Chromosome-level genome and characteristic analysis of Platax teira
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摘要:
尖翅燕鱼 (Platax teira) 具有生长速度快、肉质鲜美、营养价值高等特点,其外形奇特,尤其幼鱼更为特殊,可作为观赏鱼,是南海发展网箱养殖的重要潜力鱼类之一。由于目前缺乏尖翅燕鱼的基因组信息,其多数功能基因未被挖掘,已成为制约其遗传育种的重要因素。运用三代测序技术和组装获得染色体水平的尖翅燕鱼高质量基因组图谱,通过基因组注释获得尖翅燕鱼基因组序列的基本生物学信息。结果显示,尖翅燕鱼基因组大小为697.98 Mb,Hi-C挂载至24条染色体,挂载率为99.26%,含重复序列177.79 Mb,占整个基因组的25.47%,注释到22 851个蛋白编码基因。系统进化分析显示尖翅燕鱼与波纹唇鱼 (Cheilinus undulatus) 亲缘关系最近,分化时间距今约82.89 Ma。正选择基因主要富集在与离子通道和心脏功能有关的通路中,扩张的基因家族主要富集在嗅觉传导和氮代谢通路上,揭示了其在特定环境中的生存、适应依据和生态适应策略。
Abstract:Platax teira has the characteristics of fast growth rate, delicious meat and high nutritional content, and its strange appearance, especially for young fish, makes it an ornamental fish, being one of the important potential fishes for the cage culture development in the South China Sea. Due to the lack of genomic information, most of the functional genes of P. teira have not been explored, which has become an important factor of restricting its genetic breeding. We utilized triple sequencing technology and assembly to obtain a high-quality genome map of P. teira on chromosome level, and obtained the basic biological information of P. teira genome sequence through genome annotation. The results show that the genome size of P. teira was 697.98 Mb, assembling into 24 chromosomes with an assembly rate of 99.26%. P. teira genome contained 177.79 Mb of repetitive sequences, accounting for 25.47% of the total genome and encoding 22 851 genes. Comparative genomic analysis with 11 other fish species reveals that P. teira shared the closest relationship with Cheilinus undulatus, and the differentiation time was about 82.89 Ma. Genes under positive selection in P. teira were enriched in pathways related to ion channels and cardiac function, while the expanded gene families were enriched in pathways related to olfactory transmission and nitrogen metabolism, which reveals its survival, adaptation basis and ecological adaptation strategies in specific environment.
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Keywords:
- Platax teira /
- Chromosome /
- Genome /
- Comparative genomics /
- Ephippidae
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我国的珍珠贝主要包括珠母贝属(Pinctada) 的大珠母贝(P.maxima)、珠母贝(P.margaritif-era)、合浦珠母贝(P.fucata或P.fucata martensii)(又名马氏珠母贝P.martensii)、长耳珠母贝(P.chemnitzi)、黑珠母贝(P.nigra)和白珠母贝(P.albina)等[1-2]。这些珍珠贝种类是生产海水珍珠的重要母贝或潜在贝种,具有重要的经济价值。其中大珠母贝是生产大型珍珠的重要母贝,珠母贝是生产黑珍珠的重要母贝,经济价值尤其巨大。但有的种类之间形态差异小,如射肋珠母贝和合浦珠母贝仅凭形态描述很难鉴定[2],因此形态分类比较困难。有的种形态变异大,分类鉴定也十分不容易,因此出现了很多同物异名[3]。此外在幼体阶段种类鉴定也容易混淆。另外在珍珠贝种间杂交育种研究中杂交后代的身份检测也缺乏有效手段,是否有真正的杂交,或是雌核发育的结果?目前只有同工酶的证据[4],尚无DNA方面的鉴定标记。此外,对于没有外部特征的样品,如肌肉或内脏团组织样品,如何鉴定也是有待解决的问题。因此开发种类特异(species specific)的分子标记对种类鉴定、分子标记辅助育种等方面具有重要的应用价值。
真核生物的核糖体DNA(ribosomal DNA, rDNA),由几百个串联重复的转录单元(transcription unit)(包括18S、5.8S和28S)和内部转录间隔子(internal transcribed spacers,ITSs)(包括ITS 1和ITS 2)等组成[5]。由于协同进化(concerted evolution)作用,rDNA各部分序列在种内变异小,种间变异大[5-7],因此适合于种类鉴定。其中18S、5.8S和28S基因序列高度保守,适合于高阶分类单元的区分,而ITS 1和ITS 2的DNA序列变异大,适合于种类水平,尤其是近缘种的鉴定,目前已有许多利用ITS进行系统发育(phylogeny)分析和种类鉴定的研究报道[8-15]。但由于rDNA是多拷贝的,有的种类可能存在种内变异[16]。因此具体种类必须具体分析。He等[17]利用ITS2序列对我国珠母贝属的5个种进行了亲缘关系分析,发现有的种存在种内多态。本文拟对大珠母贝、珠母贝、合浦珠母贝、长耳珠母贝、白珠母贝和黑珠母贝的ITS 2序列和两侧的部分5.8S和28S序列进行比较分析,探讨其作为种类鉴别的分子标记的可能性。
1. 材料和方法
1.1 实验材料
共采集了大珠母贝、珠母贝、合浦珠母贝、长耳珠母贝和白珠母贝5个种各3~10个样品。其中合浦珠母贝采集自广东大亚湾、广西北海和海南三亚,白珠母贝采自澳大利亚,其他种来自海南三亚(表 1),取闭壳肌保存于95%的酒精中备用。合浦珠母贝的样品合并分析时用pfuc表示。黑珠母贝的序列来自于GenBank数据库。
表 1 样品种类、采集地点以及ITS 2的扩增片段长度、基因型及其序列号Table 1. Species, sampling localities as well as amplified fragment length, genotype and accession numbers of ITS 2种类及采样地点
species and locality代码
code基因型
genotypeITS 2(bp) 总长(bp)
length(bp)GenBank序列号
accession number大珠母贝P.maxima
(海南三亚/ Sanya, Hainan)pmax pmax1 211 525 AY877505 pmax2 211 525 AY883851 pmax3 211 525 AY877504 珠母贝P.margaritifera
(海南三亚/ Sanya, Hainan)pmar pmar1 215 529 AY877507 pmar2 214 528 AY883850 pmar3 215 529 AY877506 白珠母贝P.albina
(澳大利亚/ Port Stephens)palb palb1 251 565 AY877508 palb2 251 565 AY883846 长耳珠母贝P.chemnitzi
(海南三亚/ Sanya, Hainan)pche pche1 251 564 AY877511 pche2 251 564 AY883848 pche3 251 564 AY877510 pche4 252 565 AY877509 pche5 252 565 AY883847 黑珠母贝P.nigra pnig pnig0 254 - AY192714 pnig1 254 - AY282728 pnig2 254 - AY282729 pnig3 254 - AY282730 合浦珠母贝P.fucata
(海南三亚/ Sanya, Hainan(hn),广东大亚湾/ Daya Bay (db), Guangdong和广西北海/Beihai (bh), Guangxi)db db1 235 548 AY877581 db2 231 544 AY877604 bh bh1 233 546 AY877583 bh2 231 544 AY877605 hn hn1 237 550 AY877592 hn2 230 543 AY877597 1.2 DNA提取、PCR扩增和测序
DNA的提取用QIAamp DNA mini kit (QIAGEN)试剂盒并按其提供的操作指南进行。ITS 2的引物、PCR扩增条件和测序方法与文献[18]相同。基本过程包括PCR扩增、PCR产物纯化、测序PCR扩增、测序仪测序分析。每个种各测序3~10个样品。双向测序,选择序列峰图清晰的个体用于分析。
1.3 数据处理
测序后先用Sequence Editor (1.03)(Applied Biosystems)软件对其电泳图谱和碱基序列进行手工校对和编辑,然后用Clustral X软件[19]进行多重比对(multiple alignment)分析,再用MEGA 3[20]软件计算碱基组成、遗传距离和构建UPGMA系统发育树。用DnaSP 4.0[21]进行基因型分析,由于5.8S和28S变异小,基因型分析仅包括ITS 2。根据比对结果手工进行单碱基突变分析。
2. 结果
2.1 PCR扩增结果与基因型分析
PCR扩增产物包括ITS 2和两侧的5.8S与28S基因片段。在GenBank数据库中的同源分析(blastn)发现5.8S与28S基因片段的序列与相近物种具有高度同源性,表明扩增产物和测序结果真实可靠。通过比对分析后获得ITS 2的基因型数据,每个基因型的序列及其两端的序列已在GenBank数据库注册,所获得的基因型和序列号见表 1。不同种之间基因型不同。大珠母贝和珠母贝各获得3个基因型,白珠母贝2个基因型,长耳珠母贝5个基因型。合浦珠母贝3个不同采样点各取2个基因型用于分析。
2.2 种间ITS 2序列长度变异
扩增产物的序列长度(包括引物序列)为567~607 bp(表 1),其中5.8S基因片段长84 bp,28S基因片段长272 bp,ITS 2比对长度为270位点(图 1)。实际长度在211~254 bp之间(表 1)。不同种之间长度差异较大,大珠母贝和珠母贝的ITS 2最短(211~215 bp),白珠母贝、长耳珠母贝和黑珠母贝的最长(251~254 bp),合浦珠母贝居中(230~237 bp)。
2.3 种间序列单核苷酸变异分析
(1) 5.8S和28S基因部分序列的单核苷酸突变分析本引物扩增的5.8S基因片段较短,连引物在内共84 bp,种间序列相当保守,仅末端1个碱基发生颠换突变,大珠母贝和珠母贝为T碱基,其它种为G碱基。28S片段较长,包括引物共长272 bp,共有3个位点发生突变。在包括引物的626个比对位点中,593位点发生转换突变,大珠母贝和珠母贝为A碱基,其余种为G碱基。605位点发生插入/缺失突变,大珠母贝、珠母贝和白珠母贝为C碱基,其余种缺失(数据未给出)。本部分未包括黑珠母贝的序列资料。
(2) ITS 2的单核苷酸突变(SNP)分析与5.8S和28S不同,ITS 2为高变异区,在270个比对位点中有146个突变位点,包括72个插入/缺失突变位点,非突变位点只有114个。简约信息位点108个,单突变子(singleton)8个。每个种都有其特有的单核苷酸突变(图 1)。种间的单核苷酸突变见表 2。大部分种间的转换突变位点数略小于颠换突变数。
表 2 种间单核苷酸突变Table 2. Interspecific single nucleotide mutation种类
species大珠母贝 珠母贝 白珠母贝 黑珠母贝 长耳珠母贝 合浦珠母贝 大珠母贝 P.maxima - 8 21 21 23 20 珠母贝 P.margaritifera 6 - 25 26 23 20 白珠母贝 P.albina 31 26 - 1 19 18 黑珠母贝 P.nigra 33 31 4 - 19 17 长耳珠母贝 P.chemnitzi 22 22 34 34 - 14 合浦珠母贝 P.fucata 21 19 31 32 24 - 注:对角线下为颠换突变,对角线上为转换突变
Note:transiversion-lower diagonal, transition-upper diagonal黑珠母贝的pnig1~3个体与白珠母贝之间有5个突变位点,而pnig0和palb之间只有2个突变位点的差异,表明pnig0可能是白珠母贝,或介于两者之间的种间类型,在聚类分析中与白珠母贝聚合在一起(图 2)。
除碱基替换突变外还有大量的插入/缺失突变,大珠母贝、珠母贝和合浦珠母贝有3个较大的缺失区(25~41,65~72,170~179),大珠母贝和珠母贝还有1个缺失区,即157~164。而黑珠母贝和白珠母贝只有其中的1个缺失区,即65~72。其中,合浦珠母贝有插入/缺失位点33~40个,大珠母贝55个,珠母贝51~52个,长耳珠母贝16~17个,黑珠母贝16个,白珠母贝17个。
2.4 种内与种间的遗传距离分析
利用ITS 2序列数据进行了种内和种间遗传距离计算。种内的遗传距离在0.004~0.014之间(表 3)。不同种之间的遗传距离差别较大。白珠母贝和黑珠母贝之间遗传距离最小,只有0.034,大珠母贝与珠母贝之间(0.088)以及长耳珠母贝与白珠母贝和黑珠母贝之间(分别为0.096和0.115)的遗传距离居中,其它种间遗传距离较大(0.223~0.318)。总体来看,种间遗传距离远远大于种内遗传距离。
2.5 亲缘关系的聚类分析
基于Kimura 2-parameter遗传距离的UPGMA系统发育树表明,6个种分成4大支(图 2)。其中大珠母贝和珠母贝以及白珠母贝和黑珠母贝分别聚成1支,长耳珠母贝和合浦珠母贝各自形成1支。表明大珠母贝和珠母贝、白珠母贝和黑珠母贝的亲缘关系分别较近,而它们之间的突变位点数(表 2)和遗传距离(表 3)也最小。
表 3 珠母贝属种内与种间的遗传距离Table 3. Intraspecific and interspecific genetic divergences of pearl oysters in Pinctada种类species 1 2 3 4 5 6 1max 0.010 2mar 0.088 0.011 3alb 0.271 0.280 0.004 4nig 0.287 0.301 0.034 0.014 5che 0.318 0.313 0.096 0.115 0.006 6pfuc 0.268 0.280 0.233 0.238 0.223 0.014 注:对角线为种内遗传距离,对角线下为种间遗传距离
Note:intraspecific-diagonal, interspecific-belon diagonal3. 讨论
ITS是rDNA上的一段非编码序列,变异较大,多态性较高,适合于亲缘关系较近的种类的遗传多样性分析,已广泛应用于种类鉴定和系统发育研究[8-15]。本研究表明,5.8S和28S序列较保守,不适合于种类鉴定,而ITS 2序列差异大,种内遗传距离与种间遗传距离相差较大,是种类鉴定的理想标记。从基因型分析来看,不同种有不同的基因型,很容易将各个种分开。但仅靠基因型也不够,因为合浦珠母贝同一个种的不同种群也有不同的基因型[18],因此还必须考虑不同基因型之间的序列差异程度。ITS 2序列种间差异分析表明,不同种间序列差异不同,其中黑珍珠与白珠母贝之间差异较小,其余种类之间的序列差异较大(>8.8%),远远大于种内的遗传差异(< 1.4%),很容易把不同种区分开。白珠母贝和黑珠母贝之间的序列差异较小(3.4%),如果不考虑pnig0个体,其序列差异更小,聚类分析也表明黑珠母贝和白珠母贝的亲缘关系很近,pnig0个体采自我国海南,而白珠母贝采自澳大利亚,但pnig0个体却和白珠母贝聚合在一起。在形态描述资料中白珠母贝和黑珠母贝仅有颜色的差异[2]。这些资料表明它们可能是一个种的不同亚种。ITS 2还表现出种间长度变异。大珠母贝和珠母贝的PCR产物长567~571 bp,白珠母贝和长耳珠母贝长606~609 bp,合浦珠母贝长585~592 bp。黑珠母贝的PCR产物长度应该与长耳珠母贝和白珠母贝的差不多。因此通过长度变异检测,可分出不同的类群并可将合浦珠母贝分辨出来。Anderson和Adlard[9]对ITS序列分析也发现Saccostrea commercialis和S. glomerata为同种。由上可以看出,ITS 2的序列变异分析可以检测到种间的分化程度。如合浦珠母贝种内不同种群之间虽然基因型不同,但群体内与群体间序列变异极小[19]。因此,序列变异分析与基因型分析相结合能灵敏地区分不同的种或亚种。
从序列的变异特征看,ITS 2基因的117个突变位点中有72个插入/缺失突变,表明长度变异是种间变异的一个重要因素。种内变异远小于种间变异,表明珠母贝种类在进化过程中存在高度的协同进化(concerted evolution),使得种内变异快速同化趋于一致,而种间变异却快速积累[5-7]。这一特点使ITS 2序列特别适合作为种类鉴定的分子标记。但由于rDNA是多拷贝的,可能存在种内变异[16],因此具体的种要具体分析。对于珍珠贝类来说,克隆测序发现种内变异很小[17, 22],不影响种间鉴别。
由于测序分析仍不十分方便,且昂贵、费时,因此仍有待于开发基于PCR的快速经济的种类鉴定分子标记。
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表 1 尖翅燕鱼基因组组装和校正后组装结果统计
Table 1 Statistical analysis of genome assembly and corrected assembly results of P. teira
类型
TypeNextDenovo软件 NextPolish软件 长度
Length/bp数量
Count长度
Length/bp数量
CountN50 26 172 673 12 26 178 423 12 N90 8 653 897 29 8 656 320 29 最小长度
Min. length11 814 11 779 最大长度
Max. length33 665 020 33 669 695 平均长度
Ave. length5 207 983 5 208 839 总长度
Total length697 869 769 134 697 984 514 134 表 2 尖翅燕鱼BWA比对结果
Table 2 Results of BWA comparison of P. teira
总读长
Total reads匹配读长
Map reads匹配率
Map rate/%双端读长
Paired reads双端比对读长
Paired map reads真正的读长
Proper paired reads正确匹配率
Properly map rate/%758 790 194 755 705 560 99.59 701 824 260 698 739 626 671 793 148 95.72 表 3 尖翅燕鱼基因组BUSCO评估
Table 3 Genome BUSCO evaluation of P. teira
类型
Type数量
Number百分比
Percentage/%完整的BUSCOs
Complete BUSCOs3 595 98.8 完整的单拷贝BUSCOs
Complete and single-copy BUSCOs3 571 98.1 完整的重复序列BUSCOs
Complete and duplicated BUSCOs24 0.7 碎片BUSCOs
Fragmented BUSCOs9 0.2 未比对上的BUSCOs
Missing BUSCOs36 1.0 总的BUSCO
Total BUSCO3 640 100.0 表 4 尖翅燕鱼各染色体长度
Table 4 Length of each chromosome of P. teira
染色体
Chromosome长度
Length/bp染色体
Chromosome长度
Length/bpHiC_scaffold_1 32 115 465 HiC_scaffold_13 22 937 044 HiC_scaffold_2 28 296 500 HiC_scaffold_14 29 051 425 HiC_scaffold_3 30 192 628 HiC_scaffold_15 28 201 383 HiC_scaffold_4 24 662 710 HiC_scaffold_16 23 075 928 HiC_scaffold_5 16 755 790 HiC_scaffold_17 25 711 309 HiC_scaffold_6 29 747 999 HiC_scaffold_18 34 711 268 HiC_scaffold_7 31 770 170 HiC_scaffold_19 31 217 840 HiC_scaffold_8 30 909 847 HiC_scaffold_20 34 223 832 HiC_scaffold_9 27 971 542 HiC_scaffold_21 32 267 482 HiC_scaffold_10 28 320 680 HiC_scaffold_22 30 609 981 HiC_scaffold_11 34 468 000 HiC_scaffold_23 27 290 755 HiC_scaffold_12 26 316 500 HiC_scaffold_24 32 023 099 合计 Total 692 849 177 表 5 尖翅燕鱼重复序列注释结果
Table 5 Results of repeated sequence annotation of P. teira
元件类型
Elements type元件数量
Number of
elements长度
Length/bp百分比
Percentage/%逆转录因子
Retroelements137 738 39 456 150 5.65 DNA转座子
DNA transposons266 011 55 441 011 7.94 环状
DNA Rolling-circles2 389 682 132 0.10 无分类
Unclassified366 703 66 929 139 9.59 总夹杂的重复序列
Total interspersed
repeats161 826 300 23.18 小RNA
Small RNA700 80 729 0.01 卫星DNA
Satellites DNA660 189 494 0.03 简单的重复序列
Simple repeats317 455 12 696 083 1.82 低复杂性
Low complexity42 640 2 317 723 0.33 总的重复
Total repeats177 792 461 25.47 表 6 尖翅燕鱼结构注释结果BUSCO评估
Table 6 BUSCO evaluation of structural annotation results of P. teira
类型
Type数量
Number百分比
Percentage/%完整的BUSCOs
Complete BUSCOs3 213 88.3 完整的单拷贝BUSCOs
Complete and single-copy BUSCOs3 176 87.3 完整的重复序列BUSCOs
Complete and duplicated BUSCOs37 1.0 碎片BUSCOs
Fragmented BUSCOs155 4.3 未比对上的BUSCOs
Missing BUSCOs272 7.4 总的BUSCO
Total BUSCO3 640 100.0 表 7 尖翅燕鱼功能注释结果
Table 7 Functional annotation results of P. teira
数据库
Database数量
Number百分比
Percentage/%NR 21 016 91.97 SwissProt 18 945 82.91 KEGG 17 511 76.63 COG 5 787 25.32 Trembl 20 996 91.88 功能注释基因
Functional annotation21 104 92.35 总计Total 22 851 100.00 表 8 12个物种基因家族聚类结果
Table 8 Results of gene family clustering in 12 species
物种
Species总的
Total单拷贝
Single特异性
Specific未聚类
Unclustered眼斑双锯鱼A. ocellaris 23 035 4 787 226 185 泰国斗鱼B. splendens 22 791 4 787 245 227 大白鲨C. carcharias 19 440 4 787 1 252 1 273 波纹唇鱼C .undulatus 23 316 4 787 381 184 斑马鱼D. rerio 32 717 4 787 3 503 1 659 云纹石斑鱼E .moara 23 735 4 787 112 336 食蚊鱼G. affinis 23 135 4 787 98 166 青鳉O. latipes 22 071 4 787 460 241 尖翅燕鱼P .teria 22 851 4 787 689 2 727 深裂眶锯雀鲷S. partitus 22 589 4 787 70 363 红箭鱼X. helleri 23 921 4 787 98 138 花斑剑尾鱼X. maculatus 23 238 4 787 34 126 -
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