CN114617085B - 一种鞍带石斑鱼抗病家系的培育方法 - Google Patents
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Abstract
本发明公开了一种鞍带石斑鱼抗病家系的培育方法,包括如下步骤:(1)亲本的培育;(2)亲鱼配对及家系构建;(3)攻毒;(4)抗病关键SNP标记的筛选;(5)抗病亲本的筛选;(6)抗病家系的培育;该方法通过GWAS技术找到鞍带石斑鱼抗神经坏死病毒的显著关联的分子标记,根据显著相关标记筛选出抗病亲本,再结合家系选育用抗病亲本培育出抗病子代,最终获得鞍带石斑鱼抗神经坏死病毒尤其是抗赤点石斑神经坏死病毒的品系。
Description
技术领域
本发明属于石斑鱼育种技术领域,具体涉及一种鞍带石斑鱼抗病家系的培育方法。
背景技术
鞍带石斑鱼又名龙胆石斑鱼或龙趸石斑,隶属鲈形目(Perciformes),鮨科(Serranidae),石斑鱼属(Epinephelus)。是石斑鱼类中体型最大者,也被称为“斑王”。鞍带石斑鱼个体大、营养丰富、肉质鲜美,生长速度快,是人们喜爱的海水鱼珍品。在我国南方沿海广东、海南、福建、广西等地鞍带石斑鱼已成为海水养殖的主要对象,经济价值巨大。
目前,随着养殖量的不断增加,石斑鱼养殖过程中病虫害问题的日益显现,尤其是病毒性神经坏死病(Viralnervous necrosis,VNN),其病原是神经坏死病毒(nervousnecrosis virus,NNV)。该病毒属于β诺达病毒属,是一类细小RNA病毒,可分成4个基因型:拟鳍神经坏死病毒基因型(Striped jack NNV,SJNNV)、红鳍东方鲀神经坏死病毒基因型(Tiger puffer NNV,TPNNV),条斑星鲽神经坏死病毒基因型(Barfin flounder NNV,BFNNV)和赤点石斑神经坏死病毒基因型(Red spotted grouper NNV,RGNNV)。对石斑鱼危害最大的是RGNNV,石斑鱼在幼苗期基本都会感染此病毒,主要引起石斑鱼神经系统和视网膜损伤,仔鱼和稚鱼易受其感染,感染死亡率在90%以上,甚至达到100%,RGNNV导致石斑鱼幼苗扩大培育困难,这严重制约了石斑鱼养殖产业的发展。目前没有有效的药物能够预防及治疗此病毒病,因此开展抗病品种的培育,是应对该病毒,提高石斑鱼产量的有效方式。
分子标记辅助育种(molecular marker-assisted breeding,MAS)是借助与性状紧密相关的分子标记对具有性状优势的等位基因或基因型的个体进行直接选择育种,是分子生物学和基因组学的研究结果应用到水产养殖品种选育的技术。其中,全基因组关联分析(genome-wide association studies,GWAS)具有标记多,可以全基因组范围仔细筛查与性状相关变异位点的优点,更容易获得决定性状的分子标记和基因。因此,利用全基因组关联分析技术可以快速发现与鱼类重要性状相关的遗传变异,从而应用在分子育种中来缩短育种的周期。
发明内容
本发明的目的在于提供一种鞍带石斑鱼抗病家系的培育方法,该方法通过GWAS技术找到鞍带石斑鱼抗神经坏死病毒的显著关联的分子标记,根据显著相关标记筛选出抗病亲本,再结合家系选育用抗病亲本培育出抗病子代,最终获得鞍带石斑鱼抗神经坏死病毒尤其是抗赤点石斑神经坏死病毒的品系。
本发明的上述目的可以通过以下技术方案来实现:一种鞍带石斑鱼抗病家系的培育方法,包括如下步骤:
(1)亲本的培育:选取鞍带石斑鱼亲本,剪取亲本尾鳍后,对每一条亲鱼打上电子标记;
(2)亲鱼配对及家系构建:根据亲鱼的标记,将能繁殖的雌性亲鱼和雄鱼配对,进行人工催产和一对一的人工授精,进行孵化培育后构建全同胞家系;
(3)攻毒:选取步骤(2)中全同胞家系进行功毒,记录每天死亡鱼的数目,同时剪取刚死亡鱼的尾鳍保存,并设置易感组和抗感组;
(4)抗病关键SNP标记的筛选:选取步骤(3)中抗感组和易感组的鳍条,提取DNA后进行重测序并进行全基因组关联分析(GWAS)分析,获得鞍带石斑鱼抗病显著相关SNP位点54个;
(5)抗病亲本的筛选:选取步骤(1)中剪取的亲本尾鳍,提取DNA,进行重测序,获得每个亲本SNP位点信息,利用包括步骤(4)中的54个SNP位点信息,选取P值靠前的SNP位点,计算每条亲本的抗病性状的育种值,选取育种值前10~15%的个体作为抗病亲本进行抗病繁殖培育家系;
(6)抗病家系的培育:采用步骤(5)中筛选到的抗病亲本进行人工催产和一对一的人工授精,孵化培育后构建出抗病全同胞家系。
在上述鞍带石斑鱼抗病家系的培育方法中:
优选的,步骤(1)中挑选无病害、体格健壮、无外伤、达到5~6年龄的鞍带石斑鱼作为鞍带石斑鱼亲本。
优选的,步骤(1)中所述鞍带石斑鱼亲本中含有占亲本总数5~10%的野生亲本,剩余为养殖培育的亲本,其中所述野生亲本与所述养殖培育的亲本分开不同池养殖。
优选的,步骤(2)中所述全同胞家系中鱼苗培育到长度为4~8cm。
优选的,步骤(3)中选取步骤(2)中500~800尾全同胞家系中的鱼苗进行功毒。
更佳的,步骤(3)中选取步骤(2)800尾全同胞家系中的鱼苗进行功毒。
优选的,步骤(3)中功毒时采用病毒滴度在107~108TCID50/mL的赤点石斑鱼神经坏死病毒,攻毒方式为腹腔注射,功毒完后发现死亡鱼苗立即剪取尾鳍,同时统计死亡条数,如此直到鱼苗不再死亡。
优选的,步骤(3)中选取最先死亡的100~200尾鱼为易感组,选取最后死亡及没有死亡的100~200尾鱼为抗感组。
优选的,步骤(4)中全基因组关联分析(GWAS)分析时,以鞍带石斑鱼基因组为参考基因组结合抗感和易感性状进行全基因组关联分析(GWAS)分析,分析获得鞍带石斑鱼SNP位点1331985个,并获得抗病显著相关SNP位点54个。
其中,参考基因组为鞍带石斑鱼基因组,其来源如下:ASM528154v1https://www.ncbi.nlm.nih.gov/genome/?term=ASM528154v1。
优选的,步骤(4)~(5)中采用提取DNA时,采用常规动物组织提取试剂盒。
优选的,步骤(5)中采用GAPIT方法结合抗病显著相关SNP位点信息计算每条亲本的育种值。
优选的,步骤(5)中育种值的计算采用GAPIT(Genomic Association andPrediction Integrated Tool)方法进行计算,选取的标记中一定包含本发明中的54个显著性标记(鞍带石斑鱼抗病显著相关SNP位点),还包括1331985个SNP位点中抗病显著相关性至少前500的SNP位点,根据计算所得的育种值从大到小进行排序选取育种值排前10~15%的亲本单独分开养殖,作为抗病亲本进行培育。
优选的,步骤(6)中孵化培育时,采用常规的水泥池培苗或者高位池培苗方式。
与现有技术相比,本发明具有以下优点:
(1)本发明通过GWAS技术找到鞍带石斑鱼抗神经坏死病毒的显著关联的分子标记,根据显著相关标记筛选出抗病亲本,再结合家系选育用抗病亲本培育出抗病子代,最终获得鞍带石斑鱼抗神经坏死病毒的品系,培育的家系抗神经坏死病毒的能力显著提高;
(2)本发明方法具有较高的准确性和较大的使用价值,在石斑鱼育种方面具有显著的经济效益和社会效益。
具体实施方式
下面通过实施例对本发明作进一步具体的描述,但本发明的实施方式不限于此。
实施例1
(1)在2020年3月24号选取200条海南晨海水产有限公司养殖的无病害、体格健壮、无外伤、达到5~6年龄,大小为93.15±21.56kg的鞍带石斑鱼亲鱼,剪取尾鳍后,在每条鱼背部肌肉上打上电子标记,记录好每条鱼对应的标记号,其中包括占亲本总数5%的野生亲本个体,且野生亲本与养殖培育的亲本分开不同池养殖;
(2)在6月份繁殖季节选取了能产卵的5尾雌性亲鱼和3尾雄性亲鱼共建立5个一对一的全同胞家系受精卵,放于不同的高位池培育,最终培育出2个全同胞家系鱼苗达到4cm左右;
(3)随机选取其中一个家系鱼苗800尾,采用腹腔注射方式,注射30μL浓度为3.56×107TCID50/mL的赤点石斑鱼神经坏死病毒(分离自赤点石斑鱼,采用常规方法实验室培育获得,培育方法可参考但不限于以下文献:一株鲈鲤源鲤春病毒血症病毒的分离鉴定及其病理观察,郑李平等,中国水产科学,2019.5;或鱼类病毒性神经坏死病毒的分离和部分特性的研究,蒋方军,华中农业大学硕士学位论文,2008),注射完后每隔3小时观察一次,发现死亡鱼苗立马剪取尾鳍保存,同时统计死亡条数,如此直到鱼苗不再死亡;
(4)选取最先死亡的100条鱼为易感组,最后死亡及没有死亡的100条鱼为抗感组,用抗感组和易感组的鱼苗鳍条提取DNA后进行重测序,并以鞍带石斑鱼基因组为参考基因组结合抗感和易感性状进行全基因组关联分析(GWAS)分析,分析获得鞍带石斑鱼SNP位点1331985个,抗病显著相关SNP位点54个(表1),参考基因组为鞍带石斑鱼基因组如下:
ASM528154v1https://www.ncbi.nlm.nih.gov/genome/?term=ASM528154v1;
表1抗神经坏死病毒显著相关SNP位点信息
(5)采用前面步骤(1)中剪取的200条亲本尾鳍,提取DNA,进行重测序,获得每个亲本SNP位点信息,采用GAPIT方法结合抗病显著相关SNP位点信息计算每条亲本的育种值(表2),选取育种值前10%的个体作为抗病亲本进行抗病家系培育。
表2鞍带石斑鱼海南亲本育种值
(6)当年8月份挑选出的20条抗病亲本中3条雌鱼产卵,采用人工受精方式与一条抗病雄性亲本受精,构建了3个抗病全同胞家系受精卵,最终通过高位池培苗方式培育出一个全同胞家系6.97万尾。选取600尾进行功毒,统计死亡率发现死亡率比普通的亲本产生的子代减少16.5%(表3),抗病力显著增强,抗病家系构建成功。
实施例2
步骤与实施例1类似,其区别在于选取亲鱼的地点和时间是2021年6月15日在福建厦门选取25条抗病亲本,培育出5.23万尾抗病子代,随机选600条功毒,统计死亡率后其抗病能力比普通亲本产生的子代抗病能力提高14.5%(表3),抗病力显著增强。
表3鞍带石斑鱼抗病亲鱼子代抗病分析
综上,通过观测样本的结果表明本发明方法具有较高的准确性和较大的使用价值。本发明提供的鞍带石斑鱼抗病家系的培育方法具有高效准确,较大的实用价值。该技术在石斑鱼育种方面将有显著的经济效益和社会效益。
上面列举一部分具体实施例对本发明进行说明,有必要在此指出的是以上具体实施例只用于对本发明作进一步说明,不代表对本发明保护范围的限制。其他人根据本发明做出的一些非本质的修改和调整仍属于本发明的保护范围。
Claims (8)
1.一种鞍带石斑鱼抗病家系的培育方法,其特征是包括如下步骤:
(1)亲本的培育:选取鞍带石斑鱼亲本,剪取亲本尾鳍后,对每一条亲鱼打上电子标记;
(2)亲鱼配对及家系构建:根据亲鱼的标记,将能繁殖的雌性亲鱼和雄鱼配对,进行人工催产和一对一的人工授精,进行孵化培育后构建全同胞家系;
(3)攻毒:选取步骤(2)中全同胞家系进行攻毒,记录每天死亡鱼的数目,同时剪取刚死亡鱼的尾鳍保存,并设置易感组和抗感组;
(4)抗病关键SNP标记的筛选:选取步骤(3)中抗感组和易感组的鳍条,提取DNA后进行重测序并进行全基因组关联分析分析,获得鞍带石斑鱼抗病显著相关SNP位点52个;
(5)抗病亲本的筛选:选取步骤(1)中剪取的亲本尾鳍,提取DNA,进行重测序,获得每个亲本SNP位点信息,利用包括步骤(4)中的52个SNP位点信息,选取P值靠前的SNP位点,计算每条亲本的抗病性状的育种值,选取育种值前10~15%的个体作为抗病亲本进行抗病繁殖培育家系;
(6)抗病家系的培育:采用步骤(5)中筛选到的抗病亲本进行人工催产和一对一的人工授精,孵化培育后构建出抗病全同胞家系;
步骤(4)中获得的52个鞍带石斑鱼抗病显著相关SNP位点如下表1中所示:
表1抗神经坏死病毒显著相关SNP位点信息
;
步骤(3)中攻毒时采用病毒滴度在107~108TCID50/mL以上的赤点石斑鱼神经坏死病毒,攻毒方式为腹腔注射,攻毒完后发现死亡鱼苗立即剪取尾鳍,同时统计死亡条数,如此直到鱼苗不再死亡;
步骤(5)中采用GAPIT方法结合抗病显著相关SNP位点信息计算每条亲本的育种值,其中选取的SNP位点信息包括步骤(4)中的52个SNP位点,还包括1331985个SNP位点中抗病显著相关性至少前500的SNP位点,根据计算所得的育种值从大到小进行排序选取育种值排前10~15%的亲本单独分开养殖,作为抗病亲本进行培育。
2.根据权利要求1所述的鞍带石斑鱼抗病家系的培育方法,其特征是:步骤(1)中挑选无病害、体格健壮、无外伤、达到 5~6年龄的鞍带石斑鱼作为鞍带石斑鱼亲本。
3.根据权利要求1所述的鞍带石斑鱼抗病家系的培育方法,其特征是:步骤(1)中所述鞍带石斑鱼亲本中含有占亲本总数5~10%的野生亲本,剩余为养殖培育的亲本,其中所述野生亲本与所述养殖培育的亲本分开不同池养殖。
4.根据权利要求1所述的鞍带石斑鱼抗病家系的培育方法,其特征是:步骤(2)中所述全同胞家系中鱼苗培育到长度为4~8cm。
5.根据权利要求1所述的鞍带石斑鱼抗病家系的培育方法,其特征是:步骤(3)中选取步骤(2)中500~800尾全同胞家系中的鱼苗进行攻毒。
6.根据权利要求1所述的鞍带石斑鱼抗病家系的培育方法,其特征是:步骤(3)中选取最先死亡的100~200尾鱼为易感组,选取最后死亡及没有死亡的100~200尾鱼为抗感组。
7.根据权利要求1所述的鞍带石斑鱼抗病家系的培育方法,其特征是:步骤(4)中全基因组关联分析分析时,以鞍带石斑鱼基因组为参考基因组结合抗感和易感性状进行全基因组关联分析分析,分析获得鞍带石斑鱼SNP位点1331985个,并获得抗病显著相关SNP位点52个。
8.根据权利要求1所述的鞍带石斑鱼抗病家系的培育方法,其特征是:步骤(6)中孵化培育时,采用常规的水泥池培苗或者高位池培苗方式。
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