Combining genome-wide association study and transcriptome analysis to identify molecular markers and genetic basis of population-asynchronous ovarian development in Coilia nasus

Coilia nasus,a migratory fish species found in the middle and lower reaches of the Yangtze River and along offshore areas of China,possesses considerable aquacultural and economic potential.However,the species faces challenges due to significant variation in the gonadal development rate among females,resulting in inconsistent ovarian maturation times at the population level,an extended reproductive period,and limitations on fish growth rate due to ovarian prematurity.In the present study,we combined genome-wide association study(GWAS)and comparative transcriptome analysis to investigate the potential single nucleotide polymorphisms(SNPs)and candidate genes associated with population-asynchronous ovarian development in C.nasus.Genotyping of the female population based on whole-genome resequencing yielded 2120695 high-quality SNPs,39 of which were suggestively associated with ovarian development.Of note,a significant SNP peak on LG21 containing 30 suggestively associated SNPs was identified,with cpne5a determined as the causal gene of the peak.Therefore,single-marker and haplotype association analyses were performed on cpne5a,revealing four genetic markers(P<0.05)and seven haplotypes(r2>0.9)significantly associated with the phenotype.Comparative transcriptome analysis of precociously and normally maturing individuals screened out 29 and 426 overlapping differentially expressed genes in the brain and ovary,respectively,between individuals of different body sizes.Integrating the GWAS and transcriptome analysis results,this study identified genes and pathways related to hypothalamic-pituitary-gonadal axis hormone secretion,extracellular matrix,angiogenesis,and gap junctions involved in population-asynchronous ovarian development.The insights gained from this study provide a basis for a deeper understanding of the molecular mechanisms underlying ovarian development in fish and may facilitate the genetic breeding of C.nasus strains exhibiting population-synchronous ovarian development in the future.

Otolith microchemical evidence revealing multiple spawning site origination of the anadromous tapertail anchovy(Coilia nasus)in the Changjiang(Yangtze)River Estuary

The estuarine tapertail anchovy(Coilia nasus)is a high-value commercial fish.Estimating the spawning site or hatchery origin and habitat is essential for its conservation.This study aimed to determine the habitat use and life history characteristics of C.nasus from the Changjiang River Estuary.We investigated the environmental signatures of strontium(Sr)and calcium(Ca)in the otoliths of the collected specimens using electron probe microanalysis;additionally,we examined their gonadal maturity stage.Our results indicate that the 31 adult C.nasus specimens used in this study could be classified into two types based on their otolith Sr:Ca concentration ratios and their gonadal maturity stage.The long freshwater early life history type(Type LF)had wider central region in the otolith with low Sr:Ca concentration ratios ranging from 1.24±0.62 to 1.92±0.78 and a bluish pattern of low Sr content level.These fish are of riverine origin and had a relatively long early life history in freshwater and low gonadal maturation when captured.The short freshwater early life history type(Type SF)had a shorter central region in the otolith with low Sr:Ca concentration ratios ranging from 1.35±0.5 to 2.82±0.97 and a correspondingly bluish pattern.These fish also had a relatively short-term early life history in freshwater and high gonadal maturation when captured.The results of the otolith microchemical analysis indicated that Type LF and Type SF originated in spawning/hatching sites far from and close to the estuary,respectively.The mature gonads of Type SF fish indicated that they may breed in areas close to the estuary,whereas the immature gonads of Type LF fish indicated that they may breed in areas far from the estuary.This study is the first to reveal that the Changjiang River Estuary contains stocks of anadromous C.nasus originating in different spawning sites during the same season.The estuarine habitat plays a critical role in the connectivity between freshwater recruitment and the marine resources available to adult spawners of this commercially valued species.From a conservation perspective,this study provides important information for identifying anadromous C.nasus stocks originating in different spawning sites in the Changjiang River Basin.

基于线粒体Cytb基因和D-loop区序列的高邮湖湖鲚(Coilia nasus)遗传多样性分析

本研究利用线粒体细胞色素b(Cytb)基因和控制区(D-loop)序列作为分子标记,调查了高邮湖湖鲚(Coilia nasus)种群遗传多样性。结果显示,Cytb基因和D-loop区序列碱基A+T含量均高于G+C含量,显示碱基组成具有偏倚性。38条Cytb基因序列检出26个变异位点,定义13个单倍型,单倍型多样性和核苷酸多样性分别为0.716±0.078和0.002 70±0.000 57;40条D-loop区序列检出53个变异位点,定义21个单倍型,单倍型多样性和核苷酸多样性分别为0.906±0.034和0.006 27±0.000 99。13个Cytb基因单倍型之间的遗传距离在0.001~0.014之间,NJ系统进化树显示单倍型聚为1支;21个D-loop区单倍型之间的遗传距离在0.001~0.019之间,NJ系统进化树显示单倍型聚为2支。中性检测结果和歧点分布图均表明高邮湖湖鲚种群稳定,近期没有发生种群扩张。整体来看,高邮湖湖鲚种质资源遗传多样性水平较高,具有高单倍型多样性和低核苷酸多样性的特征。

Unravelling habitat use of Coilia nasus from the Rokkaku River and Chikugo River estuaries of Japan by otolith strontium and calcium

The migratory history of the engraulid fish Coilia nasus in the Rokkaku and Chikugo River estuaries of the Ariake Sea, Japan was assessed using otolith strontium（Sr） X-ray intensity maps and strontium：calcium（Sr：Ca） ratio life history transects by an electron probe microanalyzer（EPMA）. The results showed that seven of the ten specimens from the Rokkaku River Estuary（LJC） and all 15 specimens collected in the Chikugo River Estuary（ZHC） had low Sr：Ca ratios（≤3） at the central otolith area, indicating their riverine origin and initial freshwater residence. After the first regime shift adjacent to natal regions, the Sr level mapping displayed a wide variety of color patterns, and the Sr：Ca ratios obtained by the line transect analysis could be divided into one to six significantly different phases indicative of gradual life history transition. The other three specimens from the Rokkaku River Estuary had high Sr：Ca ratios（3–6.7） at the central otolith area but showed alternating changes between low and high values outside the natal region, suggesting that estuarine-origin individuals occurred in the Rokkaku River Estuary. The two-dimensional maps of the Sr level and average of the otolith Sr：Ca ratios along the life-history transects could be used as effective tools for reconstruction of past habitat use of the tapertail anchovy in estuaries of the Ariake Sea, Japan.

Comparing different spatial interpolation methods to predict the distribution of fishes:A case study of Coilia nasus in the Changjiang River Estuary

Spatial-temporal distribution of marine fishes is strongly influenced by environmental factors.To obtain a more continuous distribution of these variables usually measured by stationary sampling designs,spatial interpolation methods(SIMs)is usually used.However,different SIMs may obtain varied estimation values with significant differences,thus affecting the prediction of fish spatial distribution.In this study,different SIMs were used to obtain continuous environmental variables(water depth,water temperature,salinity,dissolved oxygen(DO),p H,chlorophyll a and chemical oxygen demand(COD))in the Changjiang River Estuary(CRE),including inverse distance weighted(IDW)interpolation,ordinary Kriging(OK)(semivariogram model:exponential(OKE),Gaussian(OKG)and spherical(OKS))and radial basis function(RBF)(regularized spline function(RS)and tension spline function(TS)).The accuracy and effect of SIMs were cross-validated,and two-stage generalized additive model(GAM)was used to predict the distribution of Coilia nasus from 2012 to 2014 in CRE.DO and COD were removed before model prediction due to their autocorrelation coefficient based on variance inflation factors analysis.Results showed that the estimated values of environmental variables obtained by the different SIMs differed(i.e.,mean values,range etc.).Cross-validation revealed that the most suitable SIMs of water depth and chlorophyll a was IDW,water temperature and salinity was RS,and p H was OKG.Further,different interpolation results affected the predicted spatial distribution of Coilia nasus in the CRE.The mean values of the predicted abundance were similar,but the differences between and among the maximum value were large.Studies showed that different SIMs can affect estimated values of the environmental variables in the CRE(especially salinity).These variations further suggest that the most applicable SIMs to each variable will also differ.Thus,it is necessary to take these potential impacts into consideration when studying the relationship between the spatial distribution of fishes and environmental changes in the CRE.

Comparative proteomic analysis of olfactory rosettes in anadromous Coilia nasus and resident Coilia nasus

The Japanese grenadier anchovy (Coilia nasus) undergoes upstream migration to spawning annually but can also be observed in freshwater resident populations. It has been hypothesized that anadromous adult C. nasus may utilize olfactory cues to locate spawning grounds. We firstly performed a comparative proteome analysis about olfactory rosettes in two populations to hunt for the proteomic changes. Among 5 408 identified proteins, 1 515 proteins (629 up-regulated and 886 down-regulated) were differentially expressed. Especially, several proteins and pathways associated with olfactory signaling were found to be significantly differential. Compared with resident C. nasus, the expressions of Golf protein and the sodium/calcium exchanger were significantly up-regulated in anadromous C. nasus. The expression of adenylate cyclase and regulator of G-protein signaling (RGS) were decreased. Our findings suggest a decrease in the expression of cGMP-dependent protein kinase (PKG) in anadromous C. nasus compared to resident C. nasus. The expression of Calmodulin (CaM) was increased and CaM-dependent protein kinase Ⅱ(CaMKⅡ) was decreased. In addition, KEGG pathway enrichment analysis of up-regulated proteins indicated statistically significant difference not only in olfactory transduction but also in the cGMP-PKG signal pathway. Furtherly, we sought out some proteins expressed in the same trend occurring in DEGs (differentially expressed genes) and DEPs (differentially expressed proteins) by doing the integrative analysis of proteome and transcriptome in olfactory rosettes of C. nasus.

Overview on Genetic Diversity of Coilia nasus

Coilia nasus is an important economic migratory fish in China, and it is known as ＂Three Delicious Fish of the Yangtze River＂ with puffer fishes and reeves shad. However, because of water pollution, blocked migration and overfishing, wild resources of C. nasus have suffered severe damage. This article gives an overview on genetic diversity of C. nasus from morphology, protein and DNA levels, to supply a theoretical foundation for protection of C. nasus resources.

Evolutionarily conserved boule and dazl identify germ cells of Coilia nasus

Coilia nasus is an endangered fish species in the Yangtze River,and there is urgent need to know the genes related to its reproduction and fertility.The DAZ family including boule,daz and dazl,plays an important role in germ cells development.In this study,the cDNA sequence of boule(Cnbol)and dazl(Cndazl)genes were cloned and their expression patterns were characterized in Coilia nasus.RT-PCR showed that the expression of Cnbol and Cndazl mRNAs was restricted in adult gonads.The section in situ hybridization indicated that the temporospatial expression patterns of Cnbol and Cndazl were significantly different.In the testis,Cnbol was mainly detected in spermatocytes and spermatids,while Cndazl was prominently expressed in s permatogonia.In the ovary,Cnbol and Cndazl were highly expressed in the early stages of oocytes.Interestingly,Cndazl was also concentrated in perinuclear speckle and then localized to the Balbiani body in late stages of oocytes.In addition,Cnbol and Cndazl 3′UTR can specifically label medaka Primordial Germ Cells(PGCs).According to our results,Cnbol and Cndazl are marker genes of germ cells and may play a vital role in the development and differentiation of germ cells in Coilia nasus.

Full-length transcriptome of anadromous Coilia nasus using single molecule real-time (SMRT) sequencing

This study was designed to generate the full-length transcriptome of Coilia nasus using single-molecule long read isoform sequencing(SMRT-seq)technology.RNAs of brains,ovaries,and testes of C.nasus conditioned in both seawater and freshwater except for gut which was only from the fish conditioned in freshwater,were isolated.All the RNAs were pooled together equally for SMRT sequencing.One SMRT cell produced 64.18 Gb clean data,including 765,184 circular consensus(CCS)reads and 616,126 full-length non-chimeric(FLNC)reads.Finally,93,793 non-redundant transcripts were obtained after clustered and polished.Structural analysis of the 93,793 non-redundant transcripts predicted 8242 alternative splicing events,a total of 229,825 SSR and 72,037 complete CDS.A total of 8437 lncRNA were identified.84,617 transcripts were annotated in NR,Swissprot,GO,COG,KOG,eggnog,Pfam and KEGG database totally.This is the first full-length transcriptome of Coilia nasus,including transcripts appeared in the case of seawater,which will facilitate exploration of genetic data and life story of Coilia nasus.

刀鲚、凤鲚和湖鲚矢耳石的形态学比较研究

选取凤鲚(Coilia mystus)、湖鲚(Coilia nasus taihuensis)以及采自崇明、黄海和瓯江口3个水域的刀鲚(Coilia nasus)的矢耳石为对象,描述了这些鱼类耳石的形态特征,并进一步使用基于12组形态数据的框架测量进行了种间和种内比较和判别分析。结果表明耳石形态的差异种间较种内更为明显。本研究中,刀鲚与凤鲚、刀鲚与湖鲚、凤鲚与湖鲚矢耳石形态间的差异极其显著,相互间的判别准确率分别可达100%、94%、100%。不同水域刀鲚间虽亦有不同程度的差异,但判别的准确率仅为56%～83%,其中崇明和黄海刀鲚的耳石形态较为接近,瓯江口刀鲚则与前两者差异相对较大。本研究还就影响耳石形态差异可能的因素进行了探讨。

大洋河刀鲚苗种池塘培育试验

为摸索辽宁地区刀鲚(Coilia nasus)苗种培育条件,以大洋河刀鲚亲本繁育的幼鱼(全长2 cm左右)为试验材料,设置了7500、15000、30000尾/hm^(2)和37500尾/hm^(2)四个不同养殖密度,利用4口池塘进行了刀鲚苗种培育试验。结果表明,经过2个月的池塘培育,刀鲚苗种规格由全长2.21~2.30 cm、体质量0.06~0.07 g分别增至全长7.60~15.12 cm、体质量1.18~6.03 g,成活率超过70%。随着养殖密度增加,培育规格呈下降趋势,当苗种放养密度小于30000尾/hm^(2)可以获得较好的生长效果。

两种耳石分析法在鲚属种间和种群间识别效果的比较研究

采用耳石传统形态测量法和傅里叶形态分析法,对281尾2龄长江凤鲚(Coilia mystus)和刀鲚(C.nasus)个体的矢耳石形态学作了分析,结果表明,采用传统的耳石形态测量法对凤鲚与刀鲚种间的正判率达90.9%,但2个刀鲚生态型种群之间的判别成功率仅为76.9%。而运用傅里叶耳石形态分析法,凤鲚和刀鲚物种间的识别率高达100%,2个刀鲚生态型间的识别率也提高至86.8%。可见,两种耳石形态分析法对鲚属种间的识别效果均很好,但对种群分析而言,傅里叶形态分析法可以取得更好的识别效果。

刀鲚的生物学及人工养殖研究进展

在对刀鲚（Coilia nasus）的形态与生长、生活习性、食性、繁殖习性等生物学特性进行总结的基础上，对其资源、市场现状及人工繁殖与养殖技术研究进行了综述，并展望了刀鲚产业化发展的前景。

长江口刀鲚幼鱼耳石碳、氧同位素特征初报

为了解长江口刀鲚(Coilia nasus)耳石中碳、氧稳定同位素的特征及其可能反映出的生态学意义,作者利用稳定同位素质谱分析技术,首次对长江口刀鲚幼鱼的耳石进行碳、氧稳定同位素的初步研究。结果显示,δ13C和δ18O分别为–12.1±1.8(‰VPDB)和–7.9±1.2(‰VPDB),总体上δ13C波动较大,而δ18O波动较小。基于个体的δ13C和δ18O散点分布可将本研究中的刀鲚归为两个组,可能起源于长江中两个不同孵化场水域的群体,其中δ18O显示出两组刀鲚所经历环境积温上的差异,反映出两者起源水域温度环境的不同;而δ13C的差异说明两组刀鲚在饵料组成上的有所差异。

长江刀鲚鱼种驯食浮性颗粒饲料试验研究

采用灯光诱鱼协同缓沉饲料驯食过渡至浮性颗粒饲料投喂的方法,高密度驯养长江刀鲚（Coilia nasus）鱼种,建立了8~10 cm刀鲚鱼种转食浮性颗粒饲料的人工饲养技术,为刀鲚的集约化养殖提供技术支撑。结果显示,经16 d的驯食,刀鲚在投饵后1 h之内吃完饲料,驯食期间刀鲚平均成活率为75.05%。闪光诱鱼比常光诱鱼更利于刀鲚鱼种驯食浮性颗粒饲料;先期对浮性颗粒饲料处理成缓沉投饲驯食并逐步过渡至完全摄食浮性颗粒饲料的操作能让刀鲚鱼种在短期内（16 d）完全摄食浮性颗粒饲料。

海洋生境刀鲚菌群PCR-DGGE指纹图谱构建及分析

为了分析海洋生境刀鲚（Coilia nasus）体内及生长环境菌群结构,作者采用免培养16S rDNA 的PCR-DGGE 指纹图谱技术,对刀鲚鳃、肠道壁、肠道内容物及水体环境中的菌群结构进行了对比分析。结果表明：变性梯度为35%-55%、浓度为8%的聚丙烯酰胺凝胶,150V 60℃下电泳10 h, DGGE带谱的分离效果较好；刀鲚鳃、肠道壁、肠道内容物及水体样品指纹图谱上分别显示出24、19、14和29条信号强度不同的条带；相同样品重复组细菌结构相似度在80%以上,差异不显著；不同样品之间,刀鲚鳃与海水聚为一支,具有较高的相似度为52%,肠壁与肠内容物相似度为41%。样品菌群主要以未培养菌为主,主要包括变形菌、放线菌和厚壁菌。本文首次成功构建海洋生境刀鲚菌群16S rDNA 的PCR-DGGE 指纹图谱。

不同群体刀鲚形态差异研究

基于多变量分析法对辽河、大洋河、鸭绿江和太湖的刀鲚(Coilia nasus)地理群体形态学可数性状、可量性状和框架数据,以聚类分析、判别分析、主成分分析三种多元分析法对其进行综合比较。结果显示:聚类分析将鸭绿江和太湖群体聚为一支,说明在形态上两群体较接近;主成分分析发现大洋河、鸭绿江和太湖3个刀鲚群体形态差异发生在背鳍以后的尾部;判别分析建立的判别函数对3个群体内判别准确率均为100%,群体间综合判别准确率为96.74%,有效判别各群体刀鲚归属。3个群体刀鲚在形态上存在差异,主成分分析、判别分析和聚类分析方法能够不同程度区分出它们的形态差异。

大洋河刀鲚繁殖生物学特性

为了解大洋河刀鲚繁殖生物学资料,本研究对2020年4-9月采集于大洋河下游河段4处点位的253尾刀鲚进行了繁殖生物学分析。结果显示:大洋河刀鲚样本体长范围175~345 mm,均值为(245±27)mm,体重范围27.7~220.4 g,均值为(67.7±26.7)g,年龄以2龄为主,占74.1%,雌雄总体性比为1∶1.13;雌雄鱼初次性成熟体长和年龄分别为229 mm、1.5龄和212 mm、1.2龄。通过成熟系数(GSI)和卵径大小分析,繁殖时间集中在6-7月份,GSI和卵径最高值分别为(15.08±3.94)%、(0.89±0.20)mm,9月GSI降至最低点为(0.65±0.20)%,10月前后刀鲚入海摄食准备越冬。绝对繁殖力范围在10853~84717粒,均值为(44617±26417)粒,相对繁殖力为(364±133)粒/g。绝对繁殖力与体长和体重呈正线性相关,但体重相关优势更为明显。

东平湖不同年龄结构湖鲚食性研究

为研究南水北调东线东平湖外来湖鲚(Coilia nasus)对湖区的生态效应影响,于2017年在山东省东平湖采集湖鲚样品,通过鉴定其胃含物组成,从食物网层面剖析湖鲚对湖区产生的生态影响。调查共采集湖鲚340尾,其中300尾为1^+龄湖鲚,其余40尾为2^+龄湖鲚。通过湖鲚饵料生物组成可以看出,1^+龄湖鲚主要摄食淡水浮游动物中的桡足类和枝角类,而对轮虫的摄食数量较少;2^+龄湖鲚的饵料生物中,游泳动物是最重要的种类,相对重要性指数为10 365.04,其次为桡足类,相对重要性指数为4 773.59,2^+龄湖脐的食性更偏向于肉食性鱼类。结果表明,湖脐对东平湖生态安全存在较大的隐患,需及时捕捞。

刀鲚的全人工繁殖及胚胎发育

通过对刀鲚（Coilia nasus）全人工繁殖试验和胚胎发育观察,研究了刀鲚全人工繁殖的特性和其胚胎发育的时序和特点。结果显示,对人工繁养的F1刀鲚亲鱼（2龄）进行模拟自然洄游的培育,采用注射促黄体素释放激素A2（LHRH-A2）对性腺发育良好的亲本进行催产,剂量为：雌鱼30μg/kg,雄鱼减半;在22~24℃条件下,经过18~33.5 h的效应时间,亲本自然交配产卵,催产率为91.7%;获得受精卵25.8万粒,受精率为80.6%,获得22.3万尾的初孵仔鱼,孵化率为86.4%;受精卵透明、浮性、无粘性、球形,卵径为（909.86±24.02）μm,油球大且单个;胚胎发育分为胚盘形成阶段、卵裂阶段、囊胚阶段、原肠期阶段、神经胚阶段、器官形成阶段和出膜阶段7个阶段及30个发育时期,在（23.64±0.36）℃条件下,历时28 h仔鱼出膜;初孵仔鱼全身透明,靠油球浮于水面,全长（2056.37~2074.74）μm,卵黄囊大且近椭圆形,油球呈球形,心跳90~110次/分,肌节48~50对;研究结果对刀鲚资源的保护和规模化全人工繁殖工艺流程的建立具有重要的意义。