Variation Sites on EGFL9 Associated with Growth of Channel Catfish
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摘要:
目的 研究斑点叉尾鮰表皮生长因子样结构域蛋白EGFL9(Multiple epidermal growth factor-like domains protein 9)基因与其生长性状的相关性,为斑点叉尾鮰的分子标记辅助育种奠定基础。 方法 采用靶向基因测序技术对EGFL9基因进行测序,与参考基因组比对后筛选变异位点,并将获得的变异位点与生长性状进行关联分析。 结果 在EGFL9上共发现27个多态位点,经过滤,获得22个有效突变位点,其中4个变异位点(g.142、g.573、g.3079、g.7409)对斑点叉尾鮰的生长性状具有显著影响,位点g.142、g.3079、g.7409位于内含子中,位点g.573位于第2外显子上;g.142位点的A/A型个体的平均体长显著高于A/G型个体(P<0.05);g.7409位点A/G型个体的平均体长显著高于G/G型、A/A型个体(P<0.05);g.3079位点C/C型个体的平均体质量和体长均显著高于A/A型个体(P<0.05);位于外显子上的InDel位点g.573使EGFL9基因编码蛋白减少了一个苏氨酸残基,对蛋白质三级结构产生较为明显的影响,该位点TACC/T型个体的平均体质量显著高于TACC/TACC型个体(P<0.05),平均体长显著高于TACC/TACC型与T/T型个体(P<0.05)。 结论 获得的4个斑点叉尾鮰生长相关标记(g.142、g.573、g.3079、g.7409)与其生长性状显著关联,可以应用于后续斑点叉尾鮰育种芯片开发和分子标记辅助育种。 Abstract:Objective Correlation between the variation sites on the multiple epidermal growth factor-like domains protein 9 gene (EGFL9) and the growth of Ictalures punctatus was investigated. Method The targeted gene sequencing of EGFL9 was performed to identify the variation sites by alignment with the reference genome. Correlation between the sites and the growth traits of the fish was statistically analyzed. Result There were 27 sites with polymorphic variations on EGFL9 with 22 effective mutations obtained after filtering. Among which, 4 sites, i.e., g.142, g.573, g.3079, and g.7409, were significantly associated with the growth traits of channel catfish. The SNP sites, g.142, g.3079, and g.7409, were in introns, while the InDel site, g.573, in exon 2. The mean body length of the fish with A/A type at g.142 was significantly longer than that of fish with A/G type (P<0.05), while that of the fish with A/G type at g.7409 significantly longer than that of fish with G/G type (P<0.05). Both mean body mass and length of the fish with C/C type at g.3079 were significantly greater than those of the fish with A/A type (P<0.05). The InDel site, g.573, reduced one serine residue in the protein encoded by EGFL9 significantly altereed the tertiary structure. Consequently, the mean body mass of the fish with TACC/T type at this site was significantly heavier than that of the fish with TACC/TACC type (P<0.05), and the mean body length significantly longer than that of the fish with TACC/TACC or T/T type (P<0.05). Conclusion The 4 growth-related markers, g.142, g.573, g.3079, and g.7409, revealed by this study showed significant associations with the growth traits of I. punctatus. The information would possibly lead to the development of breeding chips and molecular marker-assisted breeding on channel catfish in the future. -
Key words:
- Ictalures punctatus /
- EGFL9 /
- SNP /
- growth traits /
- association
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图 1 斑点叉尾鮰EGFL9二级结构预测
A:EGFL9蛋白质二级结构;B:EGFL9(g.573)蛋白质二级结构。在二级结构中蓝色表示螺旋;红色表示折叠;黄色表示未知结构,在表面残基可溶性中蓝色表示暴露;黄色表示埋藏。
Figure 1. Predicted secondary structure of EGFL9 protein in channel catfish
A: secondary structure of EGFL9 protein; B: secondary structure of EGFL9 (g.573) protein. In secondary structure, blue indicates helix; red, strand; yellow, others. On solvent accessibility, blue indicates exposed; yellow, buried.
表 1 用于EGFL9基因变异位点筛选和分型引物
Table 1. Primers used in screening and genotyping variation sites on EGFL9
位点
Locus变异位点位置
Variation location引物序列
Primer sequence变异类型
Variation type产物大小
PCR size/bpg.137 Intron1 F: GGGGAGCGTTTGTAGTAGACG A/T 193 R: GTTTCAGTTACTCTTGCTCGCG g.142 Intron2 F: GGGGAGCGTTTGTAGTAGACG A/G 193 R: GTTTCAGTTACTCTTGCTCGCG g.339 Exon1 F: TGCATGCAGAATGTTAGTTTTTC A/G 192 R: GTTTCAGTTACTCTTGCTCGCG g.573 Exon2 F: CTGACCTCAGCGCAATTTC TACC/T 195 R: AACAACAACAACCACCATCACC g.716 Intron3 F: GATTTTACCACCACCACCAC C/T 187 R: TGACAGGTCTATTACTCTTGTACACTTC g.913 Intron4 F: CCGCCTGACCTCAAAACCC A/G 191 R: TCCTATGTATTGAGAACCTCAAGTC g.928 Intron5 F: CCGCCTGACCTCAAAACCC A/C 191 R: TCCTATGTATTGAGAACCTCAAGTC g.1877 Intron6 F: CTGTAAGTGTTTGCCCGG C/T 199 R: CACCTTACAAAGCAGTCGCC g.2998 Intron7 F: TAACTGGGTGTGAAATTTCAAC A/AGGATGAT 194 R: AAAGCAGAATGTTGCGGGAC g.3027 Intron8 F: TAACTGGGTGTGAAATTTCAAC G/T 194 R: AAAGCAGAATGTTGCGGGAC g.3029 Intron9 F: TAACTGGGTGTGAAATTTCAAC G/T 194 R: AAAGCAGAATGTTGCGGGAC g.3031 Intron10 F: TAACTGGGTGTGAAATTTCAAC G/T 194 R: AAAGCAGAATGTTGCGGGAC g.3045 Intron11 F: TAACTGGGTGTGAAATTTCAAC A/G 194 R: AAAGCAGAATGTTGCGGGAC g.3079 Intron12 F: TAACTGGGTGTGAAATTTCAAC A/C 194 R: AAAGCAGAATGTTGCGGGAC g.5185 Intron13 F: TGCCATGCGAATGCAATG A/G 203 R: GCAAGGCCTTCTGAATGCC g.5819 Intron14 F: GTGTGTGCCAATCAAACTGGG A/C 192 R: GCAAGGCCTTCTGAATGCC g.6171 Intron15 F: TAACAAAATGCCAAGCTTAAAATG C/T 197 R: AGCCCCACTGCCAACG g.6202 Intron16 F: TAACAAAATGCCAAGCTTAAAATG C/CAT 197 R: AGCCCCACTGCCAACG g.6305 Exon3 F: TAACAAAATGCCAAGCTTAAAATG C/G 197 R: AGCCCCACTGCCAACG g.6477 Exon4 F: CGCTCCTCTCCGGCCTG C/T 199 R: CAACAGCTATCACGACAGCCTG g.7409 Intron17 F: CAGACATGCACTGATGCTTTAG A/G 193 R: TGGAATAGAAACACATGCAGTTAC g.7669 Intron18 F: GGGGCGCTGCCTTATGG A/G 196 R: AATCGAATTAGTTTTGAATCAGTCC 表 2 斑点叉尾鮰EGFL9基因变异位点遗传多样性参数
Table 2. Diversity indices of variation sites on EGFL9 of channel catfish
位点
Locus变异位点位置
Variation location有效等位基因数
Ne期望杂合度
He观测杂合度
Ho多态性信息容量
PIC哈迪-温伯格平衡
HWEg.137 A:0.815 1.4294 0.301 0.358 0.255 NS T:0.184 g.142 A:0.616 1.8963 0.474 0.637 0.361 *** G:0.383 g.339 A:0.077 1.1677 0.144 0.156 0.133 *** G:0.922 g.573 T:0.261 1.6285 0.387 0.204 0.311 *** TACC:0.738 g.716 C:0.86 1.3154 0.240 0.179 0.211 *** T:0.139 g.913 A:0.567 1.9646 0.492 0.527 0.370 NS G:0.432 g.928 A:0.159 1.3656 0.268 0.259 0.232 NS C:0.84 g.1877 C:0.606 1.9136 0.479 0.269 0.363 *** T:0.393 g.2998 A:0.38 1.8914 0.473 0.365 0.360 NS AGGATGAT:0.619 g.3027 G:0.38 1.8914 0.473 0.365 0.360 NS T:0.619 g.3029 G:0.377 1.8869 0.471 0.370 0.360 NS T:0.622 g.3031 G:0.38 1.8914 0.473 0.365 0.360 NS T:0.619 g.3045 A:0.768 1.5526 0.357 0.326 0.293 NS G:0.231 g.3079 A:0.315 1.7607 0.433 0.581 0.339 *** C:0.684 g.5185 A:0.915 1.1832 0.155 0.169 0.143 *** G:0.084 g.5819 A:0.484 1.9982 0.501 0.556 0.375 NS C:0.515 g.6171 C:0.636 1.8607 0.464 0.468 0.356 NS T:0.363 g.6202 C:0.378 1.8878 0.471 0.249 0.360 *** CAT:0.621 g.6305 C:0.803 1.4616 0.317 0.353 0.266 NS G:0.196 g.6477 C:0.937 1.1327 0.117 0.105 0.110 *** T:0.062 g.7409 A:0.44 1.9716 0.494 0.470 0.371 NS G:0.56 g.7669 A:0.206 1.4862 0.328 0.342 0.274 NS G:0.793 哈迪温伯格平衡检验,NS代表不显著,***代表极显著偏离。
Hardy-Weinberg equilibrium, NS: not significant; ***: significant at 0.001 level.表 3 斑点叉尾鮰EGFL9基因变异位点与生长性状的关联分析
Table 3. Correlation between variation sits on EGFL9 and growth traits of channel catfish
位点
Locus基因型
Genotype频率
Frequency体质量
Body mass/g体长
Body length/cmg.142 A/A(60) 0.298 955.165±273.451 a 46.200±3.602 a A/G(128) 0.636 944.849±284.504 a 45.055±3.756 b G/G(13) 0.064 988.585±189.377 a 46.154±2.882 ab g.573 TACC/TACC(128) 0.636 921.955±281.729 b 45.211±3.588 b TACC/T(41) 0.203 1025.578±261.659 a 46.524±3.521 a T/T(32) 0.159 970.100±252.701 ab 45.145±4.141 b g.3079 A/A(5) 0.025 722.880±285.012 b 42.200±4.086 b A/C(115) 0.580 939.037±267.002 ab 45.300±3.655 ab C/C(78) 0.393 983.433±283.021 a 45.799±3.636 a g.7409 A/A(41) 0.205 955.485±328.208 a 45.456±4.376 b A/G(94) 0.470 985.556±268.803 a 46.034±3.518 a G/G(65) 0.325 904.335±239.218 a 44.755±3.319 b 同列数据后不同小写字母表示同一位点不同基因型之间差异显著(P<0.05)。
Datas with different lowercase letters on the same column indicate significant difference of different genotypes of one same SNP site at P<0.05 level, respectively. -
[1] LIU Z J, LIU S K, YAO J, et al. The channel catfish genome sequence provides insights into the evolution of scale formation in teleosts [J]. Nature Communications, 2016, 7(1): 1−13. [2] ZHONG L Q, SONG C, CHEN X H, et al. Channel catfish in China: Historical aspects, current status, and problems [J]. Aquaculture, 2016, 465: 367−373. doi: 10.1016/j.aquaculture.2016.09.032 [3] APPELLA E, WEBER I T, BLASI F. Structure and function of epidermal growth factor-like regions in proteins [J]. FEBS Letters, 1988, 231(1): 1−4. doi: 10.1016/0014-5793(88)80690-2 [4] HAY D C, ROSS J A, GALLAGHER R, et al. The complexities of engineering human stem cell-derived therapeutics [J]. Journal of Biomedicine & Biotechnology, 2010, DOI: 10.1155/2010/654964. [5] 尹凯, 申传安, 尚玉茹, 等. 人表皮生长因子基因修饰细胞在创面修复中作用的研究进展 [J]. 中华损伤与修复杂志(电子版), 2014, 9(6):672−675.YIN K, SHEN C A, SHANG Y R, et al. Research progress of human epidermal growth factor gene modified cells in wound repair [J]. Chinese Journal of Injury Repair and Wound Healing (Electronic Edition), 2014, 9(6): 672−675.(in Chinese) [6] SUN D, BULLOCK M R, ALTEMEMI N, et al. The effect of epidermal growth factor in the injured brain after trauma in rats [J]. Journal of Neurotrauma, 2010, 27(5): 923−938. doi: 10.1089/neu.2009.1209 [7] WANG Y, SONG H J, WANG W F, et al. Generation and characterization of Megf6 null and Cre knock-in alleles [J]. Genesis, 2019, 57(2): e23262. doi: 10.1002/dvg.23262 [8] LI C C, VARGAS-FRANCO D, SAHA M, et al. Megf10 deficiency impairs skeletal muscle stem cell migration and muscle regeneration [J]. FEBS Open Bio, 2021, 11(1): 114−123. doi: 10.1002/2211-5463.13031 [9] LLOYD D L, TOEGEL M, FULGA T A, et al. The Drosophila homologue of MEGF8 is essential for early development [J]. Scientific Reports, 2018, 8(1): 1−10. [10] JOHNSON E B, HAMMER R E, HERZ J. Abnormal development of the apical ectodermal ridge and polysyndactyly in Megf7-deficient mice [J]. Human Molecular Genetics, 2005, 14(22): 3523−3538. doi: 10.1093/hmg/ddi381 [11] ZHANG S Y, ZHANG X H, CHEN X H, et al. Construction of a high-density linkage map and QTL fine mapping for growth- and sex-related traits in channel catfish (Ictalurus punctatus) [J]. Frontiers in Genetics, 2019, 10: 251. doi: 10.3389/fgene.2019.00251 [12] BRANDT-BOHNE U, KEENE D R, WHITE F A, et al. MEGF9: A novel transmembrane protein with a strong and developmentally regulated expression in the nervous system [J]. The Biochemical Journal, 2007, 401(2): 447−457. doi: 10.1042/BJ20060691 [13] 梁红玲, 李洪胜, 黄健清, 等. Sanger测序法和Snapshot法检测乳腺癌BIM缺失多态性比较分析 [J]. 循证医学, 2018, 18(6):370−375.LIANG H L, LI H S, HUANG J Q, et al. Study on the concordance of BIM deletion polymorphism tests by Sanger sequencing and snapshot methods in breast cancer [J]. The Journal of Evidence-Based Medicine, 2018, 18(6): 370−375.(in Chinese) [14] 贾子冬, 徐高连, 钟华燕, 等. PCR-LDR-核酸试纸条检测法在结核分枝杆菌katG基因315位密码子突变检测中的应用 [J]. 临床肺科杂志, 2011, 16(11):1721−1723. doi: 10.3969/j.issn.1009-6663.2011.11.031JIA Z D, XU G L, ZHONG H Y, et al. Application of PCR-LDR-nucleic acid detection strip in detection of KatG315-mutation in TB [J]. Journal of Clinical Pulmonary Medicine, 2011, 16(11): 1721−1723.(in Chinese) doi: 10.3969/j.issn.1009-6663.2011.11.031 [15] 张世勇, 王明华, 钟立强, 等. 斑点叉尾MSTN基因4个SNP位点及其与生长性状的相关性 [J]. 江苏农业科学, 2017, 45(1):30−33.ZHANG S Y, WANG M H, ZHONG L Q, et al. Four SNP loci of MSTN gene and their correlation with growth traits [J]. Jiangsu Agricultural Sciences, 2017, 45(1): 30−33.(in Chinese) [16] CHEN Y X, CHEN Y S, SHI C M, et al. SOAPnuke: A MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data [J]. GigaScience, 2018, 7(1): gix120. [17] WATERHOUSE A, BERTONI M, BIENERT S, et al. SWISS-MODEL: Homology modelling of protein structures and complexes [J]. Nucleic Acids Research, 2018, 46(W1): W296−W303. doi: 10.1093/nar/gky427 [18] YANG J Y, ANISHCHENKO I, PARK H, et al. Improved protein structure prediction using predicted interresidue orientations [J]. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(3): 1496−1503. doi: 10.1073/pnas.1914677117 [19] 卫侃韵, 谢淑媚, 王沈同, 等. 缢蛏EGFR基因内含子1内SNP位点多态性与生长性状相关性 [J]. 水产学报, 2019, 43(2):483−491.WEI K Y, XIE S M, WANG S T, et al. Polymorphism of SNPs in EGFR intron 1 and its association with growth traits in Sinonovacula constricta [J]. Journal of Fisheries of China, 2019, 43(2): 483−491.(in Chinese) [20] DE GOBBI M, VIPRAKASIT V, HUGHES J R, et al. A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter [J]. Science, 2006, 312(5777): 1215−1217. doi: 10.1126/science.1126431 [21] 李哲, 李雨, 敬庭森, 等. 长吻单核苷酸多态性标记与生长性状关联分析 [J]. 渔业科学进展, 2022, 43(4):127−135. doi: 10.19663/j.issn2095-9869.20210412001LI Z, LI Y, JING T S, et al. Correlation analysis of SNP markers and growth traits of Leiocassis longirostris [J]. Progress in Fishery Sciences, 2022, 43(4): 127−135.(in Chinese) doi: 10.19663/j.issn2095-9869.20210412001 [22] LIU H F, XU H W, LAN X Y, et al. The InDel variants of sheep IGF2BP1 gene are associated with growth traits [J]. Animal Biotechnology, 2021, 13: 1−9. [23] 陈静, 何吉祥, 樊佳佳, 等. 草鱼MyoD基因SNP和Indel标记的筛选及其与生长性状的关联分析 [J]. 江苏农业学报, 2018, 34(3):612−616. doi: 10.3969/j.issn.1000-4440.2018.03.019CHEN J, HE J X, FAN J J, et al. Screening of SNP and Indel marker of MyoD gene and its association with growth traits in Ctenopharyngodon idella [J]. Jiangsu Journal of Agricultural Sciences, 2018, 34(3): 612−616.(in Chinese) doi: 10.3969/j.issn.1000-4440.2018.03.019 [24] MAO C, AKHATAYEVA Z, CHENG H J, et al. A novel 23 bp indel mutation in PRL gene is associated with growth traits in Luxi Blackhead sheep [J]. Animal Biotechnology, 2021, 32(6): 740−747. doi: 10.1080/10495398.2020.1753757 [25] JIANG L F, ZHOU X D, XU K, et al. miR-7/EGFR/MEGF9 axis regulates cartilage degradation in osteoarthritis via PI3K/AKT/mTOR signaling pathway [J]. Bioengineered, 2021, 12(1): 8622−8634. doi: 10.1080/21655979.2021.1988362 [26] ALFONSI M, PALKA C, MORIZIO E, et al. De novo 9q33 microdeletion identified by array-comparative genomic hybridization in a foetus with sex reversal and congenital heart defects [J]. Clinical Dysmorphology, 2013, 22(3): 132−134. doi: 10.1097/MCD.0b013e328363023b [27] WANG W F, ZHENG X L, SONG H J, et al. Spatial and temporal deletion reveals a latent effect of Megf8 on the left-right patterning and heart development [J]. Differentiation, 2020, 113: 19−25. doi: 10.1016/j.diff.2020.03.002 [28] 陈雷, 姚锋, 唐爽, 等. 刺参多功能表皮生长因子6(megf6)cDNA克隆及在肠再生过程中的表达分析 [J]. 大连海洋大学学报, 2020, 35(2):239−246. doi: 10.16535/j.cnki.dlhyxb.2019-320CHEN L, YAO F, TANG S, et al. cDNA cloning and expression of multiple epidermal growth factor 6 (megf6) gene in sea cucumber Apostichopus japonicus during intestine regeneration [J]. Journal of Dalian Ocean University, 2020, 35(2): 239−246.(in Chinese) doi: 10.16535/j.cnki.dlhyxb.2019-320