CN114058718A - 一种鉴别荷斯坦牛优质高产的分子标记及其制备方法 - Google Patents
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Abstract
本发明提供了一种鉴别荷斯坦牛优质高产的分子标记及其制备方法，所述SNP分子标记位于荷斯坦牛基因组的13号染色体上，核苷酸序列如SEQ ID NO.1所示；所述分子标记位于第33012位点，突变碱基为T或C，同时利用该分子标记设计引物进行PCR扩增检测。同时利用Sequenom MassARRAY对992头荷斯坦牛进行基于SNP的基因分型；采用最小二乘法分析SNP与泌乳性状和体细胞评分(SCS)之间的相关性。本发明发现了牛ACSS2对产奶性状的多效作用，针对SNP等位基因发现了不同的优势基因型，对后续荷斯坦奶牛泌乳性能的分子遗传学研究提供理论依据。
Description
技术领域
本发明涉及基因技术领域，特别是涉及一种鉴别荷斯坦牛优质高产的分子标记及其制备方法。
背景技术
荷斯坦奶牛是我国奶牛的主要品种。泌乳性能是荷斯坦奶牛的主要经济特征之一，作为评价奶牛场管理最直接的指标，可以直接反映奶牛管理中存在的许多问题。奶牛泌乳性能受多种因素的影响，包括遗传因素、生理因素和环境因素。一些关键因素直接影响产奶量水平和生产潜力。泌乳性状中，脂肪含量(FC)与产奶量、蛋白质含量(PC)、乳尿素氮(MUN)和体细胞数(SCC)呈显著相关。此外，奶牛***炎是奶牛最普遍的疾病之一，使牛奶产量和质量下降，给乳品业造成了巨大的经济损失。DHI记录的体细胞数(SCC)是衡量奶牛***健康的间接指标，在一定范围内与牛乳品质和奶牛***健康呈负相关。在实际生产中，由于奶牛SSC呈偏态分布，通常将其转化为接近正态分布的体细胞评分(SCS)加以应用。
SNP(Single Nucleotide Polymorphism,SNP)，称为单核苷酸多态性，是第三代遗传标记，普遍地存在于所有物种基因组染色体上。研究发现，位于基因内部的SNP有可能直接影响蛋白质结构或表达水平，最终导致表型的差异，可作为重要分子标记，用于遗传资源的保护和重要经济性状的选育。
脂肪酸的从头合成是动物体内脂肪酸的重要来源，乙酰辅酶A羧化酶和脂肪酸合酶催化了从乙酰辅酶A和丙二酰辅酶A为底物的全过程,从而形成长链脂肪酸。而其中乙酰辅酶A则是由乙酰辅酶A合成酶催化乙酸而形成,从而用于脂肪酸的合成。编码乙酰辅酶A合成酶的基因有两个,即ACSS1和ACSS2。ACSS2基因的表达由固醇调控元件结合蛋白(SREBP)调节，而SREBP在脂肪代谢及胆固醇合成过程中起着重要作用。研究表明，ACSS2基因有可能影响动物的生长和脂质性状，促进细胞对乙酸的利用，并对维持癌细胞的生长提供了脂质底物。此外，ACSS2能够通过调控HIF-2信号通路从而影响肿瘤细胞的胞内微环境，ACSS2在牛初乳、常乳和末乳中均有表达。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊，而这种简化或省略不能用于限制本发明的范围。
鉴于上述和/或现有同多钼酸配位聚合物催化剂中存在的问题，提出了本发明。
因此，本发明其中一个目的是，克服鉴别荷斯坦牛优质高产的分子标记的不足，提供一种鉴别荷斯坦牛优质高产的分子标记。
为解决上述技术问题，根据本发明的一个方面，本发明提供了如下技术方案：一种鉴别荷斯坦牛优质高产的分子标记，其包括，分子标记位于荷斯坦牛基因组的13号染色体上，为ACSS2-g.33012T>C。
本发明的另一个目的是提供一种鉴别荷斯坦牛优质高产的分子标记的制备方法。
为解决上述技术问题，根据本发明的一个方面，本发明提供了如下技术方案：一种鉴别荷斯坦牛优质高产的分子标记的制备方法，其包括如下步骤：
提取DNA：提取荷斯坦牛血液基因组DNA；
进行标记：获取荷斯坦牛相应基因序列并对相应外显子区域做标记；
PCR扩增：使用特异性引物对于荷斯坦牛相应基因序列进行PCR
测定基因序列：将PCR产物进行电泳检测，然后测定基因序列，对于测序结果进行对比，比较不同样品之间的区别和发生突变的具***置；
分型检测：将检测到的突变位点使用质谱法进行分型检测；
关联性分析：对于日产奶量、乳脂率、乳蛋白率、体细胞评分四个形状评分进行关联性分析。
作为本发明所述鉴别荷斯坦牛优质高产的分子标记的制备方法的一种优选方案，其中：提取DNA中，所述提取DNA中，所述荷斯坦牛血液基因组DNA为尾静脉血液基因组DNA。
作为本发明所述鉴别荷斯坦牛优质高产的分子标记的制备方法的一种优选方案，其中：进行标记中，荷斯坦牛相应基因序列为荷斯坦牛ACSS2基因全序列，所述荷斯坦牛相应基因的获取方式为在NCBI网站上下载。
作为本发明所述鉴别荷斯坦牛优质高产的分子标记的制备方法的一种优选方案，其中：进行标记中，使用primer 5软胶在CDS区设计特异性引物。
作为本发明所述鉴别荷斯坦牛优质高产的分子标记的制备方法的一种优选方案，其中：进行标记中，所述特异性引物为使用primer 5软件在CDS区设计特异性引物。
作为本发明所述鉴别荷斯坦牛优质高产的分子标记的制备方法的一种优选方案，其中：分型检测中，突变位点位于ACSS2基因的33012位置。
作为本发明所述鉴别荷斯坦牛优质高产的分子标记的制备方法的一种优选方案，其中：分型检测中，ACSS2基因的33012位置包括C或T两种碱基。
作为本发明所述鉴别荷斯坦牛优质高产的分子标记的制备方法的一种优选方案，其中：分型检测中，ACSS2基因的33012位置包括TT型、CC型和CT型。
本发明研究并确定影响荷斯坦牛泌乳性能相关的分子标记位于牛的13号染色体上的核苷酸序列上，验证其对泌乳性状的影响效应，最终建立高效准确的分子标记辅助育种技术，将其应用于奶牛的遗传改良中，通过优选该SNP的优势等位基因，能够逐代增加优势等位基因频率，从而提高奶牛的泌乳性能，增加核心竞争力。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。其中：
图1为本发明中ACSS2基因33012位点几种突变。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合说明书实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用其他不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。
其次，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。以本领域技术人员能够实施为准，制定了以下实施例。
本发明实施例中使用的荷斯坦牛ACSS2基因来源为江苏六个牧场中样本统计而来，统计的牧场包括：华夏牧场，盱眙牧场，泗洪牧场，新沂牧场，申丰牧场，永浩牧场，统计的数据包括采样日期、产犊日期、产犊间隔、泌乳天数、分组号、产奶量、乳脂率、蛋白率、脂蛋比、体细胞数、体细胞分、尿素氮、奶损失、奶款差、经济损失、校正奶、持续力、WHI、前奶量、前体细胞、前体细胞分、前奶损失、高峰奶、高峰日、305奶量、总奶量、总乳脂、总蛋白、成年当量。得到的数据经过处理后如表4所示。
实施例1
本实施例中对于荷斯坦牛ACSS2基因单核苷酸多态性进行筛选。
对992头荷斯坦牛尾静脉处采血，用血液基因组提取试剂盒提取基因组总DNA，测DNA浓度和纯度，将DNA母液分装稀释至50ng/μL，于-20℃冰箱保存。
以所提取的基因组DNA为模板，通过PCR引物进行常规PCR反应，使用的扩增引物如下所示：
ACSS2-F1：CTAGATAGACATGGGTGGGTCTTG
ACSS2-R1：CCTATGTCCTAGAATTTTGGCTTG
PCR反应体系和反应条件如表1所示：
表1 PCR反应体系
PCR反应条件如表2所示：
表2 PCR反应条件
将得到的PCR扩增产物送至擎科生物股份有限公司进行测序；
使用ContigExpress软件针对测序结果进行统计分析，获得各群体基因型频率分布,筛查SNP。得到的数据记录在表3中。
表3 ACSS2基因突变位点的基因型频率与等位基因频率
ACSS2基因突变位点中包括的基因型和等位基因如上。
实施例2
采用SPSS软件的多因素方差分析与一般线性模型法对基因突变位点基因型与测定日产奶量、乳脂率、乳蛋白率、体细胞评分4个性状进行关联分析，模型如下：
Yijklmno＝μ+CSi+MSj+Pk+Dl+Gm+Fn+eijklmno
其中，Yijklmno为泌乳性能的观察值，μ为群体均值，CSi为产犊季节的固定效应，MSj为测定季节的固定效应，Pk为胎次的固定效应，Dl为泌乳天数的固定效应，Gm为基因型的固定效应，Fn为场效应，eijklmno为随机参差。
对于表3中得到的基因突变型即多种ACSS2基因单核苷酸多态性对荷斯坦牛泌乳性能的影响进行统计，得到的数据记录在表4中。
表4 ACSS2基因单核苷酸多态性对荷斯坦牛泌乳性能的影响
注：同列数据上标不同小写字母表示差异显著(P<0.05),上标不同大写字母表示差异极显著(P<0.01)；*P<0.05；**P<0.01。
由表4可得，ACSS2基因单核苷酸多态性对荷斯坦牛泌乳性能有显著影响。其中，ACSS2-c.33012T>C对日产奶量、蛋白率和305天产奶量有极显著影响(P<0.01)，TT型日产奶量极显著低于CC型和CT型(P<0.01)，TC型蛋白率极显著低于CC型和TT型(P<0.01)，CC型305天产奶量极显著高于TC型和TT型(P<0.01)。此外，ACSS2-c.33012T>C对乳脂率有显著影响(P<0.05)，TT型乳脂率显著高于CC型和TC型(P<0.05)。即在ACSS2基因中，在33012位点处伴随着不同基因型的出现有着不同的泌乳性能，其中CC型相较其他两种基因型而言有着明显的更加优秀的泌乳性能。
根据如上信息制作一种SNP分子标记，所述分子标记的核苷酸序列如SEQ ID NO：1所示，所述序列的第33012bp处的单核苷酸位点为T或C。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 扬州大学
<120> 一种鉴别荷斯坦牛优质高产的分子标记及其制备方法
<141> 2021-12-24
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 46821
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
cctcggttgg cctgttttcc gcgcctgggc gccggctgct accgccaagg cggctactgt 60
gttcggaact tgaggtgatg ggacttcccg aagagcggag ccggagcggc agcgggagcg 120
gggcccggga ggaagctgga gcccggagcc cgacacggag ctggtctccg ccgcctgagg 180
tcagccgttc ggcgcacgtc ccctcgctgc agcgctaccg cgagctgcac cggcgctcct 240
tggaggagcc gagaggtgag gcagggcccg ggcgggcctg agggacgtca gctaggagag 300
aagtgggggt tcacaggaga gggaggaatt gggggtgttc cgtcgggaaa tggaagttcc 360
gtgaggagat ggaggttccc ttgggaaatt ggggagtcgg taaggagata aaggttctgt 420
gatggaagaa tagcggttct ttaaggagat ggaaaagggg ttctgggagg agggggttct 480
ctgaggcgaa ggaggaaatg tcgttctgta ggaaatgagc gtctcctagg agttgggggg 540
ttcgtttgcg acttgggggt tcgcaggttc tgtgaacgtc tgaaagagag aaaatgtcct 600
gaaaagagag tttctcatac gaagagtcgg gttcttaggg gccaggagcg ccttgaaggg 660
aggcgggcca cgaagagaac tttctttacc gcttttcagc ccagctcagc ccgtgcctcc 720
tgacaacttt cgcctgtgag ttagtctcca ctgctgaggg gcactcgagt cccatcctgg 780
catccccaag ttagtaatgg agatctattt ttagtatttc agaaatcccg atggaaacta 840
ccatgtaaaa ctgaacaagt cactaaaata tcgcctttaa aataaaaaaa ttagggctgg 900
gattgtaata actccgtaat tctgataggt agtgccatgt ctttgagact caagctgggt 960
aaggggatgt agctgaattg ggaggcggca tgatgctggc agtgggacat gcatgaacaa 1020
tctggatttg atcttggctc tgccactcat ttgctctttg gtcttgagat tttaaaacct 1080
gtctgaaatt ccttttcttt atctataaaa tagaggtaat aagacctgcc tcgaagattt 1140
gtttgagggc taaaatgagt agtaagtgaa gtgtgcatta tagagtgcct gatgcacagc 1200
agcgttcact aaatagtggt tagtttgata accataagag caatgtggat gaagccacta 1260
ttgagtaagc aagataagat taaatcaggt gtagagctgt tgattgctgc aggcaatgtc 1320
cacgttttag tgctcagacc tctttttttt ttttaggtgt tcttatagat atttgttgac 1380
taaatttgtc tatgtagacc accccttaga caaatattac acttaatctt ttagttgact 1440
cttgtggctg gtaattcttt ccttgtcaag ttcctactct ttaaaaagat ttcatagtag 1500
gcttccttta atagggaaat gagccaatat ctttaaataa aaacacaatc acatggaagt 1560
ttcataaaat acaacacaca cctgtatcca ccaactatcc taggattctg acttcttggg 1620
atagcactga atttttgttg gaattctcta ggaataaaat ataaaggcca attcatatac 1680
tggctctaaa gcaatatcat catatcttca tctttccaat ggagacttcc tagttggctc 1740
ttagagaggt gatcttatct atagactcac ttcctaatca gagttcactt tcagattaga 1800
taaccccagc ttgtttcttt ttctttcttt ctttttaaaa ttgaagtata gttgatttat 1860
aatgtgtttg tttcaggtat acagttcaaa gtagttcagt tatttacaca tatgtccata 1920
ttctggagaa ggcaatggca ccccattcca gtactcttgc ctggaaaatc ccatggatgg 1980
aggagcctgg agggctgcag tccatggagt cgctaggagt cagacatgac tgagcaactt 2040
cactttcact tttcactttc atgcattgga gaaggaaatg gcaacccact ccagtgttct 2100
tgcctggaga atcccaggga cggcggagcc tggtgggctg ccgtctatgg ggtcacacag 2160
agttggacac gactgaagtg acttagcagc agcatgtcca tattcagact cttttccctt 2220
ataggttatt acaaaacaga gtatagtccc ctgtgctata cagtaggttc ttgttggtta 2280
tctattttat atatactaat gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgcatgt 2340
gctaagttgc ttcagttgtg tctgactctg tgaccctatg gactatagcc tgccaggttc 2400
ctctgtccat gggatattcc agaatactgg aatgggttgc catttcctcc tgcaggagat 2460
cttcctgacc cagggattga agctgcatct cttgcatcta ctgtattggc aggcgggttc 2520
tttaccacta gagctacctg ggaagcccag tactgtgtat gtgttaaccc caaacttcta 2580
atttatccct cccctcctac ctcaggcctc ttttcacttc tcttccctct ctatattttt 2640
tcctgtcatg agttcatgtt tcccatgttt tcgttttttt aaattaaggt aacattattt 2700
aagctccctg tatacaatat tgtatttcta cttctgtgta ctctatggtg tgatcaccac 2760
caaaaattta gtttccaccc atcaccatac agttaccctc tttactcatt ttaccctccc 2820
cacttcacct ctggtaacca gtaatctgtt ctctgtatgt gtgtttgttt tgtttagttt 2880
ggttgttcat ttattttgtt ttttacattg cacatatgag tgaaagcata tggtatttgt 2940
ctttgactct gtctgaaatt gtaagttttc attcttttat ggttgagtaa tattcatatt 3000
acttccctga tggctcagac aataaagaat ctgcctgcaa tgcaggagac ccaggttcaa 3060
tatccaggtc aggaagatcc tctggagaag ggaatggcaa cccactccag tattcctgcc 3120
tgtagaatcc tttggacaga agagtttggt ggactacagt ccatagggtc acagagagtt 3180
ggagacttgg agactgacac ttttttgact ttaatatttg tatatctata tgctgctgct 3240
gctaagtcac ttcagttgtg tccgactctg tgcgacccca tagatggcag cccaccaggc 3300
tccctggtcc ctgggattct ccaggcaaga acactggagt gggttgccat ttccttctcc 3360
aatgcagaaa agtgaaagtg aagttgctca gtcgtgtccg actcctagca accccatgga 3420
cttgcagcct accaggctcc tccgtccatg ggattttcca ggcaagagta ctggagtggg 3480
gtgccattgc cttctccgta tatctatata ggtaaatacg tatctctata tatcacatct 3540
tctttatcat tcatttatca gtggacactt aggttacttc catgtcttgg ctattcactg 3600
taaacataat aaacatagga gtgcatatat cttttcaaaa tagtgtttct gttttcttga 3660
gaaaaatact cagaagtgga attgctggat catctaatag tcctgttctt aattttttga 3720
gaaattgcta tactccaaag tggctgcaac agtatgcatt cccaacaaaa atgtatgaag 3780
gcttcatctc ttcacatctt tttcaacact tgtttcttgc ctttttgata atagccattc 3840
agacatgtgt gaggtgatat accattgtgg ctttgacttg cattttccta ataacaagta 3900
gtactgaaca tcttttcatg tatctgttgg ctatcagtat gtctttggaa aaatgtctat 3960
tcagcacctc tgatcacttt ttaattgggt tacttgttat tttgttgttg agttgtatgc 4020
gttctttatg tattttgaat gctaaccctt tatccgatat atgatttgca aataccttct 4080
ccccaatgat gagctgttac tgtcacagta actctttttg tgtatactta gctttgtatc 4140
caggatctca cttatgctct agtctcaaat tgcataggcc tgtagagcat tttattcaca 4200
tatcatgatg ttataccgta tttaacatta ctgtgagtca gatttaccaa ttttcccttc 4260
caaatctgct cttcttccta atgttctaat ccctgccagt ggcactatcc caaatcttat 4320
ctatctgtat atcttattta tttatatgtt gctaactgct tgctgggcac aaattctgtt 4380
tgtttgatgt agagactatt atccccattt ttcagaaaag gaaactgaca cttagagagg 4440
ttaactaaat tgaacaatgt cacagaggta cttagcggtg gagctgagat ttgaaccaac 4500
atagttttga ctgcagaatc tatactccca gtagctatgc taggcctctt ccctgatcca 4560
cagtctgcct gttatgaaat tctatcaaat actctttcca catatctctt aggttatctt 4620
ttcttccatt tccatagacc cttttaattc aagggagcag ccttctaatt ggttctctct 4680
cccctagtct cactcatctc catatttctg gtatatttag aattatctaa acactattgg 4740
actgatatgg aaatagtgct ttcatatgtc acttcaaaaa ctttcaatag ttccctattt 4800
cctacagttt aaagtgaaaa ttctgccttt ctttcagaat ctctggataa ctagcctacc 4860
ccacctttct attcttatct tctgatactc cctaccacat gcccctcatt ctacaacaga 4920
ccattcttgt tgatatgctg tatacacaaa agtccctttc ccatcaccat ggctttgccc 4980
attcccttct gttcctctct acctaatcaa acttcaccag ttccacagag catttcctga 5040
tcatgccagt ccattattac cttttctaaa cttcagtact tagaacatgt gccgaagaac 5100
catccagaat tcaacagctc caccataaaa aaaataaaag caagaactaa atgattatgt 5160
atttccctga tattccagtg gttaagattc catgcttcca ctgtaggggg catgagttcg 5220
atccctggtc atggaaccac actgcctgtg gcctggcggg ccagaaaaca aaacaaaact 5280
aagtgattat aaaactcaga tttcttcact tcttgttgat agacataaat gtggttcata 5340
tatggtttta cataatctgt gcctactact gtcacttaaa atgtattttt ttgagtatac 5400
acatattaaa tgtagttata acaacatgga acttagagtt agatcttatt ttttattcag 5460
gttactagct gtgtgattct tgggagagtt tcctatgtgt cctgagcttc agttttctca 5520
tgtattaaaa tcaagatcat cagagggcca tataaaaatt aaatgagatt agctcaatcc 5580
ttggacatta atggcattca ttaaaatgcc agttcccatt tgttctctca ttaatggcca 5640
tttagtaccc tattgtatca ctaccattaa agagatttca tctctttagt tttcatcgat 5700
tttttcattg ttgagcgtta gggtcgtttc tagttttact gttataaatg gtgttagtat 5760
aaacatctct tgagtaatca cttttttccc ctgtggtatt attttcttag ggtgtgttta 5820
cccaaagtta gcagttgaaa gaatataaaa cattttatga ctcttgtcat gcattgccct 5880
gctgttcagt aacattgtgc taatttctta caatgctgtt atcatataac agatcggttt 5940
tctagggtca cttgggcatt gagttcaata ttttcttagt ctttgctggt ctcagaggca 6000
tataatggca ccttggggtt gatttgattt ggatttcttt aattcattgc aagttgtata 6060
gaaagaaaag aaagtgaagt cactcagtcg tgtctgactc tttgtgaccc aatggactat 6120
agcctgctgg gttcctccat ccattggact ttccaggcaa gaatactgga gtgggttgcc 6180
aattccttct ccaggggaat cttcccgacc cagggatcga acccaggttt cctgcattgt 6240
aggcagactc tttactgcct gagccaccag ggaatctggt ggcaagatgt ataccctgtg 6300
atttagcttt cagtatattc tgtcttgtat actcaaaaat gttttatgtt ttagtctcat 6360
tgttgccaaa atcttgtctc tctgtgtacc aatgccaaac agaaatacgg agacagagtt 6420
atggaggaga aggaaagaat agctttatta ctttgccagg cagaggggga acacagtagg 6480
ctagtgcctc aagaactatg cctcctccct ggtgagaagg gaaaggttat atagtcaggc 6540
aggagtatgt gataaggatc aaggtaataa cagtcttgta ttctttcttc tgcaaagttc 6600
aaaagggtgg ggttcctgac aagattggat gtgtggaggg tcttaggtgg tccagtgtcc 6660
taaccttgat gagcctctct ggtcctttaa tcttgctgtt ttactgctgt tcctcctttg 6720
atgagcaact atgatgcctt ttggaactga gaggtcatgg aggctggatt cttgcctata 6780
agaaatgggg gacaaacagg cctctgtgcc caggagtccc acggggccct gctcaacatc 6840
attatctcta taatcagact gtataaacta aatcatccaa cttgaacaag tcactttact 6900
tctataaatt tgatttcttg atatcagtaa aatgagaggc cgataacctc taaatttcct 6960
tccattgtta tggttctgag tgtctctctc tctcctctag cttgtgatcc agtacttaga 7020
ctatcctagg tgttctgcaa cagcatgttt aacctgttga gtaccttttc tccagagtaa 7080
ccaggcaaat gtttcaatat tgtaagtctt ctctggttaa aactgtacaa tgactttcca 7140
ttgctcttag aaacagaaac tgttagttgc tcagtcatgt ccgactcttt gtaatgccat 7200
gagctgtagc ctgccagact gctctgtcca tgggattttc caggcaagaa tactggaaca 7260
ggttgctgtt tccttctcca gaggatattc ctgacccagg gatcaaaccc aggtctcctg 7320
cattgcaggc agattattca ccatctgagc catcaaggaa gcctgtcgct cttaggctca 7380
aattctctat gcttagaagg attgcatgtt atctgcccag tttatgcctt aatttctgct 7440
attctttctc ttattctcag tgcttcagct tgagggagtt cctcttaacg tatacccatg 7500
gttcttttct gtcccatagt aatgtttttc ctggtgggag agtggagaaa aactcgtatt 7560
catctttcag actttagcaa caatgtcttc ctcagggcag ttcaaatctt gacttagtaa 7620
atgtataagt agatatcact aagtcatgtc cgactctttg caacctcata gattgtagtc 7680
cgccaggttc ctctctccat gggattcccc agtcaagaat acaggagtgg gttgccatgc 7740
cttcctccat gggatcttcc tgattcaggg attgaacttg ggtcttgtgc ttttcaggca 7800
gattctttac ccactgaccc accttgggca aattatttag cctctctgag ccttgatttt 7860
caaaccaata aaataagaat gaaaaaccct acctcctacg gttgtggtaa gaactaaatg 7920
agataatgca tttaaagagt ttaaaagcca catctggtac ttagcaagta acacttgggc 7980
aaatagtagt aggagaaaga gatgctcatt aagtaattgc tgagagtgaa tgaacaaatt 8040
tttccttgtc atgggaggct ctgtgggtaa aagatctctt cctttcatta attaattcat 8100
tggaaaagtt actgattaac tacaatgtac taggcattgt gataggcact ggttgcagtt 8160
tgaatagatg gtgaaaaatg gagtggatat tttgagaagg gagggaagaa acttgagtca 8220
agattcagcc catgggagta aagatggttt aagagtgtgg tgggtgttaa gatttgagag 8280
gtagaagagg gacccaggtg ataggttcta gagtacaggg cagagaaatt gtgacttggt 8340
gctatgcata gaatggtaag ccatctgagg ataagtgtgc ctcagaagct aaatgatgtc 8400
tgggcttcct tttctgttgt agagttttgg ggagacattg ccaaggaatt ttactggaag 8460
accccatgtc ctggtccatt ccttcagtac aactttgatg tgactaaagg gaaaatcttc 8520
attgaatgga tgaaaggagc aactaccaac atctgctaca acgtactgga tcgaattgtc 8580
catgagaaaa aacttggcga taaagttgct ttttactggt aaaaacacct atcttatagc 8640
ctgtggcaga taaaaagtca ccctgacatt tatatagcat tttatagttc acaaagtacc 8700
ttaaaaatcc atgcatttga tgcttacaac aacccagaag aataagcaaa gtatcattaa 8760
cttaatgtct caggtgagga aaaatacctc aagttaaatg acttccctaa aatcatgtaa 8820
ctagtatgtg acagaactga cattcaaaat caagtgtcct gacttctagt ctagtgtttt 8880
caacttgtag ttggaaagaa caatgtgagg gggtaaatgg aagtaaaaag gactatgttg 8940
ttttcttatt cttgctttgg gagaaaagtg gattgtggat ttttcgctgc caccaccact 9000
gttatcatca ttacctttga ttgagctata tgccagatgc tgtgctcaag tgcttcacac 9060
atattatcct atttgatgct cacaacaact aaaaatatat tatttatttt cctcgtttta 9120
catatgtgaa aatgaaattc tgagatgtaa atagatgact tatttgaagt tatacagcta 9180
gtaagaagga ctgatattca catcctggta gcacagttcc agagttttac tcttaaccat 9240
gacattatac tatccttcta atttcttata ctagcctctc aacagctttg caaattaggt 9300
attcttagcc caattttata gctgaagaaa tgaagcatgc atctctgctt catagtgaat 9360
aagctcatag agtgagtaac tggtggagcc agggttcaaa cccaagtctg tctgattcct 9420
tgtttttaac actcagccat gctgactcct ccactgaaac taaaaccccg tcttcctagc 9480
aacatagctt agaggagatg ttactaaact gaacctgaaa taggaccttg aagttccagt 9540
tattggggtg tttctgccac caaccataag cactttgaga attaatctgt ttctttttta 9600
aatcagccag taggagcata ttggtatttc acggatgcgt taaatttgta gtcagtaaaa 9660
cgttgcccag tttgtgtgat atattaagaa aaaccctcac ctgagagata ggaaattcag 9720
tttctcactt taactctgcc atccactttc catgtaacca taggcaaggc atttaactct 9780
ctgagagtca atttccttgt ccataaaatg ggggtaatat taaatcccac ccttgcccta 9840
taactggatt gttaggagaa tcaaatatga ttttgtaaaa gtatttattg ggaaacactg 9900
tacaaatgtg agtgatctgt taccacatta ccattgctca ggttggggtc actttgccac 9960
tgaaattcac agtccattat gcctgttgag actttgttta gctcctgcca tgagacttta 10020
tgcttgagct ccagctgaca cttgggccca tctcctaggt tcctcagaga tcagaaaacc 10080
tttaaagatc tggataggag atactttgtc tgaatttgtt ccctggtttg aaatcaagtg 10140
catgatgaaa gactggaaag gcacaagcca gaattgttta ttcaatggat attgctgcag 10200
aatccttgag gcccttcttt ctagtttccc taccatctgt acttgaatag agcctgtctt 10260
tccacatatg taaaatgaaa ggagtagatt agatcagtgt tttcccaaga atgcttttga 10320
aaccactaat tactagacat attagcagga actttttttt tttttttttc cgagcagaag 10380
cttcttgatt catgaaaaaa aaacagactg agaagcactg tataataaat tacgtttcca 10440
ggattttcat tgtgtattga tacagtaaag gctccaaaaa gtcctacagt aaagatacct 10500
gattcatttt aacatgggat ttctaaagtt tatttgagcc ccaaacattt tttatagcat 10560
gcgattaaca tcacaagaag cagtattcca tggaacactg atttatgaag cacttgatgt 10620
ataatctcta catttacttc tcatgcaaaa ttctgtaact ctgtaacaag acattttcac 10680
tccattttta cattggtcag tcaaccaata agaattttaa agatattgag tactcagccc 10740
tgagtgttat ggtacagggg taagaagaat gggagacaag taccacatgg gccattatta 10800
ttcaagtctc aatctacaaa taaatcacat ttgttaaaag aaagtaaagt gcaaatatcc 10860
cagatatttg tttacttccc ttttttacct ctatatttta aaataatatg cttacactgc 10920
aatttctttt gtttttgaag aaagcattga cttcctttta tggtaaatga gaattcagtt 10980
cttatatact actccaactt ctttctctta atctcaacat aattatacct ctatttttta 11040
gttaatttaa aatcaatgct tattttagaa taatatatga ttgtatttcc ttttatataa 11100
agatttttgt tttcaaagaa gttaaaaatc gcctaaaatt ttcttatact atttgccttt 11160
atcatatact caatattttt caaactatca tataatttat tctgtgaagt tctctttttc 11220
cccttggtcc tagagcctac cttctttctg ctctaatttt tattaatgtt tttaaagtga 11280
catcttttca gtgatttgct ccagttgata cacttcttat ggagcgatag tatgactttc 11340
ttttcagcct agacatttag ataactcaag tggctttctt ttcttttttc tgagcaaact 11400
cctggaaata gtgaaggaca gggaaatctg gagtgctgca gtccaagagg tcacagagag 11460
ttggacacaa cttagaactg aacagcaaca gcatagtggc attagaaaat cacatttagg 11520
gcagatctta tatacaaact gaagtaagcc actaacaaag tgcttcagtt agccaagata 11580
gaacactatt aagaagaatt tgccttttaa cttttgactt gacacatggt aaatattcat 11640
ggaagaatag tatttttaga atttgaagtg attgttttat tagaatccat ctaggccaac 11700
tgtccttaca gatgtaaagg gtttatgcag ggatcaattt attcaataaa tacttattaa 11760
ggacttacta tgtaccaggc agtattctga acaaacaaac aaactctcta ccctcttgga 11820
ggttatattc cagcttaagg agacagacaa acaaatcaat aaatataaga caggtagtga 11880
taagtgctgt agaaaaatac aaagcagttt aaggaaaaag gaatgacagt gaaaaatggg 11940
gattgatatt ggcagccctc tctgatcaag gtgacatttg agacgcaata tgaaggaaat 12000
gcagaatgtc atgcatatag ttggttggga aaggaaacta ttccaggtac atggaagagt 12060
aagtgtagag gccctgggac aggagtgttt tgctacctag tgtgtttgaa tactagctag 12120
gaacttagcg tagctgaagc agtattagtt agaaaaacag agtgatacgg aatgaggtca 12180
gcaggacact agattgtata gattgaaaga tttgggtttt ttttttttcc acagtgaaat 12240
ggggaaccac tggaaaatct tgagcgtaga agtgacagtg atttgactta tgtttagaac 12300
ctgtctttat gttgagaata gaccgttgaa gggcaagatt agaagcatgg agacttatga 12360
ggctattata gaaatccatg tgaacaatga tagtgattga aggtgcttca gttcagttca 12420
gtcgctcagt tgtgtccgac tctttgtgac cccatgaatc gcagcacgcc aggcctccct 12480
gtccatcacc aactcccgga gttcactcag actcacgtcc atcgagtcgg taatgccatc 12540
cagccatctc atcctctgtc gtcctcttct cctcctgccc ccaatccttc tcagcatcag 12600
agtcttttcc agtgagtcaa ctcttcacat gaagtggcca aagtactgga atttcagctt 12660
tagcatcatt ccttccaaag aacacccagg gctgatctcc ttcagaatgg actggttgga 12720
tctccttgca gtccaaggga ctcccaagag tcttctccaa caccacagtt caaaagcatc 12780
aattcttcag cactcagctt tcttcacagt ccaactctca catccataca taaccactgg 12840
caaaaccata gctttgacta gacggacctt tgttggcaaa gtaatgtctc tgcttttgaa 12900
tatgctatct aggttggtca taactttcct tccaaggagt aagcgtcttt taatttcatg 12960
gctgcagtca ccatctgcag tgattttgga gcccccaaaa ataaagtctg acactgtctc 13020
cactgtttcc ccatctattt cccatgaagt gatgggacca gatgccatga tcttcgtttt 13080
ctgaatgttg agttttaaat caaccttttc actctcctct ttcactttca tcaagaggct 13140
tcttagttcc tttctgccat aagggtggtg tcatctgaat atctgaggtt attgatattt 13200
ctcctggaaa tcttgattcc agcttgtgct tcttccagaa ggtgcttaga aatggtcaaa 13260
atctggatct attttgagtg aagagcccat ggaatttgct gacttgaatt ggatgtaagg 13320
tgtgaaagaa aagggaatca agattgtatt caacagtggt gaggtccagg actaggacat 13380
cttgaccatt tttggacacc tatgaaaaga ttttatgcag gggagtgatg tcacagctgg 13440
gctcaagaag attaatctgg attcatatgg aagtgagaat ataaaggcag agagctgaga 13500
gttttgttat tcaaggtaga gatgcctaaa ctgggatatt ggttgtggga atggaattga 13560
acaataaaat tatacagaaa tttagaggag agaaaccgat ataggtaact ggttgtgtag 13620
atgtaggaga gacaggcaaa gaaagtgaga gaagagtcaa ttgttccctc agtctttata 13680
gcactttacc tttttcaaag gcacatggtc atctgatcca aaagctaaat ttaccctcta 13740
gacaagacta tggtttcaac ctctcttata ctttcttccc cctaccatga caggtagggg 13800
tgttggtggg gatttatgtg tatatgtatg tgaaagaaat cagtgacaaa attattagaa 13860
cagtcattca cagagagata gtcagtgagg gacattagag gtagctttgg gagtggggcc 13920
tgactagtta aagggataat ggcaaacaat gagaaattgg aggattaagt aaagggtttc 13980
tgaggagagg gtagaaagag tggttggtga ttgtttttcg agtgatttat acaagctgct 14040
cagtctctag gttattccag gagaggaccc caatctctca agcccccaga atttggttta 14100
gtccattcct tctacttttt gctttggatt gcctctggct atcattgtct ctgaaccatc 14160
tcctacctca gtctcaactg aagtctcttt ccacaggctc tggctatttt tcaatgcttc 14220
cttagaaact tctgagaagc aggtgtcttt aagagtgatc tcagggcaga ggctctgctt 14280
tcaggatccc tttatgttcc cgaatatgaa ttcgaaagta tgctatttag agctctccag 14340
atcctggtgg ggtatgagtg agtgtgtgta cttgaggaga tggggatggt tgaaaagagg 14400
agatgagccc atggtctgac aaggagaaga ctgactgtca aggtgacatg gaaaactaca 14460
gctctaaagt gtgttgtctt agcattcatg tccagggact ctattcatta tctaggtctg 14520
tgtcctggag ttcatttcag tgatattgga atggacagac ttttttgtcc tctatatctt 14580
cactagtttc acaatagtgt atttacttat taaaatatac catttgaccc ttgaacaatg 14640
tgagtttaaa ctgcatgggt ccacctacaa tgcaaatatt tttcaacagt aagttctaca 14700
gtactgcatg gtctgtggtt gattgaatca gcagatacgg aggaatcaag gatatggaga 14760
ggcaactgta agttataagc agattaaccc ccatgttgtt caagggtcaa ctgtacttgc 14820
taaggaaaaa cattcattga gaaggaatat ggcctcagac cattcccttc ctctctctgg 14880
gccttccctt tgtgagagtg atgggaggtg aactactgat actctgcaac gccccttcct 14940
gcactgatgt gtcaggattc tacgagtgtt tgaagaggaa gtggttctta gaaggtctgt 15000
tgcaccgtgt agtggctgtt tggtgactca gtaggcaggc tggtcttctg ttgatacggt 15060
gaagaaaagg aggattttag aaggtttggg gtgaagaggc tgaggaagag tctactcccc 15120
ttgcctggta acatgaacac tttagcacaa atactttaaa ctaatgggag atatgtgggg 15180
aaataactat gctgggaaca gtctttctcc acttgaatca gatgggcaga ttgtggagga 15240
gggcaggggt atttcatgtt taaggtgtat tgttaatttt cttctcttac ttctccctga 15300
gttttatgac cattgatgcc ttaaagagta ataagtggtg aaaatgaaac aactaagcta 15360
tgctgtccaa gacattagtc actttttgaa agtaggtggg atctgaataa catgtataat 15420
aataataaac agaatatgtt atatattaat atattattat atgtactaac atatgtaaca 15480
ataagtgtgt aataaatgga gactgtcttg aggcctgaca cagtgtcctg agccacatca 15540
actacctgtg tttgtgtctg ggctgacttc ttatacacgt gtattgagta aatatttaat 15600
gaagagaatg actttagata atgcctattt tttgagccct cttaatctca cttggagaag 15660
gcaatggcaa cccactccag tactcttgcc aggagaatcc catggacggg ggagcctggt 15720
gggctacagt ccatggggtc gctgagagtc ggatccgact gagcgacttc actttcactt 15780
ttcactttca tgcattggag aaggaaatag caacccactc cagtgttctt gcctggagaa 15840
tcccagggac agaggagcct ggtgggctgc catctatggg gttgcacaga gtcggacacg 15900
actgatgcaa cttggcagca gcagcagcaa tctcactata aacagaggta aacaggatac 15960
ttgttaaagt tacttctgtc actgatcttc agaatttcta tcacaaatct aggaaactgc 16020
agtggcaagg cagcaaggag gaaagttggg gaaggggagg aacccagtac tggcctcgtg 16080
cctttgatga gagctggtag ttaggtaggg gcacaggttc ttgccttagg gtagcctcac 16140
atttacaaca gtaaataaga aggcttagag ttgtctgcct tttcctggat ttggtatgaa 16200
agagttgggt cactggtaat gatacttagt gtttgataat aatactttga acctttatga 16260
gaatttgaga ctcccagacc tttgaggtcc acttcctgta caaagcagga gttgtagccc 16320
ctgaatcttc attcccaaca attctcatag tgatcattct gattctggaa cattaccaaa 16380
accacagagt tcacacaata tgtatcacct tgttaggcac ctctgttaca aaattcttag 16440
atatattccc tccctataac tttatcccta cttatgctct tcccatggtc ttaatcctat 16500
gttctggggt cacatgaata atcctattct ttttttccat gagacatctc ttcaggcagt 16560
ttgaaggcag ttattatatc cctttgccaa gctgaatgta ggaatctaaa tctagaagac 16620
gccttgaaga ttccttccat tgtttcccca tctacttgcc atgaagtgat gggagcagat 16680
gtcataatct tagttttttg aatgttgagt tttaagccag ctttttactc tcctctttct 16740
ctttcatcaa gaagctcttt agtacatctt tgctttctgc cataaaggtt gtatcatctg 16800
catatctgag gttattgata tttctcctgg caaataaaga acttagtgtt tggcaaacaa 16860
tatattagct aattttatca ttatgccaaa gcctcattcc taaaacaaga aagagcagag 16920
ctgatctgat tgaaatatag attgaaggac aagagccaca ttctgttggc ttctttacct 16980
tgtcattgtg ttccctgtga gatatgtgtg tggaacctct ggggatttgc aaaactcagt 17040
ttggaaaccc ctgatctagt cagtgccctt gttttatgga tgaggtaatc ttaaagttcc 17100
ttgtatagta tagtttctgg aacaactcta tcgtgcatgt tggtttcctg tagacattct 17160
ctagttggcc agtatcactt attaagtact ctacctatgg tatgccttgg acatggaatt 17220
attaccattc tttgctctga ccttgcatat taaaatcaca ttagacccat tgacagccat 17280
actctaccac gcagtctccc ataatgccta agacttttct gtttgttcta ataataaact 17340
atatctcatt tgcattgtat gtgtataatt tgttcccttg ttttttgaac ctgggtatgg 17400
ttcacagcaa tacttgacag atgaggaact taaggctcag agtggaagtg acttggccag 17460
atcacatagc agttgaactt gttctagaga gaacacttac attctttgtt atagacacta 17520
tgcttctcct ttcatggacc tagctccttt gactgtctgt cacttattta gccagtagtt 17580
tacttagatg atttagggtt ggcttaagaa atgttgacca caatttataa tgataattta 17640
taaggctaaa cacattaaag ggcaaacctc aaggaagtaa aatccttttc ttagctggga 17700
tgtgctcatt catccattca ttaaacaatt tttaaaaact gaatatttac tacatatagt 17760
ctgcatgggg cttcccaggt ggtgctggtg gtaaagaacc cacctgccaa tgcaggagat 17820
gtaagagatg tgggttctat tcctgggttg ggaagatccc ttggaggagg gcatggcaac 17880
tcattccagt attcttgcct ggagaatccc acggacagag gagcctggtg ggctacggtc 17940
cagagagtca caaagagttg gacacaactg aagcgactta acacagcatg gcacatagtc 18000
tgcatactac cttgagaata gtgggtggga ggcacagggc aatagcaggg gaaccagtaa 18060
gggggcatgg cagcaattga gataagatat gataatggag ataacagtag aagctgtgaa 18120
aagtggtcag aatttgaaag ttttgttttt gttttttgcc gtgctgtgag gcttatggga 18180
tcttagttcc ctgagaccag gggttgaacc caggccctca acagtgagag catcagggag 18240
tttctcagat tttttgaatt tgtcaatatt ttgaaggcag aaaccagtag aatttactga 18300
tgaacttgat gtgtgggtat aagagaatgg agtcaagaaa taaaggttgt tttttttttg 18360
gtctgagtac tagaaaaata taatttccat tagttgagtt ggagaagaaa ccgggaggaa 18420
aagctttgag gaaggatcag aaatttgtgt tcatgcacag ctgtaaaatt gcagctgtga 18480
catgtgttag gtagccaata aagaatttca agtagagata tgacatatca agagttatac 18540
tttgaaaaga tcactctctt gatcgtgtat atatttaaaa aaaaaaaaaa taataacgcc 18600
cccccccaca aagcctattc aaggatctgc aggcagaggt gctgaccatt gctttatgac 18660
ctttggcaag tgatgaaacc cttctgagcc tcagtttcct gacctttcaa atgggaatag 18720
caaatcttgt cctgccagat tcaaaaaatt cctacaagga ttagctgaga tgaaagacaa 18780
gaaaacacac cgaaaagatg tgttattatg aattggtgct aatactcaca tactcacaca 18840
tattacacac atctaagggt attattaggc agaaggattc aggactggta gccacacatg 18900
acttttctat cctttccaaa atgtccttct ttcctctgat aagcatagcc tcttttctca 18960
aacttggctc agaatccacg gtttctagga agggtttcat gattgatcat gcttaagtta 19020
tttctttgca tcctttttgt taatttccta cttctgcatt ttcttcttcc cctttctcgc 19080
tgtttaccaa tattggcttg atatggtggg tggtttagat ctctctaatt agagatttta 19140
agcatctgaa agtaaggaat agcctcttac tcccttagtt tcgtaatttg gcaaacagaa 19200
ggtgctgatc gttgggtggt cactctggcg aagatcatcc atttccagtc ctctgtggct 19260
tgggagttta gtaggtaaaa agttttgaag taggggaaga acctttagtc taggaacctc 19320
tccgactctt ctttacctac agaataaaat ttaaactcaa catttgcagc cctatatatg 19380
tatgagctgt ctctagcctg tattattaat ctttaaagtt ttgaaacata cttaaactga 19440
tagaaaagtt acaagtacag ctctttccca gttaccgggg cctattttta agcagaggca 19500
ggaatgtgga agctgacagg ccagtggcct ggtgctgggt ctgtagcaaa atgtgacctg 19560
ttgataaccc tttaaatccc acttcaacca agaggtgaag ttaatcagcg aatctggggc 19620
tctgaacaca catggagtct ggagggtcag cttttctctg gccaagagct gctgctgcac 19680
acctctattt gaaagcaaca ctttgctgca gtgttgatgc aacagcccct ggatgcaggg 19740
gctgtacaag ggcaaggtaa agctgaatta cctgggcctg actgtcaagg gttttttgga 19800
gatgttttca tgcttgcctt ggctgagtcc atccacctca cctgagtgct gatctgccag 19860
caccatatca gagtctgtaa gcaggtagtg aacatagaat ccttcattgt ctgcgaggcc 19920
tcccaggaag acatttgtct ttttcccctt ttttccttac caggaggcct cacgaagagg 19980
aagaaaaaga aaggccaagc tggggctgga cctggcaata aagagcaagt tcaagctcgt 20040
ccattaccac ctggactggt ggattgtcta attttctagt cagataataa aacaggatca 20100
acacttaaag gaaaaaaaaa aaaggaaaaa gaaaaattgc acgtacaata caaatagctt 20160
tttttttgaa ccatttgaga gtaagatgca gaccttgaat tgtgtacatt tccaacaaag 20220
actttctcct gtataactgt aacatagcta tcaaaatcag gaaatcaata ctgatccata 20280
attatcatct aatcttcaga ttccatagaa gttttgccag ttgtcttaat aatctcctta 20340
atggcagaag gatctagttc agaatcacac atgacgtgca gttgtcgtgt ctttttagtc 20400
tccttcaatc tagaatagtc ccttagtctt ttctggattt tcatggcctt gacattctat 20460
catttcaatc ttataggcta caaaatacag cttaagggtc taaattgaat tggctgatct 20520
ttcattttat accaatgatc gagatttttt ttttaaatta tgaatttttc agcaaggact 20580
tccaaagagt tgccaattta aaattgtatg aagtaagggt tgctgcttct gctgctgcta 20640
agtcacttca gtcatgtcca actctgtgca accccataga tggcagccca ccaggctccc 20700
ccgtccctga gattctccag gcaagaacac tggagtgggt tgccatttcc ttctccagtg 20760
catgaaagtg aaaagtgaaa gtgaagtcgc tcagtcgtgt ctgactcttc tcgactccat 20820
ggactgcagc ctaccaggct cctccgtcca tgggattttc caggcaagag tgttggttgc 20880
cattgccttc tccgaagtaa gggtactttt gtgcaaattc atacattgaa ttagaggtaa 20940
acagtaatct aggtctggtg acccaaatat ttacctgcgg atattgttaa tatccagatt 21000
cctggatccc acccagccta ttgaatcaga attttcaggg gataaggttt ctgacttgta 21060
tttttaaaac ttgctcacat gtttttgata tggtgggcag tggcaggatc tgtttgaaaa 21120
gtaattattt atgttagtga tctaataccc agaagaattt tgatatgtaa ctcttcataa 21180
attttaaagt tcagatttaa acattttatc aaaagattaa ataactgcaa agaatctaat 21240
ttccaatgtg tttttattgt atatatatat atgttatata tctttttcaa aatcttgaag 21300
ctgcacattt agctcatata atttatagat aatgttcatc atataaccta atttgcaccg 21360
acagttctgt caacatgtta ctcctttcaa tattgatttc ttatagaagc tctctggttt 21420
gcttccttgg ggctctccct ctgaatttat gcattctcaa attgtccctt tgtttggcac 21480
tctagagggt tttttttttt ttttttttaa acctcagcat tgtataagca taataaagtt 21540
tgggataaaa gagagacatg gcattttttt gtaaagtggg agaaagaaag tgggaaagga 21600
aaaccaactt agatcactgt taggaaagag tacatgtgaa agttgaaact ctggacactt 21660
aactctctta aaacttttcc tgctcaggta cccataggaa tagagtgcta taccttaaaa 21720
attactaatt taagctatgt ctacctaaat agcaaagaac tattgccatg aatctttttg 21780
catgtgtctg ttatacttga gagacttgtt gtacatgtag tgtgttgtcg ctggctgttc 21840
ttcccactcg cttttcctaa tctaggcata ttcttatcta aggcagcatt ccaactaaag 21900
tggtaataaa ggagctgaga gggctgccct cattcattta ttttggggga gactagcatg 21960
gtaggaacaa ctggatgata ggaatgagta tgattttccc cctgtaggaa ggcatgtagg 22020
catcgggaga aaggttacct tctatactag cccaggcctg ccttcctgac agagaagatg 22080
tgtagaaacc aactggcacc aaagccactt tggccaaggg gaacaggcgc aaggtctctg 22140
gctcaaggcc taaaaatagc tggcaggaga acagggtgta ttaatagctc agattgcaaa 22200
gcatgtgtcc catagcctga acctcatatt caggggcttc ttccagtctc tttcatggca 22260
tagagtccag gattgagtcc actgtagggg ctcaagaaac ttttctatat atgtttccaa 22320
atcccatgcc acaagtttta aacttttccc ctctgtctac catttccccc tgcgtgttta 22380
gcatctgtct tcttcatttg tatcctctat aatcccctct ttccttccat gtttcctcac 22440
tctgatcacc ctagatccgg ctgatcatgt aggcagagaa gtttattcat tcactctttt 22500
atccactcat ttcacagtta tgtattaagt gcctactatg tgtcaaagtt tatgcttcgc 22560
actgaggagg aaacagatta aaagatatcc tgtctgtctt taagaaactc acaatccagt 22620
gagagaaaca ggtgtatgct atgactataa tgctaaagat aaaataaata aatgccaagt 22680
ctttaggtaa atagacagta tgaatgaatg aatagtgcta aggtacaatc tttcaggaaa 22740
gaaagatttg taaagatgtc tgttttttcc aaattattct gtgtgcttat tgcagttcca 22800
atcagaattg caataagttt ttaaaatttg agatataatt cataaaatac aattgctcat 22860
ttaaagcaca cctcagtgat ttttggcata ttcacagagt tgtacaaaca tcatcacaat 22920
ctattttaga acattttcac caccccaaaa agaaacctca tactcattag caatcactcc 22980
tcattttctt ccaactttcc ttgctgtcag acctaggcaa tcactaatct attttttgtc 23040
tctattctgg acagttcata tgaatgaaat catacaatat gtgacctttt gtgtctagct 23100
tcttaaactt agaataatct tgtcaaggtt catccatgtt gtagcatgtg tcagtacttt 23160
attccttttt atacaaaata atactccatt gtatggatat gccacatttt atttatccat 23220
ttttcagttg atgtattctt ggattgtttt cactttttga catcatgaat ggtgctgctg 23280
tgaatctttg tgtacaagtt tttgtatgga tgtatgtttt cagttctctt aggttattat 23340
agttttacta aaatccacgc ttggtggact ttaaccccac agttctcagg actcctgcac 23400
tttgattttc ttctttctgc taatcctggg ttttggaaac atcactcccc ttatgtaggc 23460
cagttcattt agcaggagaa tgccctgagg catatagact ctagagctag ccacaggtga 23520
ctgttaatat ttaaaataga ttaaattaaa ttaaaaattc agttccttca caacatttta 23580
agtgttcagt agctatatgt ggcctgtggc taccatactg aacagctctg ctttacaata 23640
atagtgctgc taagtgctga ggaacctcca ggccatcact tcccatctgt gcgtttggtt 23700
tctcatctgt gaataggggt ggtgaacatc aaaagcccca tcttcctctg gcattctaag 23760
tgatttatga ttcagtcttt ggtacttgcc tcctcccacc tcctcctcca acccttttcc 23820
ttgttcagcc aggagtatcc agagggaaca ttaagctgct aatgtgtcaa gctatgttgc 23880
tcccacaccc cgctcccttg gtgaatggga aagtgacttg aggatatact tatgtctaag 23940
tgcttcacat ttgtgcagtg cctcatggat catagaacac attatttctt aattttccca 24000
aatcaaagaa tctcaaaact ggaagcatct taaaggtcat ctgaaagttc actgaacatg 24060
aatcacatgg gttgtttata aaatgaaatt ccaaggaccc cactccctcg agaatgatca 24120
aatagacaac agggtttccc tgttccaacc tgcacaatcc tatgcttctg gacaattctg 24180
gcatttagaa aaagtattaa tcactcaatt gtgtccgact ctttgcaacc ccatggacta 24240
taggccgcca ggcaactctg tccatggaat tctccaggca agaatggagt gagtagcaat 24300
tcccttcctc aggggatctt cctgactcag ggattgaacc tgggtctccc ccattgcaga 24360
cagattcttt gccatccaag ccaccaggga agcctgacat ttggaaaggc tttctttata 24420
ttgcactctc ccaattgacc tagctcttcc aattatagtt atgctatgct atgctatgct 24480
aagtcacttc agtagtgtcc gactctgtgt gactccatag acggcagccc accaggctcc 24540
cccgtccctg ggattctcca agcaagaaca ccggagtggg ttgccatttc cttctccaat 24600
gcatgaaagt gaaaagtgaa agtgaagtcc ctcagtcatg tccgactctt agcgacccca 24660
tggactgcag cctaccaggc tcctccgtcc atgggatttt ccaggcaaga gtactggagt 24720
ggggtgccat tgccttctcc aaccaattat agttacaaaa tactaatttg tttcccataa 24780
taatccttca ggttcattca ttcattcaga aagtatttat tgagcaccta ttatatacaa 24840
ggttctttgc aaggtcttgg ggattcagta gtgaagaaga ctaacttggt ctctgccttc 24900
aggaagctta cagtctagta ggtgagacag acaattagca aaaaacccca aaaaacagta 24960
actaaataag acaattacaa cttgaaaaaa atgctataaa aaatgaacaa ttttgtgatg 25020
aagaatgtca ggaaggactg atgctggtca tggaaagctt ccctaggaaa gtgacagtta 25080
aactgagatc tgaaacatga ggtagagcaa accattcgaa gagttcagga aagacagtaa 25140
aagaccaagg catgttctct cagtcagaag gctaatgtgt ttggagtctg gtaggcagaa 25200
tggaacaatg gcacataatg agttggagaa gatactgtag cctggtttgt gtttataata 25260
gttcattttg gtttatgtat agattggaag ggggcaggag tgtagtggga ggccagcgtg 25320
gcgtcactgt ggtcatctag gcaagacatg acggtggctc atactagggt tggcagcaac 25380
agagatacag gtgtttgaga cttactatca ggtttctcat tatattctca cctccaggct 25440
aaacagataa gtctggtctt ccttttataa cggttttgac tcctctcatc tgggtacttc 25500
tcccctggaa gacaactcac taggtttgat cctgccttct gatgcttctg tgaggaaccc 25560
agttttcccc atctattttt tttttttttt tttgctcagc tccttcctcc tatcttgtgc 25620
tttacttttc tgctagttta gggaactaaa atctccaatt caatgactat ccttatacaa 25680
aaagaaagag ttccagtctt cccttgtcat taattagtta caagtttttc actgatttga 25740
ccatgaacct taagtattta ctgtgccaac tgtgcttaga gaaaacagaa aggaagaaga 25800
cacaattcct tcccttcaag agcccagggt ataatgggaa caataaggcc ccaataaatg 25860
taatacaggt agaaccaata gatcacaagg aaagtgagat aaagggactt ggaaggaaga 25920
aagaattttc cactttgagg taggtgagga aaagagaaat tagggaagac tttctggaag 25980
atgttacctt ggagtatctg ggactggggg tggacaagtg cttttttata tggcatggtc 26040
aaagatgacc tcactgagaa gccaacattt gagcaataac tcaaaagata gggaataaat 26100
catgtggctc tctgggggaa gaatactcaa gtggagggca aaagttctga ggttaagtgt 26160
gattggtgtt gccaaacagt aaggaggcta tgtggctgga gcagagaggt ggggagagga 26220
agagagaatg ggagtatttg aggatagaga ggtaatgagg gagtcattta gtaggtcagt 26280
gtgaggactt ttactcaaaa tgatatggga tgacatctga acattttgat cagaggaatg 26340
acatgatctg acatacttaa tggagacttt ggactatgat gatagtggat gtgaagggaa 26400
gtagtctgat tctgaagata ttttgcatca gtaatggata acagcgaata tggagagtgt 26460
aaagataagc tagttgtaga agttttcctt taaaaaaaga ttataagaga cacagacgta 26520
tagaacagtc ttttggactc tgtgcagggg gtgatgattt gggagaatgg cattaaaaca 26580
tgtataatat catataagaa aaaaaagaag agtatatgag atataaaaat ttcgattctt 26640
tggtatgtga aaaaaaaagt aaaaaagatc atacatagac ccgataaaac attacaatta 26700
aaaaaaaaaa aaagattata tgccttgagc tgtcccatac catatggctg ccaggtaaaa 26760
gggaagtagc tgggtctgct gcagggttcc caatccgtag cccatcagct aaatctttga 26820
ttaatagttt tgtttgacca cagtgtttaa atttaaagtt aattgcatca aacttaaaaa 26880
ttcagaaaca gattccaggt ctctcttgaa aaactgaaaa attgggcagt attgagacct 26940
tattcccaga gggcaaaagg taataggcgc tgcgtttaag tgctagatgc ctcctttcca 27000
gagcacgtat tctcaagttt tccagtcccc gcacctctct attttttccc cctaaaacta 27060
agtgtcattg tgcacatgct gctgtttgtt tttttctttt tatttttttg atatagtaaa 27120
atattttatt tacttatttt attcaaattt taaaattatg aaatattctg aatatataaa 27180
atgtagagat ttatattata ggcactcact tgttcatttt atatgcttat tacgtactga 27240
tatttcactt agtcttttta ttatttattt atttaaaatt tttttattat tttttatttt 27300
ttaatataaa tttatttatt ttaattggag gctaactact ttacaatatt gtattggttt 27360
tgccatacac tgacatccga catgggtgta catgtgttca cctgctgctg ctgctgctaa 27420
gtcacttcag tcatcttttt taaattgaag tataatttgc ttacaatatt ggttttaggt 27480
gtatagcata gtgattcaat atttttatac attacaaaaa tgatcatcaa aataagtcta 27540
gttaccatct gtgaccatac aaagttgtag tattattact atattttcta tgctatacct 27600
tacatccaca agacatttac tttatagctg gaagtttgta cctcttaagc tccttcacct 27660
gtttcaccca ttccccactc tcctctgccc ccagcaacaa ccagtttgtt ttctatatct 27720
atgaatctgt ttctgtttca ttatgtttgt ttatttttag agtccacata taagtgaaat 27780
catagggtgt ctgtctttct ctgatttact tcatttagca taatatcctc aagtctgtct 27840
atattatcac acatggcaag gtttcactct tttcttatgg ctgagtaata ctacattttg 27900
ggggtatgtg tgtgtcccca tgcacatgtg tgtgtgtgca tgcgtgctcc ctcccctctc 27960
ccaaccagcc gtggccccag ccgggtgttt atgtcagaga aagaagacac tggaaggaat 28020
aaggtgcctc tatcaataat gtataacttg agcacagagc cagggagaga gatgtacact 28080
ctgtgtgtgt gtgtaactgg aagtgggctc aagtaagctg ttctcgtatt cttgtatgct 28140
gctgctaagt cgcttcagtc gtgtccgact ctgtgtgacc ccatagacgg cagcccacca 28200
ggctcccctg tccctgggat tctccaggca agaacactgg agtgggttgc catttccttc 28260
tccaatgcgt gaaagtgaag tcgctcagtc gtgtcctact ctagcgaccc catggactgc 28320
agcctaccag gctcctccgt ccatgggatt ttccaggcaa aagtactgga gtggggtgcc 28380
attgccttct ctgattcttg tataggtttg ttcttatctg tgcctgtgca ccttctatgt 28440
accagtgtct attctaggta ttgaatctca tcgctaggga tgtagcaatg aacaagacag 28500
acaaggtcat tgttatcatg ggatttatat tccagtgaaa gatacagata atacacaagt 28560
gaataaaata catgagctaa tttcaaatca tagaaggttc cacaaagaaa ataaaacagg 28620
ataatgtgat agagagtttc tggaaggtgc acgtaggtag atacatacgt gtgtggtgca 28680
tatacttgca gatgggccca tgttcgagtc ctatatccat atattaatgc tgtttgttcc 28740
tatgtgtgca aatgagcagt tcctatgtct acaatgtttt ttatattagt atatatgttt 28800
ttatgtatgc aggggtacat aggtcagcat acatatgggg acacgtatgc atgggtgtga 28860
ggtgtacatc agcatttaac tgtaactaga gatgttcctg tttgccactt gtccgtgagc 28920
caattaagga agtgactttt tctgcatccg gcattcaggg ccatttgcca gcttcctctc 28980
tcccagttcc ttcccacttt aaacagctct gtgcttttct cctgtcagct ctaggaaggt 29040
gaagccaatg agaggatagt aactaactca ttgagccagg agctggaaag aagctgataa 29100
tcttctgtga aatcgccaaa gacctggctt ccctgctagt ccctgtcctt cacactatga 29160
ggagacagcc tgacttgaag tcagaagacc tgaggatcta ttcttggttc tgccgcttat 29220
tattgtgacc ttgacaagct cctacctctc tctgggtttt agttacctcc ttcgtaagtt 29280
ctctgggtgg taaggaagga aaggaagcac agtgaccttt atggagggct acgtggtgct 29340
gcccgaaaga ggtgggaagg cattctgagc tggggcattg gtgtgattaa gattgtggaa 29400
gtggcacttt gtgtataagg gaagccagga agaatctcct taggataaaa ttcaaatttc 29460
ttaccgtgcc tacaaagcct tatgtaatct gactgctgcc tacttctgat tcttactctc 29520
cttcttgttc actacactcc agccatatta gcttcctttc tgttctttga ataggccaag 29580
ctctttacta cctgcccagg atactcttct caagaactca tacatttgcc tgctctcatt 29640
gctcaggtct tagctcaaat gtcacctctc cagagagagg ccttccctga tcatcctact 29700
tagggaactt atcctctagt atttatcagt atttcaaatg tccatatctt tgacccaaca 29760
atttcagttc tagtaattca tccttcagat atattttcaa gtgagtaaat gacatattaa 29820
taaagatact cattgaaata tttatttgtt aaataaatta tggcacacac aaatagtaga 29880
acattatgca tatgacaaag aaaatgaaat agcactgtaa gtcttaatgg aaaatgatat 29940
ccagatatat tgttaaattt aaaaattaag caagatgatc aagctctata tttaagattt 30000
atgcagttta ttgaatgcat attatattca ataaaaaaca agatacagaa aaatgcatat 30060
tgtatgccac catttgtata ggaaaaaaag agagtatata aacacagaag catgtttata 30120
tgcttgtcta ataggcctgt cctgtgctta aagctcttct ttcccacctc cccattctat 30180
gcttttttgg ggactttgaa gtatttattt ctcttctgaa cagcgttaaa attcaatttt 30240
caaattcact taaaaattta catatctttt tatgttcagt tcaggtcagt tgctcagttg 30300
tgtccgactc tttgcgaccc catggactgc agcacgccag gccgccctgt ccatcaccaa 30360
ctcccagaga atgctcaaat tcatgtccat tgagtcggtg atgccatcca actgtctcat 30420
tctctgttgt ccccttctcc tcctgccttc aatcttacct agcgtcaggg tcttttctaa 30480
taagtcagtt tttcgcatca ggtggccaaa gtattggagt ttcagcttca gcatcagtcc 30540
ttccaatgaa cattcagaac tgatttcctt caggattgac tggtttgatc tccttgcagt 30600
ccaagggact ctcaagagtc ttctccaaca ccaccgttcg aaagcatcaa ttctttggcg 30660
ctcagctttc tttatggtcc aactctcaca tccatacatg actactggaa aaacaatagc 30720
ttcaactaga cagacctttg ttggcaaagt aatgtctctg ctttttaata tgctgtctag 30780
attagtcata gcttttgttc caaggagcaa gcgtctttta atttcatggc agcagtcacc 30840
atctgcagtg attttggagc ccaaaaagat aaagtctgtc actgtttcat ttccccatca 30900
atttccatga agtgatagga ctagatgcca tgatcttagt tttctgaatg ttgagtttta 30960
agccaacttt ttcactctcc tctttcactt tcatcaagag gctctttagt tcttctttgc 31020
tttctgccat aaatgtggtg ccatctttgt atctgaggtt attgatattt ctcccagcag 31080
tactgattct agcttgtgtt tcatccagtc cggcatttca catgaagtac ggagaaggca 31140
atggcacccc actccagtac tcttgcctgg aaaatcccat ggatggagga gcctggaagg 31200
ctgcggtcca tggggtcact gaaggtcgga cacgactgag cgacttcact ttcacttttc 31260
actttcatgc attggaggag gaaatggcaa cccactctag tattcttgcc tggagaatcc 31320
cagggacggg ggagcctggt gggctgccgt ctgtggggtc gcacagagtc aaacatgatt 31380
gaagcgactt agcagcagca gcagcactct gcatataagt taaataagta gggtgacaat 31440
atacagcctt gacatactcg tttctcaatt tggaaccagt ccattgttcc acgtccagtt 31500
ctaattgttc cttcttgacc tgcatacaga tttctcagga ggcaggtaag ttggtctggt 31560
attccaatct gtttaagaat tttccacagt ttgttgtgat ctacacagtc aaaagctttg 31620
gcatagtcaa taagcagaag tagatgtttt tctggaattc tcttgctttt tctatgaccc 31680
aacagatgtt ccaatttgat ctttggttcc tttgcctttt gtaaatccag attgaacatc 31740
tggaagttct tggttcacat actgttgaag actagcttga agaattttga gcattacttt 31800
gctagcatgt gagatgagtg caattgtgca gtagaattat ttaaaaatca taaaattgaa 31860
aaataatact gaaggggata ttcttacagt taaatctttg tatacagtct gaatccttaa 31920
gatgtctttt taaaaatttc tcggtcatta ttatgtcatt ctttatatct ttcataatgc 31980
ttatcataat tggaaattaa attatctgtt atatttttac ttgtatattt cctgtactat 32040
cattggaata taagcttcat gttgggaggg attttgctta tcttattctc agtgccttat 32100
ttctattcat atatatagca catagtagtt gaataagcaa atgaatcgat gaagaaatga 32160
ccgaaggaaa ccaggctgaa gcctagaaag gagtaggagg caaggctgag gaggtagaca 32220
gcaagaagtt gatagagggt ggtgattctc aactttaaag gtgcatctga tttacctatt 32280
gattctttat accaacttgt agtaaagaag cagattcttg gatcttgtga ttgtgattct 32340
gagtttgggc tcaggaatct acattttgac aagttcccta ggtgacttgg gtacaagtaa 32400
tctagagacc ccactttgaa taacaccagt gcaatgctga catcactttc aggaagaatc 32460
acattcgtga taccatttct ccttaataca tgcctcgaat ggaagagaga agaccatgat 32520
cccattttat caaatgaggt tcagagatgg cagtgcattt tccagtgata cacaggttgt 32580
gtataaagta tatcttgtaa gcccagtgta tctgatcagt cagctcaagg tgtggtggca 32640
gatggctcac cctcacagaa tattcccagg ccttggtgtt aatgaatgga ttttatgact 32700
gttcctattt ttgtttgttt gtttggggaa gaagtcatat tccctaatga ctggctcttt 32760
ctgatacatc tcagggaggg caatgagcca gaggaaacca cccagatcac ataccgagag 32820
cttctggtcc aagtgtgtcg gttcagcaat gttctccgaa aacagggtga gtgaggggat 32880
acagggagag gactggagag cccagcctgg gggtatctga ggacttgggg gaggttgaca 32940
aggaaaagaa ggaatctgct aacagaagaa caggaaaggt cacctgggag gggaggtagg 33000
tgggtggcag ataattggat gagaaggaac acgtagcagc aggtgggatc cactgtggtc 33060
catgacctgt gtcctttacc gcaggcattt gcaagggtga ccgagtggcc atctacatgc 33120
ccatgatccc ggagctcgtg gtggctatgc tggcgtgtgc ccgccttggg gctctgcact 33180
ccattgtggt aggaattggg gctggagaaa ggggcctggt tgggggtagg gctcaggata 33240
agtaggttga gcctggatga tggagaatct gaaaaacaag actaaagcat ttagaatgtt 33300
tgtgtcatag gttccttatt ccttttcttg ttcactcgat ccttgggttg ccatctttct 33360
cagtctttct tcccttttct agtttgcggg gttctcttca gagtctcttt gtgaacggat 33420
cctggattcc aattgtagcc ttctcatcac tacaggtgac taagtctctc acccctcccc 33480
caaaaccccc acactcaaag tcttggtctt caaccagcta gctggtattt ggggctgctt 33540
aatgtaaagg gtaggggcag cctacccttt atttcctgct tccacctcag ccttccctct 33600
gttagctcaa attagctgga agatccagag acaagaggcc tatccaggaa ggactgaaca 33660
ttgtacccac tagggataaa ggggtagaag tgtgatgcct tcctaaggct tggaatatct 33720
gttgctcccc aaagatgcct tctacagggg ggaaaagctt gtcaacctga aggagctggc 33780
tgacgaggcc ctggagaagt gtcaggagaa gtaagtgtgt ttggtctgct ggctactgcc 33840
ctgagttgac tgggcatttc cccagtgcca ggtttccttt gtccctccta ccctagatag 33900
acatgggtgg gtcttgccaa attctccttt gagccaacca gcccaccact tcaggctttt 33960
ctttcctcca ggggtttccc agtgaaatgc tgcattgtgg tcaagcacct ggggcgggca 34020
gagctggtca caggtgactc tcctagccaa tctcctccga ttaagaggcc atgcccggat 34080
gtgcaggtga gtctggtacc attatgccct cctctgggct ccaactggct ccctcttcct 34140
ccccaggaag ttcctgatgt ccatctttgt ttccttatca taatgctgag gatctaggag 34200
cagtcctcgg aatggcctcc ctggcagggc tggctggaga taggggactt tgaccttgga 34260
tgtaggtagg gagctttgga aacttagccc aagagattgt gaggaagact cctgtgctgg 34320
ttggagtatt ctgatgggca tcagggatct ttagggattg ggcttgggtt agaaatgggc 34380
tgacctgggt gactatatgt agtcatctta aatactggtc tttggtttgg ggtgggctac 34440
ctatgtggtc ccaccatgga aaaaccagga tctgcccctc aatagatggc ccttgatagc 34500
tttggataca tgcttctgat gaaaaagccc agtggtggcc tccaggtcta agaggcatga 34560
aggataaaag ctccagggtc cagaaggaca ggggtttctg aaattctgtg acttgagagg 34620
ctttgagtag tcttagagct agacagactt gagtctggtc taggaacatt ggagaccctg 34680
tcagcatttg gggttctgag tgtatagagg ctatgaacag aatcaggaaa atggaggctg 34740
ggaggaccag tgagagagga accagaggga aaggaagcag ccagaggttt ggggcacatt 34800
gacccttccc tccccctctc ctatccttgg accctgctca ccaccctttc cgtgggctct 34860
taacagggta agccgaaaga gaaacccaag cacatccggc cccaggtatc cacctctgca 34920
ttgccttggg cactgctttc ctgcatgctt gtttgcttgt ccaactgctc tttgcctgtt 34980
cgccccattg aggtgactgc cagacctaac ccccttctct ctacccttag ttagtctaag 35040
tcactctgta gggtcatgag accaaatgct tattgttctg aattggcata gagcatggat 35100
ctcagagagg tagcaacaga catcccattt cactcctgct tctcaggacc attgtgggag 35160
agaggaccaa gctgtctgtc agaaagtttt tactgtgcac ttagctgagg cagaaccagg 35220
aaagacagga ggaacaggta gaggggatgc agaccttgac gttgggaatc accatcaagt 35280
tggcagctga gaagaaggct cctaaatata tttataaact actttgggct ttacaaacta 35340
cacataaact ctgatataat taaatgctaa attgaacaaa agttatattg taggtgattc 35400
taagaagaga gactcttaag tggggactca aagctgatta gtatagagga gagactgggt 35460
ttggagtcca gagaccaatt tttagctata tgaccttaaa tgagtcaccc cttctctttg 35520
gacttcagtt cccttatcag taatggagac atgatagtaa atgctttaaa agaacctctt 35580
tgcttactaa aacaaaacat aaaagccact attcctcttg agttttacca tcctcatagg 35640
atgtcttact ctaaatctgc tatgatcaac ctcttaggct aatggtaaat ataatacacc 35700
ttttctctat tctcattagc acggtggaga cagatctgag tcttagggaa aaatgttttc 35760
atttgaaaac tcaggcaggg acctaagact tttctcttga aagaggaaac aaacttagtg 35820
gttagaatat aggctctgga gtcagaccac ttggcttcac atcttggctc tgttacctgc 35880
tatgtgggaa cttgggcaag tttcttaagc tcctttactt cagtttcccc atctctaaaa 35940
taaggataaa attgtaccta cccgacaggg ttttgtcagt ataggtaaag tgttcgacac 36000
atagtaactg ctccataaat gttagctttt attatttcta atatagcaga caggctggca 36060
tttctaaaag ggagtttaga aaagaaataa actcttttaa ttcttaatac cagaggttgt 36120
taggccacag gctcctggaa ttttatttct tggaggtaaa aagactcagt taccaaaaac 36180
ccaaagacca tgaaaatcag cttacaaaac ccaggttggt tggcctgaga actgtagtca 36240
aaacagtgga aatcttggca gagctccaaa cccctcattc ttaagcagtc ttcaagggag 36300
ggcctgttac agttggcagg gaccttcctc aaacatatta cctataagct tcctcatatt 36360
cctctctcac acctggcagc tgccctccct aaggctttcc tggtccaata acaaaggcca 36420
gctccttgat tcttatttca gaaaatgcaa accttttcct ggttccaagc cctgggccct 36480
cataggtata cacctaattc acaaccaaca tgtgtgccac tgattgctct cagcctgctt 36540
cctggtgctg gttatcagct ccccacctgg agtatccacc ccaccgtcct tacccacacc 36600
cacaaaggaa gcagttttac aagattacaa gagtcccctc actgccacag tgtaactgtg 36660
ctttgccctt gcgtgtacct agtgcctttt ctgtgaccat ccacaccagc accatagact 36720
agctgcattt gctcaaagca ctattgacac acttcttctt aaacacatta gtgccctcat 36780
ctcaagtaga tcatctggtg ggcaaaaaat tattcatgtg acaatgtatg tgtacacctg 36840
tatggctatt ccaattttta aaaaaagtag ggtgtatttt tgtattttct agcttgtggg 36900
gaccttctct ctgttctttt tcttcttaat ggtgtctctc ttgacctgga cttgtcatgc 36960
cagaaacttc aaattctcca gagtaagaga cagatagttc agaaaaaaga gaatggtaat 37020
ttttaattta tcaaggtatg tgtctaacac tgtggggctt tgaaaccaaa tatgcacaca 37080
ataaattact atataaccat ataatgagag gaataggatg tttcctctta tactatcatg 37140
tctacctgcc aaatgggact atagaacaag gaagtaatga atatgaaaca gttttgtgac 37200
agttaaacca aacccacaat agtttccatc atcacactct tatttcacct cccagaggag 37260
ttcagtggaa ttaacttttc accttcctga cattcatcag tcttctctgc ccacccagac 37320
aatccaaggc ttgatgttta ctaggtaata acagatttcc tttgatgttt ccaaaatcct 37380
gtgagactat cagggcaaga atgactatcc tcattttaaa aatgagtaac ttgaggtaga 37440
gtggcttgcc catggtcata tatatctagg aagtggcaga actgggaatc gaaatccagt 37500
gcaactttgg acgaactcag ggaatacaac ttcactcagc aaagagtaga cttaaaacca 37560
gttttcacac aggtgatata attcagaaag taaaacagtt tcaaggaaag tttacaaaaa 37620
tttcaggatg acagatctga aagacacttc agtattcgag ggacatcctt ctactgggag 37680
aaatccacct tgatcctttt ccaccaactt aaagcaaagt ttccttctga atggaacctc 37740
aaggaagtgt ctttatgaga aacccattgg taccactggt atagggtcag ggcagagctg 37800
gagtaggaac ctccagatgt ggatgatgtc cgtacatgca aaaaaagatg cttagaccaa 37860
catctgttga cttcttttgc tactaactat tggggtcagg tacccacctc acctgcaggc 37920
tctgactaat tctgcatttc tgtattgtgt ggtatctgca tccctgctga tgagccacta 37980
gtcccataag tatatatgtg tgtatacagg cctgtgtatg catattcatt catccaacaa 38040
aattttactg agtgcctgtt gagtgctagg cattttacta ggtgttggag atttaggggg 38100
gtgggcaaga aagatagaca tgccaatgta ctcatagagg tcactatctg gggagacggg 38160
aatcaagtaa acagctaaat aaaataactg gaaatatgtg ctatgtggta aagaaataag 38220
gtgctgtgat caagaattat agcaggggtg ggaattcctt ggccatccag agttaagact 38280
ctgcgctttc actgctaagg gcccacgttc aattcctggt tgggaaacta atctcacaag 38340
ctgattaaaa aaaaaagaag aagaatagta ggggtgaggg tgggtctggg agcctatcag 38400
atgctgttta ggttaaacct ttccaagaag gtgacattta agctaggaat gacccgagaa 38460
gggaccaacc aagggggccg cctccgcctg aggccaggtg agagaagttc tgaagcaggg 38520
tgctgggagt acccaagaag cctcctggtt ctgtttctgt tctccctgcc ccatttcacc 38580
ctctctgggc ccttcctccc tctcaggacc tccatctgcc actcctgccc tggcctctcc 38640
aggtgctagg taggggtgaa ttttgccccc agcctgatct gttcacaatt cctcgttgcc 38700
cattcctatt ttctgccccc tgcagatctc ctggaatgag ggggttgact tgtggtggca 38760
tgaactcatg cagaaggcag gggatgagtg tgagcctgaa tggtgtgatg ctgaagaccc 38820
actcttcatc ctgtacacca gtggctccac aggcaaaccc aaggcaagtg tgtgtgtgtg 38880
tgtgtgggta catacactgt gtgagggtga gaagtgagtt tctgaggaaa catgtaatga 38940
catgaaattt acaactctga gtttcagaaa tatcacatta ccttttctaa atcaaattga 39000
cataaatcct caatcactga tggtgaagcc caaggtcaga caattcaagt ggggagcctc 39060
agggtgccca gaaaaggagg aaaggataaa tccgtggggc tgacatctgt gtttcctgcc 39120
catacccacg tggtttggtt gaagaagagc tgcctgtaaa gtcaaggcca agagtgggca 39180
cagggatccc tctttgcctt cttgactaca gctcttagag ttcaatccct ttcacatgcc 39240
acactggtaa catctagtcc ctcccggagc agtgtgggaa ctggaaacac ttagaactct 39300
gacttaaaag atatatcact ctaccttcca ctcgccacac ccataaatta ttctctccac 39360
aagctttgga gtattcctca gtgaaccttt agagacacaa tttgggcctc tctttccttg 39420
gagaagaaaa tggcaaccca ctccagtatt cttgcctgga aaatcccatg gatggaggag 39480
cctggtaggg atgagatatt aatggagcaa gcaaagaaga ttatcttttt tccccactct 39540
cattctatca acgaataatt gtatgcgtgt gcacacacaa cagagacact attggatgtg 39600
agtgtaccga aagggttcat gtgtgtgaaa gtaatgtggg tggttcagaa ataccatctt 39660
agggtgtgag tgtggatgat ggactgacta tataaataca tgtggtgtgt gaagtgggaa 39720
cgtggagaaa gcaggatgga agagagatgg atgctgtgaa agcagtgctg gcagggccag 39780
aatcctgggg aggcttttcc tctacatttg gtagtcagcg agggtgagcc caacttgagg 39840
ccctattctc tccacagggc gtgttacaca cagttggggg ctacatgctc tatgtggcca 39900
caaccttcaa gtatgtgttt gactttcatg cggaggatgt gttctggtgc accgcagaca 39960
ttggctggat cactggccat tcctatgtca cctatgggcc actggccaat ggtgccacca 40020
gtgttttggt aagaagggtg ctgggaccca tgactacctg gcttatttag gggatggaca 40080
agatgatcct gggtgatgac ctcctaaagc caccttgcct agaggcagag ggacttatac 40140
aatgacctgt ttccttgaga aagaggagtt ctcagagggt tgtatacgct gagtgggaaa 40200
atctcttggc ttgtctggta agggtgtgaa gacagggatt tgtcagaaaa ggcaaaatac 40260
tgagcctgag ggtatggtag ggggatggag ggactattga gaagggctga aaatgaccag 40320
ccattattgg ggtcagtttg aggggattcc cacatacccg gatgtcagcc gcctatggaa 40380
tatcgtggag aagtacaagg tgaccaagtt ctacacagca cctacagcca tccgtctgct 40440
catgaagttt ggagatgagc ctgttaccaa gtgagacccc tctctggctg ctgccccttg 40500
agtgtccctc agagctggtg ctggcctttg cacagtcctt tccagcccat tattctaaca 40560
ccctggcttg gaatagggga gggggcagaa gagcctgtgc atgtcttcct ggttcctagt 40620
ggtgctcaga gcctcccttt ctccccactc ttctgcccaa ggcacagccg ggcgtcctta 40680
caggtgctag gaactgtggg cgaacctatc aaccctgagg cctggctctg gtaccaccgg 40740
gtggtaggtg cacagcgctg ccccattgtg gacaccttct ggcagacaga gacagtaagt 40800
aacaggttca gaaggctggg gctcaggcac agtgtgcgaa gactgactca aacccccggt 40860
ctctgacttc cacagggtgg ccatatgctg accccgctcc ctggggccat acccatgaag 40920
cctggttctg ctgtgagtga ggctcccctg gctggtcctg ggctaggccg gggagagtct 40980
tggggcattc agcctaggta gagggtaggg ggctggactg ataggtgtgg ggaatctggg 41040
gctctggggc tctctagagg tagagagttg agtcagagtt gtctcattcc tcgttgtgag 41100
tcctgtctcc tgtttgcttc tagaccttcc cattcttcgg ggtagctcct gcgatcctga 41160
atgagtctgg ggaagagtta gaaggtgaag ctgaaggcta tctggtgaga tcctagccct 41220
tgtgggtctt tgatgagggg gaggggagga tgctccacaa aagggcacag tgtgctttcc 41280
tttagagttt agaacctaac ctttcctggt agctgctttt ggctaaagct ccctggttga 41340
gggtttatcc tctgcagaga gaccctagtg gggcaatcct tctggatatt ttgcctgcct 41400
ctaggccttt tggcccacaa gactcttaag ggctctactc aaagatactt gatggtgcct 41460
ttttgggtct gaaagacagt ggaaaatcat ataggctcca gagtcaacct gcctgggtgc 41520
aaacctggct ctgcctcttc tgtgacttta gataagttac ctaacccctc tgaacataaa 41580
tattctcatt tgtaaaagag ttaccatagt acctttgtct ggagaaggaa atgacaactc 41640
gctgcagtac tcttgcctgg gaaatcccat agacagagaa gcctggaggg ctgtagtccc 41700
tgggatagca aaaaagagtc ggacatgact tagcaactaa acagcaatag cagcagtatc 41760
tttatcccct gtggttgctg gattgattag atgattcatg tgaagggttt atttagcaca 41820
gaacctaaga gttggtgctt gataaatgta agctgttgtg atcatttagt agttgtaggg 41880
agatcattag caccctcacc ctccaaccca ctttgttttt actaagagca agcttccctg 41940
gtagctcaag acggtgaaga atctgcctgc aatgcaggag acctgggttc gatccctggg 42000
tggggaagat cctctggaga agggaatgac tatgcaattc agtattcttg cctggagaat 42060
cccatggaca gaggagcctg tcagactaca gttcctgggg tcccaaagag tcagacatga 42120
ctgagtgact aatacacttt taaaagagca agttagtaac atgggcaaga cttgaaccca 42180
ggtcctctga ctcttagaat agagttctgt ccatcccacc aatctgctgc cacaggacat 42240
tactgattgg ggcaataatt acttacttat gtgtgggtcc cacattttta tgtaactgct 42300
ttcatttgta tcaaattgta cctgcatccc tgagggatta cctaaaagga gactcaaggc 42360
acttcccttg tggtccagta gctaagactc cacattccca atgcaggggg ctgggttcga 42420
tccccggttg gggagctaca tcctgcatgc cacaactaag atccagtgga gccaaataaa 42480
taaaaatctt ttttttttta aaaaggagac tcaaatttac taaatgctta ttaccaacaa 42540
agcatgtgct aggcagcttc cacatactat taatttaatc ctcaaaataa ctttggcagg 42600
tagaaattat tttctctgat ttacagataa ggatcctgaa actcagagac gtaaagttac 42660
ttacctgagg ccacacagta agtggcagag ttaggattcc aagcctagac tgtctccaaa 42720
gcatatactt tgttctactt cttgggaatg gcattcaggc tatttctcct aaggtttggc 42780
aaagacgtaa ctgtcttcct gtgagacagt gtagcagatg gttaccactt cctcatcctc 42840
caggtgttca agcagccttg gccagggatc atgcgcacgg tctatgggaa ccacgaacgc 42900
tttgagacca cctactttaa gaagttcccc gggtactatg tgacaggaga tggtgagcct 42960
tggctatccc tttcccacac ctcccactaa gacatgtacc atcccctttc attttcaaga 43020
agagttggtt agggatgtgt cttatgtatc aatgtgtaat atccagtcag agatacagag 43080
aagaccataa gaacaggaat gagtgttgca gaaaggctct cagcacggtg ctgagatttg 43140
atgtatgagg gaaaatgttc ttgtttccat cactgctggg ctgaaagaga ggcagcacat 43200
ggcagtatgt attaccatgt caagtgcgtc cccatgtctg gagcctatgt ttgcccatgt 43260
gtccatccac caatccattc ttccataaat atttattgag cacgtactac ttatctaagc 43320
acaggagatg ttaccatgaa taaggtgggc aaggtctctg ctctcatggg gtttacatta 43380
catcctctct agtgaagaca gacaggcagt aaattcagca aataaatact gcagggacag 43440
ccaccataca tgggatggtg agaagtttct ttgaagtgtt atttgaccag agacctaaag 43500
aagcagccag gtatgggaat atctggagta aaaatgaatg ttctgtacca gagagacata 43560
agcatgcaat gtctgatgaa cagactgaag cttgtgtagc tggaatgtag tgagcaacag 43620
caagaatgtt gtatcaagtt ggagagggag ataatacagg gccttgtgag ccatggtgag 43680
gaatttggat tttatccagt gtacagtggg agaccatcag gctttttaag cagagaagtg 43740
attataattt atatattatt tagaaaataa ctttgcttgt tttatggaga atagacttta 43800
ggtcagtata ttagttatac taattaactc caaacctggc agctaaaaac aataattatc 43860
tcatagtttc tgaggatcag gattctgtga gtggcttcgc tggtggttct tattcagaat 43920
ttcttgtggg ttgcagtcaa cctgtggttg gggctgcagt catctaaagg tctgactgag 43980
cctggaggac ccacttctaa gctcacttat gtggctgttg gcaggaacct tcagttcctt 44040
gccacatgga cctctccata gggctactca tgacacggca gctggctgac cccagagcaa 44100
ataatcagag agaatgcaac caagatagaa gccacagtgt cttctataat cttatctcag 44160
aaatgacatc ctatcatctc tgccaaatgc gattgctcag gagacaaacc ctagtacagt 44220
gagataaagg actacacagg gatacagata ccaggagacg gaggttggtg tgaccgtctt 44280
ggagcctggc tagcacagca ggaagacttg ttaggaggct gttgccatgg gtacagcagt 44340
ttggtgatgt ggatcgctgc ggtatcctac tgtggtgtat ttattttccc ctcccaggct 44400
gccggcggga caaggatggc tattactgga tcactggcag gatcgatgac atgctgaacg 44460
tatctggtga gggctaggga ccatcatccc ttctcattaa ccctgctcgc ccagcaactc 44520
acagccaaag ccttctaaga gagccagacc atacctggag tgcagtgttc atttctgggc 44580
tgaagtaata aacactgaag agtaataaac actgggagga gaggaaccag gatggtgggc 44640
aacaatgtcc cttgaggaat gaatgaaagg ctgtccctgt ttgaccagac cgggactgcc 44700
ttattgggac agatggaaca ggggctgggg tctctctcat ggcaacttcc tttcccttga 44760
caaggacacc tgctgagcac ggcagaggtg gagtcagcac tcgtggaaca caaggctgtt 44820
gcagaggcag ccgtggttgg acaccctcat cctgtgaagg gcgaatgcct ctactgcttt 44880
gtcaccttgt gcgatggcca catcttcagc ccagctctca ctgaggagct caagaagcag 44940
agtaaggagc ttccctgggc agggattgcc aggtctgtcc tgatggccca gtcaccacag 45000
caggttgttg gggaggggct gtagtgagct agtttctcaa accacaaaca gatcccaggg 45060
gctgactgcc ctcttcccac tcagtctcca tcatgttaat aatgattgac atttgtagaa 45120
ttctttcttt gtgccactga actaagtgtt ttaaatgcat taatacattt aatctctagt 45180
cacctccatg aagcttccca ggtggcacag tggtaaagaa tctgcctgcc aatgcaggag 45240
atgcagaaga catgggttcg atccctgggt cacaaagatc ccctggagta ggaaatggca 45300
acccactcca gtattcttgc ctggaaaact ccatgaacag aggagcctga tgggctacag 45360
tccatggggt cacaaagagc tggacacaac tgaacaactg agagcacaca cctccatgaa 45420
atagatccta ttgttactct ccttctaaag atgaagaaat tgaggctcag agaatttagg 45480
aataaatcac tggttcgagg ccacactgca agtaaagtga atttcaactc tagcaggctg 45540
attctagagc ttgagttcca tgagggtgaa ttaatataca atgagagagt aaagagacaa 45600
tgtgttaaaa agagaggaag acaaaaagtg agcctgagaa tgattaggat gggaacaaag 45660
ctatctctgc cttggttttg ggcagcctgg ctaagggtac tggaaaaatg tgtaatgatt 45720
tgttggttac agttagagaa aagattggcc ccattgccac accggattac atccagaatg 45780
cacctggatt gcctaaaact cgctcaggta tgttcagagg cctctaggga ttgggattgg 45840
atgggccaag gcccactagg ggcgagtatg cgtggatgga agcttttggc agggccggag 45900
tgggtcagtg tgttgtcacc aggtaaccag aagcgtgtgg acccccaggg aaaatcatga 45960
ggcgggtgct tcggaagatc gctcagaatg accacgacct gggggacacg tccactgtag 46020
tcgacccatc catcatcacc cagctcttca gccaccgctg cttgaccatc caatgaataa 46080
gatcctgcct ggaacccagg attaccccct gcttggcttt ccaaagtttt gcccaccctc 46140
cctgccccca tccaggagtg ctgagggcca gtgctgaccc acaacacttc ccttgtccag 46200
ctgtctggga ccgaggacca tctttgcttc tgggtggagg caacttcctg cgaccaccag 46260
gcagatgcga gagggcccag gtcagcctca ggctgtgccc ctagactgca aagagccctt 46320
ggagccctga agggacctga aggtcatgtc ccaaacccaa gtgttcagtg ggcaaatgag 46380
ggcatggact gaaacaggga agcaactcta atcctatgtc atctctctca ggaagcacca 46440
gtacttatct tgggcatgca cttgccccga gaaatgcctg tttgtgagtc ctggaagaat 46500
gattattttt taaaacgttt tgttgcttct cttgtgcgtc tgaattccct tttgtaccgg 46560
atggcagtgc ctgtctgcca tctgttccag gggctctgca ggcagcctca gtttccggag 46620
ggctttcaga tcatttgtgt ccttgaagga atacctgtgt tatgctgcca ggaccagagg 46680
agctcacttt tcttgtcctc tgttcccagc ccccctgaac agtcttgctc atgagagatg 46740
ttctcagatg aagctctttt ttcctaccca agttgtactc aggccctgtg gttgtaggaa 46800
taaagtctgt gacttcatga a 46821
Claims (9)
1.一种鉴别荷斯坦牛优质高产的分子标记，其特征在于：所述分子标记位于荷斯坦牛基因组的13号染色体上，为ACSS2-g.33012T>C，如SEQ ID NO.1。
2.一种鉴别荷斯坦牛优质高产的分子标记的制备方法，其特征在于：包括如下步骤：
提取DNA：提取荷斯坦牛血液基因组DNA；
进行标记：获取荷斯坦牛相应基因序列并对相应外显子区域做标记；
PCR扩增：使用特异性引物对于荷斯坦牛相应基因序列进行PCR
测定基因序列：将PCR产物进行电泳检测，然后测定基因序列，对于测序结果进行对比，比较不同样品之间的区别和发生突变的具***置；
分型检测：将检测到的突变位点使用质谱法进行分型检测；
关联性分析：对于日产奶量、乳脂率、乳蛋白率、体细胞评分四个形状评分进行关联性分析。
3.根据权利要求2中所述的鉴别荷斯坦牛优质高产的分子标记的制备方法，其特征在于：所述提取DNA中，所述提取DNA中，所述荷斯坦牛血液基因组DNA为尾静脉血液基因组DNA。
4.根据权利要求2中所述的鉴别荷斯坦牛优质高产的分子标记的制备方法，其特征在于：所述进行标记中，荷斯坦牛相应基因序列为荷斯坦牛ACSS2基因全序列，所述荷斯坦牛相应基因的获取方式为在NCBI网站上下载。
5.根据权利要求2中所述的鉴别荷斯坦牛优质高产的分子标记的制备方法，其特征在于：所述进行标记中，使用primer 5软胶在CDS区设计特异性引物。
6.根据权利要求2中所述的鉴别荷斯坦牛优质高产的分子标记的制备方法，其特征在于：所述进行标记中，所述特异性引物为使用primer 5软件在CDS区设计特异性引物。
7.根据权利要求2中所述的鉴别荷斯坦牛优质高产的分子标记的制备方法，其特征在于：所述分型检测中，突变位点位于ACSS2基因的33012位置。
8.根据权利要求7中所述的鉴别荷斯坦牛优质高产的分子标记的制备方法，其特征在于：所述分型检测中，ACSS2基因的33012位置包括C或T两种碱基。
9.根据权利要求8中所述的鉴别荷斯坦牛优质高产的分子标记的制备方法，其特征在于：所述分型检测中，ACSS2基因的33012位置包括TT型、CC型和CT型。
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| CN113832240A (zh) * | 2021-11-04 | 2021-12-24 | 华南农业大学 | 一个与中国南方荷斯坦奶牛305天产奶量显著相关的snp分子标记及应用和选育方法 |
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| CN113832240A (zh) * | 2021-11-04 | 2021-12-24 | 华南农业大学 | 一个与中国南方荷斯坦奶牛305天产奶量显著相关的snp分子标记及应用和选育方法 |
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