccagga agcctggcag aaaggagagg cggaagaaat 900
tgactggggg ccaccagtga gagtgcagag gatccaagac agagaagagg ctacaatcct 960
agacacagaa gccacagtgg gtgaccccac gccccttgga catgcagcta caaaggcctt 1020
tccatccttc tcgaacactg ggcattcttt ccctgaccat accacgagca tgagcaacac 1080
ttgccattt 1089
<210> 20
<211> 1188
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tgcgactgaa aaacatcaat caggtaaata aagaagcatc tgaagtgtgt cacaccactt 60
aaatactgga tgttgataaa ctactgtatg tatttgaaag gatcgttgag gaagcacttg 120
tgcattaatc agcactgaaa gaacggatgt gtgtattaac ccagagtagt taaggaagga 180
ctgtagtatg tcacagcatg atagaataaa tggtgatgaa acagttgctg agtttcacct 240
ctggggatca aacaagcgcc tactatatgc caccacttac ggccctgaaa ccttggcaat 300
gataaaatgc tgggaatgtc caactgatgt ggactattaa gcacttgaag catatccctg 360
tactgaatac tggatggtga tacactaatg agtatgtcta cagcagaaga tgaagcacat 420
gaagcatatc acagcactga aactctgaag ggtggtaaac cccaaggagt gacgggccag 480
gattgacaaa ggagcgatgc agcgattcac agtactgaaa tatagatgtt gaaaaactat 540
tgagtatttg acaccaggtg gacaaagaag cattcggagc atgtcctacg tcaagcactg 600
agcactgcat ggtgataaac tacagagtgt gtttttccag agagtggatg aataggcgct 660
tgaatcacgt catagcactg aaacactgac tttgtaaagt actggatgta tttttccagt 720
ctggatgata cagcagtttg tgtcacagct tggaagggcg ggatggtgat aagctcctgg 780
tgtgtttgat aagatgcact tgaagttgtg acagcagtga aaagcagcaa gtgataagct 840
ggtgaggcta ttttacctgg ggtggagaaa gatgtggctg aacctcctct cgaagtgtgg 900
ataataagct acttagtatg tttgaccagg ggtggatgaa gacacactga aagcatgtcg 960
cagcagtgaa acactggatg gagatacaca ctggatgtgt ttgacccagg gtgcatgaag 1020
aataactcga ggtgtcactg cactgaaaca gtagatgatg atcatccatt ggatacattc 1080
aatgagtaac aatgaagaag cacatggaat ggcactgcat tgtgataaac acctgcttgt 1140
atttgcccag tgtggatgaa gacgttcttg aagcgtcccc aatctttg 1188
<210> 21
<211> 1485
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
aggggatgaa ggttcaatcc ctgactggga aactaacatc ccatatgcta cagaaaaact 60
aaacctgcat agctgcaaat actgaagttc tcacactccg agactgcatg ccatggttac 120
agttggcttg tctcaaggac tatcttacgt gacacgacta agacccaaca cagccaacta 180
cataaattaa gaagaatccg tcacaggtga aacactggtg acaaactgct gagtgtgctt 240
tacctggagg aaatgaagaa acgcttgaat catctctctg cattgaaaca tgggatatga 300
taagctactg ggtacatggg gtgcatgaag aagacttgcg ggatgttccg cagttaaaac 360
agtggatgat gatagtctac tgagtgtatt agacctgagt ggatagaagc atgtcaccaa 420
atgaaacatt aagggtggta aaccactgct gtgtttaact aggggaggat gaagaaacac 480
ttaacctgtc acagcctgaa tactagatat gtttgtcccg gagtgagttg aacaagcact 540
cgaaacaggt tacagaactg aaagaaacac tggatggtgg taaacgacac agtttgacca 600
gagatgaaga agcaatggaa acatatcatg acagcgaaac actagctgat gagaaacagt 660
attatcagtg gcaagatgcc tgctccagag ttcggttcaa gacgcagcat gtttgcacta 720
gaatgcgggc ttaataagtt tgtgatgcct cccctaaagt gcaaattcag cagttaccac 780
agcctatcct ggtcgtgatg tcagaagggg taaaatgaga gctggaggct gcaagctcag 840
aatgtgcatg atgtgcgcta gaatgagggc tcattggtgg ctgatgcatg cttagactgc 900
aatttcagga gttgtgacat gggtatccta tatcacccat gatataaaac ctcacactgg 960
tcttctgttt ttcgcatatg gtaccgtacc aattaaacgc gcttttctca aatcatccca 1020
ccttcacctt ctcccactga gtcaaacgtc tgttcttgag atctttgtct cttctccttg 1080
cctgcgtatg cccatctttc taaattccac atatgcgagt taatatacag ttttgtctct 1140
ctctttctta gttccttcat tctgtgtaat aggtttcgtg ctcttcccct cattagaaca 1200
gactcaaatg catgacttct tataactgag taacatttca tatgcatatg tagcataact 1260
tccttaacca ttcatctgct gatggactcc tggtcacttt cacatcctag gtattgcaaa 1320
caagtgcttc aataaacatt ggagtactgt gtttcttacc attctagttt ccttgaagtt 1380
catgccaaga gtgggatcgc tgggtcatat gatatttctc tttccaactt tctaaggagt 1440
ctccataatg gttgtaccag tttgcattcc aatcaacagt ggaaa 1485
<210> 22
<211> 1089
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
agttgatgct caagtaaaca ttaggttgaa cattaccctt caattgctag aagaccagtg 60
actttgtcat gtcacaacta accttcgatt gctagcagac actgattttg tgatgtcaca 120
gctttttgac tgggaggacc tctgtgattg tccagagagt ttctctctgt aggccagcca 180
aacatcattt gaagttgctt gtgtaaaacc agacaaatga gaaaattatc tgaagaaaga 240
tttccctgag atggcacata tttcttcaga aattcaagat ggacctccta aagatgatcc 300
actggttcag aaacctccat tagatcttat tggtatgatt atacaactgg ggactcagtt 360
ttgagatcta cgattgaaga atatgctttt caggcttgag gatctcatga tcaaaaggcc 420
gcactacaca tattctgtct ctgaaacaga tgatgtgaat gattttcttt ccacaaaaac 480
tttggaatag agttgaagtg atcagtttga atctgtttgg tgggatgagt caggcacatg 540
tagagtgatc aaagaagaac tctttaagaa agaagtcttg gaaagaaagc ctttagaata 600
gttgaaacca agagtatgaa aagtttaatt caccagctca atctttatgg atttaatcaa 660
aaccaacaaa cggttcaaag attggcttca ccactgtctt tctggaagaa gaaaacaaca 720
tctctctttt gagtaaggta ttccagaatt ttcacttatt ggcaatatgt cagattaaat 780
catgtgacca agaagcatgt tcatatatgt agctaaactg gagattaaac ttgagataac 840
aaattattga aaaagtttta tttttttgaa gttgagaaca gctggagcta aaatattgga 900
tgtttacaaa accttcctag caacttgaaa ccagatacaa gttctgaagc tacttaaagt 960
ggtatttact gtatatatac aacatatttt agttaagcat atacgttcta acatgttacc 1020
tgttaaaact accaaacaga gatattttat ttgatgttat gtcattgctt ccaataaagg 1080
aaacttata 1089
<210> 23
<211> 2178
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ctggcctgat ccattgcctg tgcacaaaca tgcacgtgta cacaaaacat ccctgagagg 60
aaacttgtcc atatgcagaa gtacaagttt tcttttccaa aacatgaact ttggtaagca 120
ctcaaacttc tatgcgcacg tgtgcacaga catatgcgtg tacacagtaa catcgctgag 180
aggaaacctg tccatatgcg aagtaaaagc tttctttttc aggaacatga atttgggtaa 240
gcacccacat ttctttgtcc ttgttttttc acttgctcat cgatcatgtc atgttacctt 300
ctaactgtgt gccttatcct caaaggttgc aaaaccttgg catttgctat tgacagggac 360
tggcctgcag agcagcgggc agggttggaa tttcttccat gaacagctgt ataacatgtg 420
cctgccttct gcattctccc atcaccttgg acagcttcat agtaccagtc acgttggcct 480
gatccattgc ctgtgattcc ttcaggggac cagaaaacaa ggacctctga gctcagctga 540
cttgaagaac tgctttctcg gtgtgcacct ctctaggtca gtccatgtca gtgtgtgccc 600
ctcttgctag cctaccaccc cttacagccc acagcattct gaaggaggcc ccctgttctt 660
cctatctgtg tagcgttcta tgtgcaccat ctactgtgcc cccatgtgct atctccatgt 720
agcaggaaaa agcccgaaac gcctgagcct ccaaaagccc ccagagactg tgacaggcgt 780
gggtctgatc caggttgtga agaagatgca catggataaa gggtctcttc cgatacaaag 840
gatggaaccg gaggccataa aagtcaggtg gcatccctac acgtcttcct tctgtttaac 900
actacccatg cacacggagt ccccaaacag atgatgccct gggaaagctg cagtttctgg 960
gcccagagct aatgcctccg ttggacagaa gcagtgttac tatctcaaat ggaccaagag 1020
ggcttgaacc acccatgcct cagtgatttg ctaaatgcca agattctctt tcaggtgctc 1080
tcatcaggcc cggtaggact ccctacagtc acgcatccct gcttccctac acagcacccg 1140
tgagaaggaa gccagtggct tgttgggccg agggacaccc aagacggtac ctggaaggca 1200
gcatccctct gagtgtcacc tgggtgtaga tcccctacaa cacaatcaga gattctgctc 1260
cagcaagact accagaatcc tccttcagtg cagacgtgca cctttgcccc tcaggccccc 1320
aaagccagtg gcaagacccc aaagaaaaga aggaattcat gtcagaactt tcagcacgga 1380
ccatgggtat ctttggcagg accgctactg cctagagtct ggaccctgaa aaccagcagg 1440
ccctgaactg ccccaggggc ttgtctgtct cccactctcc tgaggctctt gggacatgca 1500
gtttacccat cgcccttgtc ttttctctgt tccctccctc acaagcatta cccaaatagg 1560
gtcacacagg taaacccact tgcgtttcct cgtctttggg cactgtcagg gaggcatgcc 1620
aggcttacag agcatgcctc tcctaggatg cctgtggttt tgcagataca gcatacttta 1680
gcagtctcat gttttgtgtc atatcgtgcc agtgtgtgcc accctcaggg agggcgtgtg 1740
ctggctgctg ggctgatctg gggcagcact ggcccaggac ctcccagttc tccttttcac 1800
atgtgtgtga aacaagtctc cagtgcagca ggcagtctgt gactccttct ccttcagctc 1860
tctgcccatc tcagcaggac ctagcctcac catcccgggt cttttcatag ccacgtccat 1920
catttccaca ggccagccca tggccccaca cactgagggc ttcacaggca gtaatttcaa 1980
gccccaccca atgtgatttg ctctgtgtga atccttagac catgaaaccc tcttcctgat 2040
ccaaacacac acaacaacac agtagaactg atgagcgtgt ttgtacctgg tctaattgca 2100
gatatctgga gctacagtct ggaggtatca ctggccatgc tttcctcctt cccatcctgg 2160
gcagggtcaa tgcagaga 2178
<210> 24
<211> 2574
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
cgcataaata agcacgtgtg tatacacaca catgctcagc atggggacac aaatgcaaac 60
aacgaacata ggtgtttcag gaggagaaga ggactgacgc agcaatgtcg gagatggtga 120
cagtaggcaa catgccctgc ccttgggggt ccttgttcat cttctccacg aactcttcca 180
tattttacca aggcagccga agccgcttcc agcgcagcat gtttgctggt tgtgagacac 240
tgagtggtga atgtgggaat tcacacaaga tcccaaagtc agcacatagg tgcactagaa 300
cccggagagg acaccttttc aatgacaggc cttcccaggg taaggggcgg gagtgaagtg 360
cccggcaatc tcaaactcag ggagcagcag gaggccgggg atggaaggac tcacaaagga 420
tgattctgac aaagatcctt actacacact gcctctagta gtatcacctg gattctggtg 480
ctgccaccac agctgtttga tggagacttg cttcactcat ggacacacca agtccccaaa 540
tagacagtct caggaggtcc cctgctacat gcacgaccca aattcaggct ccacgccagc 600
ttgcatgagt ctgagcatcc ccatttggga aaggacagtg tccctgtagt atcacacgag 660
tgtcccttca gggcccacct tcagacccca tctcacatgt ttctgtcagt tactctcatg 720
gaagaacctt cctgcccagg gtactacttc aggggatcgt cccacacatc ttggccgaca 780
gtctgctggc aaacaagcaa ggtgagccag cagccaagaa tcccaaaagg aattgctaac 840
tgtgttcagg cccaaagcaa ccacacctca gcaatcctgt tcagttctga gagttgtgac 900
ctaaccacca gccgagaaag ttaaggctcc tggtgtccat gctgcggcag gatgctcccc 960
gttcaaagtc ccagaaccag tggacaggag ggaacaacgt gccctatacc ctacagagag 1020
acaggggaga tgggtgtgga tcccacaggt ggacaatcca caaccttgca cacccacaca 1080
tcaccccaga accacgtttt acaagtgatg tcaaggtaat tctacctagt ggtcactgtg 1140
ttcaagaagt tggggagtcc tgaaaggaaa atatcaactt gcagtgggcg atggatggct 1200
ccgcaacagc acctgtcagg ctcgggagga gggtaaaggt tcctgcacct cctcctcttc 1260
ctgagcagca gaattcaaag agcagaaaaa ggatacagca atggcccaga agcaagggat 1320
gccccaggtg atggcgctcc tccaagccaa gtcaggcttc ctcctctgat ggttcttgca 1380
catgaatgga agtaggccct gcagtttttc ttatgctcag agctcagttc caccagcagg 1440
gcagccagtg cctgcagcgc gtcttgccct ggaagctcaa tagcgtcccg ggcctttcct 1500
ggacctgctc ctccactgtc ttctcagctc ctggaggaag ccctattgct gctcctcatc 1560
tgccacaacc tccacctcct ccatacgtct tccacaagca gctagaacaa ccacccaatc 1620
cccatcacgt ctctgtcctc caggggctca ccgtcgtcca ctgcctccac cctgcatagt 1680
gcgctaatct gcctggcatg acaacttgtg gctgcaaggt cccacctcaa aaagcctgag 1740
gatggcaggc caccaacccc agcgcttgaa accggggctc ccactaggaa ggtttgcagt 1800
cccaggatgc tcctgcattc ctcttactgc ctttcacact cctcttggcc ctggtcgtga 1860
ccccaggctg gggcagatgc aagggatggg acataatagc acaacaagtg gtgcgcaaat 1920
gcggctcaga gctgtgggac ccactagctc tttgggggcg gggagtgagg gggatggtag 1980
gaagcggggg ctggtgtgtg tccagtcccc tgaggctgaa gaaaggggag gcagacagcg 2040
tgggacatgg tgaaggggcc ctggggaaat cagccgaatg gcggatccct aggctgtgag 2100
ccacgcacag gctgctggct caaagctagt ccaatggccg agagcgtggt gggaggcacc 2160
cttatggaca ctcccctcaa cgtagggtat cttccagggt tcctgggcta cagcgcaccc 2220
acatggcata cactgctcca tggaagtaat gggttttgca tctgctgacc ccccagggct 2280
gtcattgtcc aggctctgga aattccaacg acgggttcct gcttcgcagt ctgaacagca 2340
cactcctgtg tccatcagga cccgcaataa ctgagactct ccctgagtat ccaaaggata 2400
aacaggtgct ccacgtggct ggatactttt ctccatggga cacagaacct gttgttgtcc 2460
agatgctagg ggagagggaa gaaccgcagt ttgccacagt tgatctccac tgttcaggga 2520
caccagagcc ttagggtaca gagctggctc tgtgtctctt catctctgct tctg 2574
Claims (1)
1.山羊Y染色体特异性核苷酸在山羊性别鉴定中的应用，其特征在于，所述特异性核苷酸的序列如SEQ ID NO.1~24所示，包括以下步骤：将未知性别的山羊的全基因组重测序数据与所述的特异性核苷酸序列进行比对，若比对率≥0.04%，则鉴定为雄性个体，若比对率为0.00%，则鉴定为雌性个体。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1978664A (zh) * | 2005-12-01 | 2007-06-13 | 新昌县大东种畜发展有限公司 | 通过pcr鉴定山羊早期胚胎性别的方法 |
| CN102094088A (zh) * | 2010-11-26 | 2011-06-15 | 中国农业科学院兰州畜牧与兽药研究所 | 一种羊早期胚胎性别鉴定的试剂盒 |
| CN102181523A (zh) * | 2011-03-09 | 2011-09-14 | 塔里木大学 | 一种鉴定山羊早期胚胎性别的方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1978664A (zh) * | 2005-12-01 | 2007-06-13 | 新昌县大东种畜发展有限公司 | 通过pcr鉴定山羊早期胚胎性别的方法 |
| CN102094088A (zh) * | 2010-11-26 | 2011-06-15 | 中国农业科学院兰州畜牧与兽药研究所 | 一种羊早期胚胎性别鉴定的试剂盒 |
| CN102181523A (zh) * | 2011-03-09 | 2011-09-14 | 塔里木大学 | 一种鉴定山羊早期胚胎性别的方法 |
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