SNPS IDENTIFICATION IN RNA-SEQ DATA OF LARGEMOUTH BASS (MICROPTERUS SALMOIDES) FED ON FORMULATED FEED AND ASSOCIATION ANALYSIS WITH GROWTH TRAIT
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摘要: 为开发人工饲料代替冰鲜杂鱼养殖大口黑鲈的分子标记，以食用冰鲜鱼和配合饲料的同批大口黑鲈为研究材料，利用RNA-Seq（RNA sequencing）技术挖掘SNPs（Single nucleotide polymorphisms）标记，并以关联分析筛选可用于育种的候选标记。转录组进行测序共获得174 M数据，8681个SNPs位点。挑选其中具有表达差异的50个SNPs位点进行SNaPshot分型，结果39个分型成功，其中有4个为假阳性，通过转录组技术开发出SNPs标记35个，成功率为70.0%。为进一步检验这些标记是否可用于评估驯食饲料的大口黑鲈选育研究，研究以327尾摄食人工配合饲料的大口黑鲈为试验材料，SPSS软件进行一般线性模型分析SNPs的不同基因型与生长性状的相关性，结果显示有2个SNPs位点与体质量、全长和体高等生长性状存在显著相关性（P<0.05），可作为候选标记用于大口黑鲈的分子辅助育种。由于转录组数据直接反应基因的表达情况，从中挖掘与性状相关的优势基因型与分子标记的成功率高，效果较好。同时也为解决大口黑鲈选育研究中标记缺乏提供了有效途径，为选育提供遗传依据、加速育种进程。
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关键词:
- 转录组测序(RNA-Seq) /
- 大口黑鲈 /
- 单核苷酸多态(SNPs) /
- 生长性状
Abstract: It is a hot research area to substitute frozen trash fish (FF) with artificial compound diet (CD) in feeding the carnivorous economic fish. Largemouth bass (Micropterus salmoides) is one of the important aquaculture species with an annual output of about 340000 tons in fresh water in China. Effect of artificial compound diet at the transcriptome level of largemouth bass is rare reported. The current study utilized RNA-Seq (RNA sequencing) technology on the Illumina sequencing platform (Illumina HiSeqTM 2000) to screen polymorphic SNP (single nucleotide polymorphism) markers of largemouth bass in two diets and analyzed the association of SNPs with growth trait. As a result, a total of 174M reads were assembled to generate 95024 unigenes. We used BWA (http://sourceforge.net/projects/bio-bwa/) and SamTools (http://source forge.net/projects/samtools/) to detect SNPs. Several filtering steps were performed to reduce the false positive of SNPs, 8681 SNPs met the selection criteria. 50 SNPs sequences in different genes were validated by PCR amplification and genotyped by the SNaPshot assay. 39 out of 50 SNPs were confirmed. Of these, 4 SNPs were monomorphic for all individuals and 35 SNPs were polymorphic. Validation of selected SNPs was 70%. 327 largemouth bass with marketable sizes fed by CD were used to evaluate the correlation between these validated SNPs and growth traits including body mass, total length, head length, body width, withers height and caudal peduncle length. Genotyping data were computed and analyzed by SPSS 19.0 statistical software package. Two SNPs were associated with body mass, total length and withers height, which could be used as important candidate molecular markers for breeding selection of largemouth bass. This study shows that RNA-Seq is an efficient approach to uncover gene-asso-ciated SNPs, which could facilitate genetic and functional genomics research in the artificial domestication of formulated feeding of largemouth bass. -
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图 1 大口黑鲈的SNPs位点分类
Figure 1. The distribution of SNPs identified from the largemouth bass
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图 2 大口黑鲈群体体质量的正态分布图
Figure 2. Normal distribution curve of body mass of largemouth bass
表 1 大口黑鲈随机群体的生长性状测量值统计
Table 1 The parameters of the random group of largemouth bass
性状Character 体质量Body mass (g) 全长Total length (cm) 头长Head length (cm) 体宽Body width (cm) 体高Body height (cm) 尾柄长Caudal pedunclelength (cm) 均值mean 428.36±8.19 28.37±0.16 7.34±0.08 4.11±0.03 8.62±0.06 8.49±0.07 
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表 2 大口黑鲈生长性状的主成分分析
Table 2 Principal components analysis on growth trait of largemouth bass
成分Component 初始特征值Initial value of components 提取平方和载入Sum of squares of extracted components 合计Total 解释的方差的Explained variance ratio (%) 总方差累积Accumulation variance ratio (%) 合计Total 方差贡献率Variance contributionratio (%) 累积贡献率Accumulated variance contribution ratio (%) 体质量Body mass 4.36 72.64 72.64 4.36 72.64 72.64 全长Total length 0.85 14.15 86.79 0.85 14.15 86.79 头长Head length 0.62 10.31 97.10 0.62 10.31 97.10 体宽Body width 0.11 1.77 98.87 体高Withers height 0.04 0.64 99.51 尾柄长Caudal peduncle length 0.03 0.49 100
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表 3 大口黑鲈几个生长性状的相关矩阵
Table 3 Correlation matrix on some growth traits of largemouth bass
体质量Body mass 全长Total length 头长Head length 体宽Body width 体高Body depth 尾柄长Caudal peduncle length 体质量Body mass 1 全长Total length 0.887 1 头长Head length 0.428 0.450 1 体宽Body width 0.943 0.835 0.410 1 体高Withers height 0.957 0.908 0.442 0.912 1 尾柄长Caudal peduncle length 0.511 0.759 0.178 0.481 0.511 1
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表 4 大口黑鲈2个SNPs位点的等位基因和基因型频率
Table 4 Genotype and allele frequencies of two SNPs of largemouth bass
Unigene编号Unigene number 位置Position 基因型频率Genotype frequency 基因频率Gene frequency H-W平衡Hardy-Weinberg eguiliberum 多态信息含量PIC 有效等位基因数Ne 杂合度H Unigene0022436 297 CC: 0.33CT: 0.46TT: 0.21 C: 0.56T: 0.44 Χ2=1.976(P=0.160) 0.5631 2.7584 0.4557 Unigene0031044 1332 GG: 0.45CG: 0.46CC: 0.09 G: 0.68C: 0.32 Χ2=0.674(P=0.412) 0.4900 2.3880 0.4587 均值Mean 0.5266 2.5732 0.4572
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表 5 SNPs位点与生长性状的相关关系
Table 5 Correlation analysis between SNPs polymorphism and growth trait
Unigene编号Unigene number 分型Genotype 数目Number 体质量均值Mean of body mass (g) 全长均值Mean of total length (cm) 头长均值Mean of head length (cm) 体宽均值Mean of body width (cm) 体高均值Mean of withers height (cm) 尾柄长均值Mean of caudal peduncle length (cm) Unigene0022436 CC 108 427.77ab 28.28 7.48 4.08ab 8.48ab 8.53 CT 149 442.64a 28.61 7.33 4.17a 8.62a 8.72 TT 70 400.08b 28.02 7.15 4.02b 8.26b 8.55 Unigene0031044 GG 146 448.83a 28.73a 7.88a 4.17a 8.69a 8.71a CG 150 417.88ab 28.20ab 7.29a 4.07ab 8.40b 8.60ab CC 31 382.66b 27.47b 7.28b 3.95b 8.02b 8.29b 注: 表中上标为平均值的差异显著性(最小显著性差异法, LSD), 同一列数值中, 上标含相同字母表示两种基因型之间差异不显著(P>0.05), 不同小写字母代表差异显著(P<0.05) Note: Values with different superscript letters within a column indicates significant difference at P<0.05
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[1] 董金和. 中国渔业统计年鉴. 北京:中国农业出版社. 2014, 31 Dong J H. Chinese Fishery Statistical Yearbook[M]. Beijing:China Agriculture Press. 2014, 31
董金和. 中国渔业统计年鉴. 北京:中国农业出版社. 2014, 31[2] 雷霁霖. 中国海水养殖大产业架构的战略思考. 中国水产科学, 2010, 17(3):600-609 http://www.cnki.com.cn/Article/CJFDTOTAL-ZSCK201003027.htm Lei J L. Strategy consideration for industry construction of Chinese marine culture[J]. Journal of Fishery Sciences of China, 2010, 17(3):600-609
雷霁霖. 中国海水养殖大产业架构的战略思考. 中国水产科学, 2010, 17(3):600-609 http://www.cnki.com.cn/Article/CJFDTOTAL-ZSCK201003027.htm[3] Habji Y. Microbial and sensory quality changes in refrigerated minced goatmeat stored under vacuum and in air[J]. Small Ruminant Research, 2000, 36(1):75-84 doi: 10.1016/S0921-4488(99)00106-6
[4] 王广军, 吴锐全, 谢骏, 等. 大口黑鲈投喂两种不同饲料对水质指标的影响. 水产学杂志, 2009, 22(1):35-38 http://www.cnki.com.cn/Article/CJFDTOTAL-SCXZ200901008.htm Wang G J, Wu R Q, Xie J, et al. Effects of feeding two kinds of fooeed on the water quality in largemouth bass (Micropterus salmoides) aquarium[J]. Chinese Journal Fisheries, 2009, 22(1):35-38
王广军, 吴锐全, 谢骏, 等. 大口黑鲈投喂两种不同饲料对水质指标的影响. 水产学杂志, 2009, 22(1):35-38 http://www.cnki.com.cn/Article/CJFDTOTAL-SCXZ200901008.htm[5] 仲如. 真核基因体外转录. 中国生物化学与分子生物学报, 1987, 3:193-198, 287 http://www.cnki.com.cn/Article/CJFDTOTAL-SWHZ198703000.htm Zhong R. Eukaryotic gene in vitro transcription[J]. Chinese Journal of Biochemistry and Molecular Biology, 1987, 3:193-198, 287
仲如. 真核基因体外转录. 中国生物化学与分子生物学报, 1987, 3:193-198, 287 http://www.cnki.com.cn/Article/CJFDTOTAL-SWHZ198703000.htm[6] 孙效文. 鱼类分子育种学. 北京:海洋出版社. 2010, 26-30 Sun X W. Fish Molecular Breeding[M]. Beijing:China Ocean Press. 2010, 26-30
孙效文. 鱼类分子育种学. 北京:海洋出版社. 2010, 26-30[7] Martins D A, Valente L M P, Lall S P. Effects of dietary lipid level on growth and lipid utilization by juvenile Atlantic halibut (Hippoglossus hippoglossus L.)[J]. Aquaculture, 2007, 263(1/4):150-158 http://cn.bing.com/academic/profile?id=2133534899&encoded=0&v=paper_preview&mkt=zh-cn
[8] 付世建, 曹振东, 谢小军. 鱼类摄食代谢和运动代谢研究进展. 动物学杂志, 2008, 43(2):150-159 http://www.cnki.com.cn/Article/CJFDTOTAL-BIRD200802032.htm Fu S J, Cao Z D, Xie X J. Feeding metabolism and locomotion metabolism in fishes[J]. Chinese Journal of Zoology, 2008, 43(2):150-159
付世建, 曹振东, 谢小军. 鱼类摄食代谢和运动代谢研究进展. 动物学杂志, 2008, 43(2):150-159 http://www.cnki.com.cn/Article/CJFDTOTAL-BIRD200802032.htm[9] 刘浩, 李胜杰, 白俊杰. 鱼类食性的基因调控与遗传改良研究进展. 中国农学通报, 2015, 31(11):78-82 http://www.cnki.com.cn/Article/CJFDTOTAL-ZNTB201511016.htm Liu H, Li S J, Bai J J. Research advances in gene regulation and genetic improvement of fish feeding[J]. Chinese Agricultural Science Bulletin, 2015, 31(11):78-82
刘浩, 李胜杰, 白俊杰. 鱼类食性的基因调控与遗传改良研究进展. 中国农学通报, 2015, 31(11):78-82 http://www.cnki.com.cn/Article/CJFDTOTAL-ZNTB201511016.htm[10] Pratoomyot J, Bendiksen E A, Bell J G, et al. Effects of increasing replacement of dietary fishmeal with plant protein sources on growth performance and body lipid composition of Atlantic salmon (Salmo salar L.)[J]. Aquaculture, 2010, 305(1-4):124-132 doi: 10.1016/j.aquaculture.2010.04.019
[11] 王国梁, 何晓群. 多变量经济数据统计分析. 西安:陕西科学技术出版社. 1993, 33-376 Wang G L, He X Q. Multivartate Statistical Analysis of Economic Data[M]. Xi'an:Shanaxi Science &Technology Press. 1993, 33-376
王国梁, 何晓群. 多变量经济数据统计分析. 西安:陕西科学技术出版社. 1993, 33-376[12] Hubert S, Higgins B, Borza T, et al. Development of a SNP resource and a genetic linkage map for Atlantic cod (Gadus morhua)[J]. BMC Genomics, 2010, 11(12):1853-1858 http://cn.bing.com/academic/profile?id=2067944243&encoded=0&v=paper_preview&mkt=zh-cn
[13] Xu J, Ji P, Zhao Z, et al. Genome-wide SNP discovery from transcriptome of four common carp strains[J]. PLoS One, 2012, 7(10):e48140 doi: 10.1371/journal.pone.0048140
[14] Xu J, Zhang X, Zheng X, et al. Development and evaluation of the first high-throughput SNP array for common carp (Cyprinus carpio)[J]. BMC Genomics, 2014, 15:307 doi: 10.1186/1471-2164-15-307
[15] Frazer K A, Ballinger D G, Cox D R, et al. A second generation human haplotype map of over 3.1 million SNPs[J]. Nature, 2007, 449(7164):851-861 doi: 10.1038/nature06258
[16] Wang J, Wang W, Li R, et al. The diploid genome sequence of an Asian individual[J]. Nature, 2008, 456(7218):60-65 doi: 10.1038/nature07484
[17] Rieder M J, Taylor S L, Clark A G, et al. Sequence variation in the human angiotensin converting enzyme[J]. Nature Genetics, 1999, 22(1):59-62 doi: 10.1038/8760
[18] Botstein D, White R L, Skolnick M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. American Journal of Human Genetics, 1980, 32(3):314-331 http://cn.bing.com/academic/profile?id=1803375514&encoded=0&v=paper_preview&mkt=zh-cn
[19] 金梅, 王艳杰, 张婷婷, 等. 辽宁新品系绒山羊微卫星标记与经济性状相关性的研究. 畜牧兽医学报, 2012, 43(2):186-195 http://www.cnki.com.cn/Article/CJFDTOTAL-XMSY201202005.htm Jin M, Wang Y, Zhang T T, et al. Correlation analysis of microsatellite DNA markers with some substantial economic traits in Liaoning new line cashmere goat[J]. Acta Veterinaria et Zootechnica Sinica, 2012, 43(2):186-195
金梅, 王艳杰, 张婷婷, 等. 辽宁新品系绒山羊微卫星标记与经济性状相关性的研究. 畜牧兽医学报, 2012, 43(2):186-195 http://www.cnki.com.cn/Article/CJFDTOTAL-XMSY201202005.htm[20] 李镕, 白俊杰, 李胜杰, 等. 大口黑鲈家系早期形态性状的相关和主成分分析. 海洋渔业, 2011, 33(3):282-288 http://www.cnki.com.cn/Article/CJFDTOTAL-HTYY201103008.htm Li R, Bai J J, Li S J, et al. Correlation and principal components analysis of morphological traits of Micropterus salmoides family at early age[J]. Marine Fisheries, 2011, 33(3):282-288
李镕, 白俊杰, 李胜杰, 等. 大口黑鲈家系早期形态性状的相关和主成分分析. 海洋渔业, 2011, 33(3):282-288 http://www.cnki.com.cn/Article/CJFDTOTAL-HTYY201103008.htm[21] 余锐, 梁旭方, 易提林, 等. 鳜鱼3个养殖群体经济性状及遗传多样性分析. 水产科学, 2012, 31(9):511-515 http://www.cnki.com.cn/Article/CJFDTOTAL-CHAN201209002.htm Yu R, Liang X F, Yi T L, et al. Economic traits and genetic diversity of three cultured mandarinfish Siniperca chuatsi populations[J]. Fisheries Science, 2012, 31(9):511-515
余锐, 梁旭方, 易提林, 等. 鳜鱼3个养殖群体经济性状及遗传多样性分析. 水产科学, 2012, 31(9):511-515 http://www.cnki.com.cn/Article/CJFDTOTAL-CHAN201209002.htm[22] Rainbow S R, Kwon H, Zene L. The role of Nkx3.2 in chondrogenesis[J]. Frontiers in Biology, 2014, (5):376-381 http://cn.bing.com/academic/profile?id=2088369300&encoded=0&v=paper_preview&mkt=zh-cn
[23] Craig S N, Paul A K. The Xenopus bagpipe-related homeobox gene zampogna is expressed in the pharyngeal endoderm and the visceral musculature of the midgut[J]. Development Genes and Evolution, 1999, 209(2):132-134 doi: 10.1007/s004270050236
[24] Simon M, Campos-Xavier A B, Mittaz-Crettol L, et al. Severe neurologic manifestations from cervical spine instability in spondylo-megaepiphyseal-metaphyseal dysplasia[J]. American Journal of Medical Genetics Part C:Seminars in Medical Genetics, 2012, 160C(3):230-237 doi: 10.1002/ajmg.c.v160c.3
[25] Jerzy A, Franz J J. A guide to 17β-hydroxysteroid dehydrogenases[J]. Molecular and Cellular Endocrinology, 2001, 171(1-2):1-4 doi: 10.1016/S0303-7207(00)00383-X
[26] Luu-The V. Analysis and characteristics of multiple types of human 17β-hydroxysteroid dehydrogenase[J]. The Journal of Steroid Biochemistry and Molecular Biology, 2001, 76(1-5):143-151 doi: 10.1016/S0960-0760(00)00155-2
[27] Moeller G, Adamski J. Integrated view on 17β-hydroxysteroid dehydrogenases[J]. Molecular and Cellular Endocrinology, 2009, 301(1-2):7-19 doi: 10.1016/j.mce.2008.10.040
[28] Rantakari P, Lagerbohm H, Kaimainen M, et al. Hydroxysteroid (17β) dehydrogenase 12 is essential for mouse organogenesis and embryonic survival[J]. Endocrinology, 2010, 151(4):1893-1901 doi: 10.1210/en.2009-0929
[29] 李延恩, 周艳红. SNP功能分析的生物信息学方法及其资源. 计算机仿真, 2007, 24(4):297-300 http://www.cnki.com.cn/Article/CJFDTOTAL-JSJZ200704080.htm Li Y E, Zhou Y H. Bioinformatics methods and resources for SNP functional analysis[J]. Computer Simulation, 2007, 24(4):297-300
李延恩, 周艳红. SNP功能分析的生物信息学方法及其资源. 计算机仿真, 2007, 24(4):297-300 http://www.cnki.com.cn/Article/CJFDTOTAL-JSJZ200704080.htm
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