PRODUCTION OF YY SUPER-MALE AND XY ALL-MALE PSEUDOBAGRUS USSURIENSIS BY SEX CONTROL TECHNOLOGY
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摘要:
研究探索了17β-雌二醇(17β-estradiol, E2)的不同处理浓度和不同处理时间对乌苏里拟鲿(Pseudobagrus ussuriensis)生长、存活率和性比的影响。结果显示, 在10—70dph期间, 使用10、50和100 mg/kg E2投喂乌苏里拟鲿的雌性率高达100%, 但生长速度和存活率随E2浓度升高而降低, 投喂150 mg/kg E2组乌苏里拟鲿全部死亡。利用10 mg/kg E2投喂诱导乌苏里拟鲿100%雌性化的最佳时间为10—40dph。qRT-PCR结果显示雄性性别分化关键基因dmrt1、amh和cyp17a1在XY生理雌性的表达量显著低于XY雄性, 雌性性别分化关键基因cyp19a1a、zar1和gdf9在XY生理雌性的表达量与XX雌性一致, 显著高于XY雄性。此外, 利用全基因组重测序技术在15尾雌性和15尾雄性乌苏里拟鲿中共筛选到3777645个SNP和1287509个Indel, 其中有99601个性别连锁SNP和27614个性别连锁Indel。性别连锁的SNP主要集中在乌苏里拟鲿8号染色体6.84—23.82 Mb的位置。利用性别连锁的Indel开发了乌苏里拟鲿X和Y染色体特异的分子标记, 适用于黑龙江野生群体、河南和湖北的养殖群体。使用性别分子标记在XY雄性与XY雌性繁殖的子一代中鉴定出了YY超雄鱼, 使用YY超雄鱼为父本繁殖的乌苏里拟鲿雄性率为100%, 1年龄个体的体重是两性养殖群体的1.54倍。综上所述, 在10—40dph期间使用10 mg/kg E2诱导乌苏里拟鲿雌性化的效果最佳, 利用全基因组重测序技术开发的性别连锁分子标记能有效鉴定乌苏里拟鲿的遗传性别获得YY超雄鱼。研究创制的XY全雄乌苏里拟鲿与两性群体相比, 具有规格整齐和生长速度快的特点。
Abstract:In this study, we investigated the effects of various doses and treatment durations of 17β-estradiol (E2) on the growth, survival rate, and sex ratio of P. ussuriensis. From 10 to 70dph (days post-hatching), feeding P. ussuriensis diets containing 10, 50, and 100 mg/kg E2 resulted in a 100% feminization rate. However, the growth and survival rates decreased with increasing E2 concentration, and all fish in the group fed with 150 mg/kg E2 died. Overall, 10 mg/kg E2 treatment from 10 to 40dph was found to be the optimal approach for the feminization of P. ussuriensis. The expression levels of male key sex differentiation-related genes, such as dmrt1, amh, and cyp17a1, were significantly lower in XY females than that of XY males. The expression levels of female key sex differentiation-related genes including cyp19a1a, zar1, and gdf9 in XY females were consistent with those in XX females and were significantly higher than that in XY males. Additionally, whole-genome resequencing revealed 3777645 single nucleotide polymorphisms (SNPs) and 1287509 insertions/deletions (Indels) in 15 female and 15 male P. ussuriensis. Among these, 99601 were identified as sex-linked SNPs and 27614 as sex-linked Indels. The sex-linked SNPs were enriched in the 6.84 and 23.82 Mb regions of chromosome 8. X and Y chromosome specific molecular marker were developed based on sex-linked Indels, which proved effective for the sex identification in P. ussuriensis. These markers were well applied to wild populations from the Heilongjiang River as well as farmed populations in Henan and Hubei Province. YY super-male were identified from the offsprings of XY male mating with XY female. Subsequently, using YY super-male P. ussuriensis as the male parent, an all-male population was bred with a 100% male ratio. The all-male population at one-year old was 1.54 times heavier than the mixed-sex population. In summary, 10 mg/kg E2 treatment from 10 to 40dph is the optimal approach for the feminization of P. ussuriensis. The sex-linked molecular markers developed by whole-genome resequencing can effectively identify the genotype of P. ussuriensis and help produce YY super-male fish. XY all-male P. ussuriensis had the characteristics of uniform size and fast growth performance which will be more popular among farmers.
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水域生态系统的捕食者, 能够通过营养级联效应调节低营养级物种的组成和丰度, 影响水生生物群落的结构和多样性, 对于维持食物网的能量流动和生态系统的稳定性发挥着重要作用。捕食者除通过捕食影响猎物的丰度和分布外, 还可通过间接途径影响其他非猎物类群和生态系统过程[1, 2]。已有越来越多的研究表明, 捕食者不仅可以通过下行效应对生产者有机物质生产过程产生直接作用, 还可调节生态系统环境因素对低营养级生物造成的影响[3, 4], 在水域生态系统中具有重要的生态地位和不可替代的功能作用[5]。在近几十年来, 人类活动的影响已造成了许多捕食者的灭绝, 从而造成了生态系统一系列难以预料的变化, 生态学家对于捕食者的研究更加关注[5, 6]。
湖泊是涵盖多种生境条件的复杂生态系统。湖泊不同区域生境条件往往不同, 环境因子与饵料生物也存在差异, 进而可能导致捕食者群落特性在同一湖泊中的空间异质性[7]。有研究表明, 捕食者由于活动能力强、活动范围大, 在不同生境间食物网能量流动与物质循环中发挥了重要作用[8]。这种不同生境间的耦合对于食物网动力学至关重要[9]。在湖泊生态系统中, 沿岸带生境围绕固体湖岸基质, 一般有大量水生植物及附着藻类群落, 敞水区生境则为悬浮状态, 二者差异较大[10], 但关于二者之间食物网如何紧密联系实现物质与能量的传递与流动的研究尚不多见。不同生境间的耦合是一个重要的生态系统过程, 它通过化学、物理及生物过程的作用促进营养和能量的流动, 进而将不同的生境连接起来[9]。捕食者被认为在生境耦合的生态系统过程中发挥了重要作用[9, 11]。
稳定同位素技术是研究生态系统食物网结构和营养关系及其动态变化的重要手段, 基于稳定同位素质量平衡模型, 其可以用于消费者营养溯源, 以确定多种营养来源对消费者营养的贡献比重[12, 13]。碳稳定同位素比值(δ13C)在捕食者和食物之间的分馏较小(0—1‰), 可用于指示食物来源; 氮稳定同位素比值(δ15N)在捕食者与食物之间存在明显的富集效应 (3‰—4‰), 多用于指示消费者的营养等级[12, 14]。稳定同位素技术与胃肠含物分析等技术手段相比, 其能反映较长时间尺度内捕食者营养来源特征[15, 16]。因此, 可利用碳氮稳定同位素在食物网中的特性来指示湖泊中捕食者的食物来源与营养级, 进而探究捕食者在湖泊不同栖息地物质与能量流动中的作用。
1. 材料与方法
1.1 研究区域概况
保安湖(30°12′—30°18′N, 114°39′—114°46′E)位于湖北省大冶市西北部, 地处长江中游江汉平原东部边缘, 原与三山湖连成一片, 现以北练山、南练山为界, 山以东称为三山湖, 以西称为保安湖, 属梁子湖水系, 为长江中游南岸的一个轻度—中度富营养型湖泊。
1.2 样品采集与测定
达氏鲌(Culter dabryi)与红鳍原鲌(Cultrichthys erythropterus)均属于鲤形目(Cypriniformes), 鲤科(Cyprinidae), 鲌亚科(Culterinae), 达氏鲌为鲌属(Culter), 红鳍原鲌为原鲌属(Culterichthys)。鲌类作为湖泊和水库的高营养级消费者, 对水生生物组分具有重要的调控功能。
本研究样品于2019年6月在保安湖采集, 通过在沿岸带和敞水区设置网具, 获取饵料鱼类样品和高营养级捕食者—鲌类(达氏鲌与红鳍原鲌)样品。在样品采集过程中, 研究人员采集到达氏鲌10条(沿岸带与敞水区各5条), 红鳍原鲌采集到7条(沿岸带5条, 敞水区2条); 饵料鱼类种类主要包括湖鲚(Coilia ectenes taihuensis)、黄颡鱼(Pelteobagrus fulvidraco)、圆尾鲴(Distoechodon tumirostris)、似鱎(Toxabramis swinhonis)、䱗(Hemiculter leucisculus)、似刺鳊(Paracanthobrama guichenoti)和中华鳑鲏(Rhodeus sinensis Günther)等, 体长范围分别为湖鲚(193±38) mm, 黄颡鱼(177±37) mm, 圆尾鲴(125±12) mm, 似鱎(100±9) mm, 䱗(109±27) mm, 似刺鳊(202±7) mm, 中华鳑鲏(43±8) mm。鱼类样品放入干净白瓷盘后, 利用干净的镊子及手术剪取适量背部白肌肉, 去除骨刺与鱼皮, 然后放入冻存管密封冷冻保存, 一尾样品取样完成后即用超纯水清洗取样器材, 避免污染。蚌和螺等大型底栖动物样品通过带网夹泥器获取, 冲洗干净后, 利用干净镊子去壳取肌肉组织, 放入冻存管中密封冷冻保存。其余底栖动物样品使用彼德森采泥器采集, 底栖动物进行种类鉴定后, 分类分别放置于蒸馏水中过夜, 让其排空消化道内残留物。为保证足够的样品分析质量, 将同一物种的底栖动物混合在一起进行分析。浮游动物样品使用13#浮游生物网收集, 并将收集到的浮游动物样品过滤至灼烧过的GF/C滤膜。将以上采集到的样品放入烘箱中60℃烘干至恒重。浮游动物样品使用手术刀片轻轻从滤膜上刮取下来。所有同位素样品均利用珠磨式组织研磨器(MiniBeadbeater-16)研磨粉碎至粉末状, 放入干燥器中干燥保存待测。
利用稳定同位素比质谱仪(CE公司的CarloErba NC 2500元素分析仪与Thermo fisher公司的Delta Plus质谱仪联用)测定保安湖高营养级捕食者（达氏鲌与红鳍原鲌）以及敞水区和沿岸带饵料生物的碳、氮稳定同位素值δ13C和δ15N。分析碳(δ13C)、氮(δ15N)同位素的参照物质分别是VPDB (Pee Dee Belemnite)和空气中N2, 标准物质分别选用国际上通用的IAEA-USGS24和IAEA-USGS26。
1.3 营养富集因子与源矫正
营养富集因子(Trophic enrichment factor, TEF, Δ)由消化与代谢过程中同位素分馏引起, 其定义为Δ=δtissue–δdiet, 其所代表的捕食者组织间与其营养来源间的同位素的差异是构建贝叶斯混合模型的一个重要前提条件[17－19]。TEF的取值受到生物种类、取样组织以及食物来源等多种因素的影响[20], 对于TEF的错误选择和应用会造成模型结果的潜在问题[18, 21]。捕食者稳定同位素数据落入经TEF矫正后营养来源确定的同位素混合空间中, 对于模型得出精确结果十分重要[22]。为使稳定同位素质量平衡混合模型得出准确的结果，应当合理进行TEF的选择和源矫正[18]。
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保安湖高营养级捕食者达氏鲌与红鳍原鲌稳定同位素数据落在4种营养来源确定的同位素混合空间中, 且共线性特征不显著(图 1), 适合对达氏鲌与红鳍原鲌的食物来源组成进行进一步的分析。
1.4 建模数据质量检验
首先, 通过图 1TEF校正后的营养来源稳定同位素混合空间, 可以初步判断达氏鲌与红鳍原鲌稳定性同位素数据是否落入4种营养来源确定的同位素混合空间中。其次, 通过迭代模拟的方法来判别达氏鲌与红鳍原鲌稳定同位素数据落入4种营养来源确定的同位素混合空间中的可能性。迭代模拟即基于TEF校正后的4种营养来源的稳定同位素均值和标准偏差, 迭代生成10000次稳定同位素混合空间, 继而计算达氏鲌与红鳍原鲌稳定同位素数据落入这10000次混合空间中的频次。 达氏鲌与红鳍原鲌稳定同位素数据落在>0.05可能性区域, 可判定数据质量满足建模需要(图 2A—C)。
当捕食者样本的稳定同位素数据过多的存在于由各种营养来源确立的稳定同位素混合空间的质心区域时, 同位素混合空间中营养来源与捕食者的共线性会增加, 导致稳定同位素质量平衡模型求解无法收敛或无法较好的匹配预测值与观测值[24]。因此, 继续通过迭代模拟的方法来判别数据质量。再次基于TEF校正后的4种营养来源的稳定同位素均值和标准偏差, 迭代生成10000次高风险混合同位素空间(以质心为中心的50%不规则多边形面积内)[24, 25], 继而计算达氏鲌与红鳍原鲌稳定同位素数据落入高风险混合空间中的次数, 计算落入高风险稳定同位素混合空间的概率, 来检验数据建模的质量; 达氏鲌与红鳍原鲌稳定同位素数据落在<0.95可能性区域, 可判定数据质量满足要求(图 2D—F)[24, 25]。
图 2A—C显示了保安湖捕食者达氏鲌与红鳍原鲌稳定同位素值的变化将如何影响营养来源混合模型合理求解的概率[25]。达氏鲌与红鳍原鲌的稳定同位素数据值应处于95%概率轮廓内, 本研究中的两种鲌类样品均符合要求。图 2D—F则显示了捕食者达氏鲌与红鳍原鲌同位素值的变化将如何影响营养来源混合模型低估风险的概率[24]。捕食者达氏鲌与红鳍原鲌样品落入风险区的概率总体低于50%, 未出现高于95%概率的样本, 同样满足模型要求。
1.5 模型构建与非度量多维尺度分析(NMDS)
本研究使用R包simmr来拟合所有的同位素贝叶斯混合模型(iter=50000, burn=1000, thin=10, n.chain=4), 使用R包vegan对两种栖息地鲌类食物来源数据进行NMDS分析。
2. 结果
2.1 模型结果
模型结果表明(表 1), 沿岸带和敞水区栖息地采集到的达氏鲌与红鳍原鲌样品营养来源组成基本相似, 均以浮游动物占比最高, 以沿岸带饵料鱼类占比其次。
表 1 不同营养来源贡献比例Table 1. Contribution ratio of different nutrition sources栖息地Habitat 营养来源
SourceMean SD Median CI lower CI upper 沿岸带Littoral 敞水区饵料鱼类
Pelagic prey fishes0.234 0.013 0.234 0.210 0.260 沿岸带饵料鱼类
Littoral prey fishes0.265 0.016 0.265 0.236 0.297 底栖动物
Zoobenthos0.234 0.013 0.234 0.209 0.260 浮游动物
Zooplankton0.266 0.016 0.266 0.236 0.298 敞水区Pelagic 敞水区饵料鱼类
Pelagic prey fishes0.231 0.013 0.231 0.208 0.257 沿岸带饵料鱼类
Littoral prey fishes0.263 0.016 0.263 0.233 0.295 底栖动物
Zoobenthos0.236 0.013 0.236 0.211 0.263 浮游动物
Zooplankton0.269 0.016 0.269 0.238 0.302 2.2 模型整体性能评价
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表 2 模型整体性能评价Table 2. Model performance栖息地类型Habitat DIC pD N DICcor 沿岸带Littoral 84.02 3.83 10 56.13 敞水区Pelagic 61.23 3.83 7 57.39 2.3 两种栖息地鲌类的非度量多维尺度分析
对两种栖息地鲌类食物来源数据进行NMDS分析(图 3), 来自两种栖息地的鲌类食物来源基本一致, 未出现差异。
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图 3 两种栖息地鲌类食物来源NMDS图Figure 3. Food sources NMDS plot of culters in different habitats3. 讨论
在湖泊中, 鱼类水体空间类型包括敞水区与沿岸带等, 鱼类往往占据沿岸带或者敞水区生境二者之一, 并能形成适应于栖息生境条件下的独特形态。然而, 鱼类由于具有高度的流动特性, 并且由于捕食行为在各种生境间来回游动, 因此往往被认为是水体小生境之间物质与能量的“连接者”与“整合者”[31, 32]。敞水区栖息地为湖泊开阔水域, 食物链模式以往主要被认为是浮游植物→浮游动物→浮游食性鱼类→肉食性鱼类[33], 但由于鱼类的运动能力强且具有灵活的捕食策略, 将湖泊不同栖息地紧密联系起来, 其食物组成还应包括沿岸带、底栖等生境中生物与碎屑组分[34]。
某些鱼类群落依靠沿岸带与敞水区两种生境的能量途径, 且两种能量途径对于鱼类群落来说同等重要[35]。保安湖沿岸带与敞水区两种生境中的高营养级捕食者的不同食物来源比重基本相似, 表明保安湖鲌类在两种栖息地中均有摄食行为; 且两种生境中鲌类的食物来源较为广泛, 基本涵盖了沿岸带与敞水区的所有饵料生物, 表明其能够将来源于两种生境中的能量整合到一起。
以往的研究资料表明, 具备灵活运动能力的鱼类或顶级捕食者是水域生态系统中不同生境的“连接者”[36], 能够维持生态系统中食物网的稳定性[11, 37]。在以往的研究资料中, 捕食者(指鱼类)对于不同生境所起到的耦合作用的大小通常基于食物组成进行分析, 高营养级捕食者能够从水体各个生境中均衡地获取食物被认为耦合能力更强[38－40]。本研究运用稳定同位素质量平衡混合模型与非度量多维尺度分析, 对保安湖高营养级捕食者营养来源的分析也支持以上观点。保安湖高营养级捕食者—达氏鲌与红鳍原鲌在运动与捕食过程中, 将来自沿岸带与敞水区两种生境中的食物链的营养与能量整合在一起, 实现了不同湖泊生境间的耦合, 对于维持保安湖生态系统的营养循环、食物网功能的完整性与稳定性发挥了重要作用[34]。
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图 1 染色体 SNP 分布情况
A. 染色体SNP密度分布图; B. 染色体性别连锁SNP密度分布图; C. 性别连锁SNP在染色体上的分布; D. 性别连锁SNP在8号染色体上的分布
Figure 1. Distribution of SNP in chromosome
A. density profile of SNP in chromosome; B. density profile of sex linkage SNP in chromosome; C. distribution of sex linkage SNP in chromosome; D. distribution of sex linkage SNP in chromosome 8
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图 2 性别特异性引物PCR验证图
引物M1(A)和引物M2(B)在河南和湖北养殖群体及黑龙江野生群体乌苏里拟鲿中验证
Figure 2. PCR validation picture of sex linkage primer
Primer M1 (A) and M2 (B) validation in Henan and Hubei Province culture populations, and Heilongjiang River wild population
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图 3 性别特异性引物扩增条带序列
引物M1 (A)和引物M2 (B)扩增产生的特异性条带序列
Figure 3. Sequence of band amplified by sex linkage primer
Sequence of band amplified by primer M1 (A) and M2 (B)
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图版Ⅰ 不同浓度 E2处理后 90dph 乌苏里拟鲿的性腺表型
1. XY基因型的精巢; 2. XY基因型的卵巢; 3. XX基因型的卵巢, 波恩氏液固定后性腺形态学观察; 4. XY基因型的精巢; 5. XY基因型的卵巢; 6. XX基因型的卵巢, HE染色后性腺组织学观察; 7—9为4—6对应的放大图; SPG. 精原细胞; PSP. 初级精母细胞; SSP. 次级精母细胞; PO. 初级卵母细胞; N. 细胞核
图版Ⅰ. Gonadal phenotypes of P. ussuriensis treated with different concentrations of E2 at 90dph
1. Testis of XY genotype; 2. Ovary of XY genotype; 3. Ovary of XX genotype, fixed by bouin’s solution; 4. Testis of XY genotype; 5. Ovary of XY genotype; 6. Ovary of XX genotype, stained by hematoxylin-eosin; 7—9. the corresponding magnified picture of 4—6; SPG. spermatogonia; PSP. primary spermatocytes; SSP. secondary spermatocytes; PO. primary oocyte, N. nucleus
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图 4 性别分化关键基因在性腺的表达分析
雄性性别分化关键基因dmrt1 (A)、amh (B)和cyp17a1 (C)在XY精巢、XY卵巢和XX卵巢组织中的表达; 雌性性别分化关键基因cyp19a1a (D)、zar1 (E)和gdf9 (F)在XY精巢、XY卵巢和XX卵巢组织中的表达; ns表示单因素方差分析显示组间没有显著性差异, *表示有显著性差异(P<0.05), **表示有显著性差异(P<0.01)
Figure 4. Relative expression of key sex differentiation-related genes
Relative expression of male differentiation genes dmrt1 (A), amh (B), and cyp17a1 (C) in XY testis, XY ovary, and XX ovary; Relative expression of female differentiation genes cyp19a1a (D), zar1 (E), and gdf9 (F) in XY testis, XY ovary, and XX ovary; ns means no significant differences in each tissue, *means significant differences (P<0.05) and ** means significant differences (P<0.01) by One-way ANOVA
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图 5 1年龄乌苏里拟鲿全雄群体与两性群体生长对比
引物M2鉴定XY雄性与XY生理雌性繁殖获得的子代基因型(A)，1、2、4、6、7、12、14、16、17为XY雄鱼，8、9、10、15为XX雌鱼，3、5、11、13为YY超雄鱼；1年龄乌苏里拟鲿全雄群体与两性群体的雄性比例(B)、体长(C)和体重(D)对比分析
Figure 5. Comparative analysis of growth performance in one-year-old P. ussuriensis between all-male and mixed-sex population
Genotype identification of offspring mating by XY male and XY female with primer M2 (A), 1, 2, 4, 6, 7, 12, 14, 16, 17 are XY males, 8, 9, 10, 15 are XX females, and 3, 5, 11, 13 are YY supermales; Comparative analysis of male ratio (B), body length (C) and body weight (D) in one-year-old P. ussuriensis between all-male and mixed-sex population
表 1 乌苏里拟鲿基因型鉴定引物和定量PCR引物
Table 1 Primer of genotype identification and qRT-PCR in P. ussuriensis
引物名称
Primer name引物序列
Sequence (5′—3′)扩增大小
Amplicon size（bp）目的
PurposeM1-F TCCCTCCAAGATTACGC Y和X特异片段分别为349和424 bp 基因型鉴定 M1-R CTCGCAGGCAGACAGAA M2-F ACCATCTGCTGAAACCC Y和X特异片段分别为212和308 bp 基因型鉴定 M2-R CAGGACCAAATCAAATAAG dmrt1-F AACCACGGCTTCGTCTCG 217 qRT-PCR dmrt1-R CAGGCTCATTCTTCACCACA amh-F TTGCTTCTGCCACTAACG 285 qRT-PCR amh-R TTCGGCTCACCGTCCTTA cyp17a1-F GAGTTGAGCCTTACACCC 269 qRT-PCR cyp17a1-R CAGACTGGTCCTGTCACTTA zar1-F TGTGAAGGAAGGACCGAAGA 281 qRT-PCR zar1-R CTCCCAGCGAAGGTTGCA cyp19a1a-F AACATCACGCTGTGGAAG 199 qRT-PCR cyp19a1a-R GAACAGACGGTTGGAAAT gdf9-F TAGACCCGATTCCAGATA 255 qRT-PCR gdf9-R AAGTGATACCGCGTAGTT β-actin-F ATTGCCGCACTGGTTGTT 270 qRT-PCR β-actin-R CAGCTCGTTGTAGAAGGTATGA 
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表 2 全基因组重测序数据统计及与参考基因组比对分析
Table 2 Statistics on genome re-sequencing data and results of mapping to reference genome of P. ussuriensis
样品名
Sample滤后数据
Clean reads (Gb)Q20 (%) GC (%) 比对片段
Mapped reads比对率
Mapping rate (%)测序深度
Depth (×)覆盖率
Coverage (%)雌1 14.50 98.15 39.23 96749092 98.48 19.81 95.94 雌2 11.57 98.19 39.17 76718966 98.31 15.86 95.79 雌3 13.86 98.32 39.35 91846572 98.33 18.88 95.96 雌4 13.14 98.40 39.24 87469694 98.50 17.98 95.94 雌5 14.29 98.35 39.39 94780100 98.38 19.45 96.12 雌6 16.50 98.45 39.56 111238674 98.66 22.43 96.23 雌7 14.22 98.28 39.21 94303612 98.40 19.48 95.75 雌8 14.99 98.25 39.40 99265184 98.36 20.40 96.26 雌9 16.40 97.89 39.27 109939318 98.61 22.32 96.11 雌10 15.48 98.26 39.32 103286516 98.45 21.15 96.09 雌11 14.51 98.16 39.40 96758464 98.45 19.77 96.24 雌12 15.55 97.89 39.34 103774566 98.53 21.19 96.02 雌13 13.80 98.18 39.29 92052298 98.46 18.93 95.76 雌14 16.34 98.51 39.88 111871132 98.73 22.25 96.22 雌15 15.31 97.82 39.24 102373812 98.58 20.89 96.10 雄1 14.72 97.90 39.25 98008536 98.33 20.11 96.04 雄2 12.86 97.53 39.33 85642362 98.40 17.70 95.63 雄3 14.50 98.08 39.44 96807852 98.34 19.78 96.03 雄4 16.03 98.41 39.19 107289926 98.58 21.89 95.98 雄5 14.39 98.20 39.31 95864434 98.41 19.67 95.98 雄6 16.13 97.51 39.31 107485372 98.49 22.09 95.77 雄7 13.37 98.25 39.41 88689152 98.38 18.26 95.98 雄8 15.14 97.99 39.26 100476860 98.33 20.70 95.96 雄9 14.72 98.16 39.28 98048248 98.33 20.07 96.11 雄10 11.52 94.47 39.17 77728982 98.80 15.95 95.15 雄11 14.58 97.58 39.18 97830558 98.53 19.95 95.85 雄12 15.19 98.31 39.27 101311888 98.44 20.75 96.13 雄13 13.13 98.15 39.27 87638776 98.42 17.98 95.87 雄14 14.43 97.97 39.40 96155192 98.37 19.68 96.04 雄15 9.16 94.62 39.50 61381790 98.60 12.75 94.93 均值Average 14.34 97.87 39.33 95759597 98.47 19.60 95.93
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表 3 SNP和Indel统计分析
Table 3 Statistics on SNP and Indel
样品名
SampleSNP总数
Number of
SNP性别连锁SNP
Number of
sex linkage
SNPIndel总数
Number of
Indel性别连锁Indel
Number of sex
linkage Indel雌1 1740567 170 565254 56 雌2 1740223 761 557201 194 雌3 1687628 157 550540 68 雌4 1748462 62 568832 30 雌5 1717626 349 560197 97 雌6 1758374 45 579774 26 雌7 1687038 488 549668 140 雌8 1749949 92 579301 33 雌9 1756990 14 574583 9 雌10 1687241 156 553102 67 雌11 1682511 605 552340 130 雌12 1752807 40 571522 23 雌13 1746836 275 563831 74 雌14 1725321 159 552191 60 雌15 1739789 368 568588 108 雄1 1883954 99233 609847 27476 雄2 1773911 98768 566891 27317 雄3 1888591 99052 608598 27371 雄4 1891707 99323 612275 27494 雄5 1900439 99098 611746 27424 雄6 1894298 99310 610198 27484 雄7 1864146 98944 601139 27317 雄8 1808798 99250 584040 27478 雄9 1863312 99233 602056 27459 雄10 1673463 97382 519145 26630 雄11 1833216 99183 585992 27437 雄12 1830696 99236 595590 27471 雄13 1889415 98928 601542 27303 雄14 1815201 99130 585570 27457 雄15 1589405 94634 485372 25674 过滤后Filter 3777645 99601 1287509 27614
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表 4 不同浓度梯度E2投喂条件下的90dph乌苏里拟鲿生长、存活率和性逆转比例统计
Table 4 Statistics on growth, survival rate, and sex reversal ratio of P. ussuriensis treated with different concentrations of E2 at 90dph
雌二醇浓度
E2 concentration (mg/kg)体长
Body length (cm)体重
Body weight (g)存活率
Survival rate (%)卵巢比例
Ovarian ratio (%)性逆转率
Sex reversal ratio (%)对照组 7.79±1.20a 7.19±1.86a 90.00±2.50a 54.45±3.85b 0.00±0.00b 10 6.48±0.93b 4.43±1.06b 89.08±3.26a 100±0.00a 100±0.00a 50 5.81±0.96c 3.62±0.99c 41.42±3.50b 100±0.00a 100±0.00a 100 5.13±0.74d 3.07±0.63d 21.17±2.50c 100±0.00a 100±0.00a 150 — — — — — 注: 对不同浓度E2的数据显著性差异用“a, b, c, d”表示, 同列数据不同字母上标表示有显著差异(P<0.05), 卵巢比例(%)=(卵巢数量/解剖性腺总数量)×100, 性逆转率(%)=(XY卵巢数量/解剖XY性腺总数量)×100; 下同Note: The significant differences in data for different concentrations of E2 are represented in “a, b, c d”. In the same line, different superscript letters indicate significant differences (P<0.05). Ovarian ratio (%)=(number of ovaries / total number of dissected gonads)×100, sex reversal ratio (%)=(number of XY ovaries/total number of dissected XY gonads)×100; the same applies below
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表 5 10 mg/kg E2投喂不同时间组90dph乌苏里拟鲿生长、存活率和性逆转比例统计
Table 5 Statistics on growth, survival rate, and sex reversal ratio of P. ussuriensis treated with different sustained times of 10 mg/kg E2 at 90dph
持续时间
Sustained time (dph)体长
Body length (cm)体重
Body weight (g)存活率
Survival rate (%)卵巢比例
Ovarian ratio (%)性逆转率
Sex reversal ratio (%)对照组 7.69±1.09a 7.18±1.76a 88.75±1.25a 48.89±3.85c 0.00±0.00d 10—20 7.30±0.80b 6.46±1.23b 89.58±5.64a 64.44±5.09b 34.78±1.27c 10—30 7.26±1.10b 6.36±1.30b 88.75±2.50a 95.56±1.93a 92.33±3.60b 10—40 7.12±0.96b 5.46±1.54c 89.17±1.91a 100.00±0.00a 100.00±0.00a 10—50 6.44±0.75c 4.55±1.13d 88.75±5.00a 100.00±0.00a 100.00±0.00a 10—60 6.44±0.76c 4.94±1.07d 88.33±3.82a 100.00±0.00a 100.00±0.00a
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