大黄鱼异源精子诱导雌核发育及性别特异性AFLP标记筛选
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摘要
大黄鱼（Pseudosciaena crocea）是我国人工育苗和养殖规模最大的海水鱼类，但目前养殖大黄鱼已经出现了种质资源衰退和遗传多样性降低，有必要进行种质改良，培育优良品种。雌核发育是一种特殊的有性生殖方式，其子代遗传物质全部来自母本。通过诱导雌核发育，不但可以快速建立纯系、固定优良性状，还可以为探讨性别决定机制、繁育单性群体奠定基础。已有研究显示使用同源灭活精子诱导的大黄鱼雌核发育中可能会因灭活不完全导致部分子代中掺有父本遗传物质。为避免此现象，本研究以不经灭活的鮸鱼精子和紫外灭活的黄姑鱼精子作为异源激活源，通过冷休克处理诱导染色体组加倍，成功诱导了大黄鱼雌核发育二倍体（可调式精子灭活装置和温度休克处理装置均为自行研制，均已获得专利）。通过对冷休克处理起始时刻、温度和持续时间的探索，得到异源精子诱导大黄鱼雌核发育的最佳冷休克处理条件为授精后2-3min将受精卵置于3-4°C过滤海水中处理10-13min。在此冷休克处理条件下，使用不经灭活的鮸鱼精子和紫外灭活的黄姑鱼精子作为激活源均可得到大黄鱼雌核发育子代，SSR标记扩增分析结果显示两种异源精子诱导得到的大黄鱼雌核发育子代中均只出现母本条带而未出现父本条带；形态观察和染色体分析显示40-50日龄幼鱼外部形态与普通大黄鱼幼鱼相同，且均为含有48条染色体的二倍体，表明子代全部为雌核发育二倍体。
对以紫外灭活的同源（大黄鱼）精子和未灭活的异源（鮸鱼）精子诱导的雌核发育大黄鱼进行了胚胎发育时程和SSR标记分析的比较，结果显示：（1）异源组的受精率和孵化率分别为41.33±0.58%和36.67±0.58%，分别高于同源组的20.67±1.52%和24.67±0.58%；但异源组的存活率为3.28±0.09%，低于同源组的6.93±0.15%；（2）两组中经冷休克处理未能恢复倍性的胚胎发育畸形而陆续死亡，恢复倍性的胚胎在发育程序上均与普通大黄鱼相同，但由于冷休克处理导致了发育的延迟，各阶段出现时间较对照组滞后：同源组和异源组在胚盘形成时分别滞后3min和2min，至低囊胚期分别滞后35min和45min，至原肠晚期分别滞后55min和1h，至心跳期分别滞后50min和1h45min，至出膜时分别滞后1h和1h30min；（3）两对SSR引物（KPC9和KPC45）对同源组的扩增分析显24尾子代中有20尾在两个位点均未出现父本条带，为雌核发育个代，占83.3%；3尾在两个位点均出现父本条带，为正常受精个体，占12.5%；1尾仅在KPC9位点出现父本条带，可能是灭活不完全精子中的部分DNA片段进入了子代，占4.17%；SSR引物KPC43对异源组的扩增分析显示18尾子代中均未出现父本条带，全部是雌核发育产物。结果表明以灭活的同源（大黄鱼）精子和未经灭活的异源（鮸鱼）精子诱导大黄鱼雌核发育各有优缺点，需根据具体情况选择使用。
为更好的对鮸鱼精子诱导的雌核发育大黄鱼进行检测分析，同时也为今后鮸鱼这一经济鱼类的遗传多样性分析、种质资源保护以及石首鱼科的种系进化研究奠定基础，使用磁珠富集法构建了鮸鱼的SSR文库，挑选247个阳性克隆测序后获得138个含有SSR核心单元的独特序列，对其中35个序列设计了37对SSR引物，在象山港鮸鱼养殖群体进行扩增、分析，结果显示有30对引物可扩增到清晰、特异的目的条带，其中24个位点呈多态性，6个位点呈单态性。24个多态性引物的等位基因数为2-8个，平均每个位点4.33个；观察杂合度（Ho）为0.0667-1.0000，平均0.6361；期望杂合度（He）为0.3828-0.8139，平均0.6443；PIC值范围为0.348-0.823，平均0.576，其中17个呈高度多态性（PIC>0.5）；8个位点偏离哈代平衡，可能与群体内小范围的非随机交配和人工育种有关，某些位点杂合度很低，也可能与采样群体较小有关。
将筛选得到的30对鮸鱼SSR引物在象山港养殖大黄鱼群体中进行交叉扩增，结果显示有12对引物可稳定扩增且条带清晰，交叉扩增的成功率为40.00%，其中10对呈多态性，2对为单态性。10个多态性引物的等位基因数为2-8个，平均每个位点5.10个；观察杂合度（Ho）为0.0000-0.8667，平均0.5200；期望杂合度（He）为0.0655-0.8492，平均0.6053。10个位点的PIC值范围为0.062-0.815，平均0.555，其中6个呈高度多态性（PIC>0.5）；4个位点偏离哈代平衡。对大黄鱼和鮸鱼中相同引物的扩增序列进行比对，结果显示除MM007位点的相似度较低外（51.08%），其他位点相似度均较高（89.63%-99.36%）；除核心单元的重复次数不同外，微卫星侧翼序列在鮸鱼和大黄鱼间也存在差异，表现为个别碱基的转换/颠换或一段序列的插入/缺失。用4对交叉扩增成功的引物对鮸鱼精子诱导的雌核发育大黄鱼进行扩增分析，结果显示4个引物都可以在父本和母本间扩增出清晰的差异性条带，子代在4个微卫星位点均只出现母本条带而没有父本条带，表明子代是真正的雌核发育产物，同时多个位点的检测结果也证明精子中的遗传物质既没有以染色体形式也没有以DNA片段形式进入子代。
现有的核型分析显示大黄鱼没有异型分化的性染色体，目前尚无法鉴定大黄鱼的遗传性别。使用AFLP技术对雌、雄大黄鱼进行条带对比分析，结果显示64对选择性扩增引物组合中除E-ACC/M-CAT外均可得到清晰的条带，但仅引物组合E-ACC/M-CTG扩增到一条在雌、雄大黄鱼间显著差异的条带，表明大黄鱼雌性和雄性的基因组差异极小。将引物组合E-ACC/M-CTG扩增到的仅在雄鱼中出现差异条带进行回收测序，来自4个雄性个体的差异条带测序成功，结果显示虽然长度都是188bp，但为两段不同的序列，经BLAST搜索，两条序列在GenBank中均无同源序列。对两条序列分别设计引物进行PCR扩增检验，结果显示两对引物在雌性和雄性个体中都可扩增到条带，推测此AFLP条带可能是由DNA序列修饰（如甲基化等）和酶切识别等因素共同作用产生的性别差异性条带。
综上所述，本研究建立了异源（鮸鱼、黄姑鱼）精子诱导大黄鱼雌核发育的技术体系，除幼鱼外部形态观察和染色体分析外，还使用已发布的大黄鱼、黄姑鱼SSR引物对大黄鱼雌核发育子代进行了鉴定和比较分析，从个体、细胞和分子遗传等多个水平上确认了雌核发育的真实性，并对同源和异源精子诱导的雌核发育大黄鱼进行了胚胎发育时程的观察和比较；通过磁珠富集法筛选了30对鮸鱼SSR引物，并将这些引物在大黄鱼中进行交叉扩增，对交叉扩增成功的12对引物进行了鮸鱼、大黄鱼间的扩增序列比较并应用于鮸鱼精子诱导大黄鱼雌核发育子代的鉴定；使用AFLP技术对雌、雄大黄鱼基因组进行了扩增条带的比较分析，找到一条仅在雄鱼中出现的差异条带。这些结果为大黄鱼雌核发育、群体遗传结构分析、性别决定与性别分化、石首鱼科种系进化等理论研究提供资料，也为大黄鱼遗传育种、种质保护以及单性群体繁育等应用性研究奠定了基础，但雌核发育大黄鱼的生长发育特别是性腺发育情况以及遗传性别的鉴定等问题仍有待于更加深入的研究。
Large yellow croaker Pseudosciaena crocea is the largest artificial breeding and culture species ofmarine fish in China. In recent years, reared P. crocea have begun to degrade in germplasm and loss ofgenetic diversity. It is needed to improve the germplasm and breed superior variety. Gynogenesis is aspecial type of sexual reproduction whereby the chromosomes of offspring are exclusively inherited fromthe mother. It is a valuable tool both in genetic research and aquaculture, e.g., producing monosexpopulation and pure lines, revealing sex-determing mechanisms, constructing genetic maps. Althoughgynogenetic diploids of P. crocea have been induced by UV-irradiated homologous sperm, some researcheshave reported that paternal genetic material was revealed into some offspring, which may due to theinadequacy of UV-irradiation. To ensure that any surviving offspring are truly gynogenetic, using suitableheterologous sperm is an alternative. In this study, gynogenesis was induced in P. crocea using untreatedMiichthys miiuy sperm or UV-irradiated Nibea albiflora sperm, and cold-shock was performed to diploidizethe female chromosome. By altering the initial time, temperature and duration, cold shock at3-4℃for10-13min which started in2-3min after fertilization was found as the optimum protocol for retaining thesecond polar body and producing meiotic diploids P. crocea with heterologous sperm. Under the abovecondition, the morphologic of gynogenetic P. crocea induced by unirradiated M. miiuy and UV-irradiated N.albiflora sperm were all similar compared to normal P. crocea, and cytogenetic analysis showed all thegynogenetic larveas were diploid with~48chromosomes. The results of microsatellite DNA analysisshowed that gynogenetic progeny were exclusive maternal inheritance.
The embryonic development and SSR patterns were compared between the two groups of gynogenetic P.crocea which were induced by UV-irradiated homologous sperm and untreated heterologous (M. miiuy)sperm, respectively. The fertilization and hatching rate of heter-gynogenesis was41.33±0.58%and36.67±0.58%, respectively, and both higher than the rate of homo-gynogenesis, which was20.67±1.52%and24.67±0.58%, respectively. But the survival rate was opposite, which was3.28±0.09%forheter-gynogenesis and6.93±0.15%for homo-gynogenesis. In the two gynogenesis groups, the embryoswhich were failed to inhibit the extrusion of the polar body show obvious haploid syndrome; the embryoswhich were successful diploidized had the same process with the normal diploid embryos. However, thedeveloping speed of gynogenetic diploid was slower than control group: the homo-and heter-gynogenesisembryos delayed3min and2min at blastodisc stage,35min and45min at low blastula stage,55min and1h at late gastrula stage,50min and1h45min at heartbeat stage, and1h and1h30min at hatching stage,respectively. The delay may be due to the disturbance of cell division cycle caused by cold shock. Inhomo-gynogenesis, the analysis of SSR primes KPC9and KPC45showed in24tested offsprings,83.3%(20/24) had maternal bands only;12.5%(3/24) individuals had paternal bands in the two loci, suggestingthey derived from normal fertilization; and1individual showed paternal band in locus KPC9but absent inlocus KPC45, suggesting there were genetic leakage of the paternal fish. In heter-gynogenesis, the analysisof SSR prime KPC43showed18tested offsping all have maternal specific band only. The results showedheterologous sperm can avoid paternal gene inflowing to offspring. In gynogenesis induction, the selectionof homo-or heterologous sperm should accord to their relative merits.
In order to analyze gynogenetic P. crocea induced by sperm of M. miiuy, as well as manage and protect M.miiuy and study the phylogeny in Sciaenidae, SSR markers were isolated for M. miiuy using genomiclibrary enriched by magnetic beads, and subsequently these markers were cross-amplified in P. crocea andused to analyze the gynogenetic P. crocea induced by sperm of M. miiuy. In total,247positive clones weresequenced and got138unique sequences that contained microsatellite motifs. Thirty-seven primer pairswere designed for35sequences and amplified in30M. miiuy individuals. Thirty primer pairs wereeffective, and within the tested population, twenty-four were polymorphic with an average of4.33allelesper locus (range from2to8). The observed (Ho) and expected (He) heterozygosity ranged from0.0667to 1.0000(mean0.6361) and0.3828to0.8139(mean0.6443), respectively. The mean PIC value was0.576,ranging from0.348to0.823. Seventeen loci showed high polymorphism (PIC>0.5). Eight loci weresignificant deviations from HWE, which may be due to the small sample size. Twelve out of the30primerpairs showed amplification in the different genus fish P. crocea and ten showed polymorphic with anaverage of5.10alleles per locus (range from2to8). The observed (Ho) and expected (He) heterozygosityranged from0.0000to0.8667(mean0.5200) and0.0655to0.8492(mean0.6053), respectively. The meanPIC value was0.555, ranging from0.062to0.815. Six loci showed high polymorphism (PIC>0.5). Fourloci were significant deviations from HWE. Except MM007(the homologous was51.08%between the twospecies), the sequence alignment of microsatellite flanking regions of other11loci showed highlyhomologous (range from89.63%to99.36%) between the two species. The gynogenetic P. crocea inducedby sperm of M. miiuy were analyzed through four cross-amplified primer pairs, and no specific paternalbands were found in the offspring. The results of four SSR markers indicate the genetic material of M.miiuy sperm did not revealed to the gynogenetic P. crocea as neither chromosome nor DNA fragment.The karyotype showed there was no identifiable sex chromosome in P. crocea. The inability to determinethe genotypic sex of P. crocea is a problem in aquaculture. In this study, AFLP technique was used toanalyse the bands between female and male P. crocea. Sixty-three out of64primer combinations couldamplify clear bands, but only the primer combination E-ACC/M-CTG got a band wihch was present in allmales and absent in all females. The result indicates there were only a few genomic differences betweenfemale and male P. crocea. Alignment showed this male-specific candidate band was two differentnucleotide sequences which were both188bp, and BLAST showed there was no homologous sequence inGenBank. Two PCR primers were designed, but amplification showed bands were appeared both in femaleand male P. crocea. We speculate that this sex-difference AFLP band came from the interaction ofrestriction enzyme recognition sites, DNA sequence modification (e.g, methylation) and other factors.
In conclusion, this study established a technique that inducing gyonogenetic P. crocea by heterologoussperm (M. miiuy and N. albiflora) and using cold-shock to diploidize the female chromosome, verifiedgynogenesis through morphology, cytogenetic and SSR markers analysis, compared the embryonicdevelopment and SSR patterns between two group of gynogenetic P. crocea which were induced byhomologous sperm and heterologous (M. miiuy) sperm, isolated and characterized SSR markers for M.miiuy, verified the gynogenetic P. crocea using M. miiuy SSR markers which were cross-amplifed in P.crocea besides P. crocea SSR markers, screened a male-specific ALFP band. The result of this study wasuseful both for theoretical research and application research. Nevertheless, the growth, development(especially gonadal development) of gynogenetic offspring and identification of genotypic sex in P. croceaare needed to be further investigated.
对以紫外灭活的同源（大黄鱼）精子和未灭活的异源（鮸鱼）精子诱导的雌核发育大黄鱼进行了胚胎发育时程和SSR标记分析的比较，结果显示：（1）异源组的受精率和孵化率分别为41.33±0.58%和36.67±0.58%，分别高于同源组的20.67±1.52%和24.67±0.58%；但异源组的存活率为3.28±0.09%，低于同源组的6.93±0.15%；（2）两组中经冷休克处理未能恢复倍性的胚胎发育畸形而陆续死亡，恢复倍性的胚胎在发育程序上均与普通大黄鱼相同，但由于冷休克处理导致了发育的延迟，各阶段出现时间较对照组滞后：同源组和异源组在胚盘形成时分别滞后3min和2min，至低囊胚期分别滞后35min和45min，至原肠晚期分别滞后55min和1h，至心跳期分别滞后50min和1h45min，至出膜时分别滞后1h和1h30min；（3）两对SSR引物（KPC9和KPC45）对同源组的扩增分析显24尾子代中有20尾在两个位点均未出现父本条带，为雌核发育个代，占83.3%；3尾在两个位点均出现父本条带，为正常受精个体，占12.5%；1尾仅在KPC9位点出现父本条带，可能是灭活不完全精子中的部分DNA片段进入了子代，占4.17%；SSR引物KPC43对异源组的扩增分析显示18尾子代中均未出现父本条带，全部是雌核发育产物。结果表明以灭活的同源（大黄鱼）精子和未经灭活的异源（鮸鱼）精子诱导大黄鱼雌核发育各有优缺点，需根据具体情况选择使用。
为更好的对鮸鱼精子诱导的雌核发育大黄鱼进行检测分析，同时也为今后鮸鱼这一经济鱼类的遗传多样性分析、种质资源保护以及石首鱼科的种系进化研究奠定基础，使用磁珠富集法构建了鮸鱼的SSR文库，挑选247个阳性克隆测序后获得138个含有SSR核心单元的独特序列，对其中35个序列设计了37对SSR引物，在象山港鮸鱼养殖群体进行扩增、分析，结果显示有30对引物可扩增到清晰、特异的目的条带，其中24个位点呈多态性，6个位点呈单态性。24个多态性引物的等位基因数为2-8个，平均每个位点4.33个；观察杂合度（Ho）为0.0667-1.0000，平均0.6361；期望杂合度（He）为0.3828-0.8139，平均0.6443；PIC值范围为0.348-0.823，平均0.576，其中17个呈高度多态性（PIC>0.5）；8个位点偏离哈代平衡，可能与群体内小范围的非随机交配和人工育种有关，某些位点杂合度很低，也可能与采样群体较小有关。
将筛选得到的30对鮸鱼SSR引物在象山港养殖大黄鱼群体中进行交叉扩增，结果显示有12对引物可稳定扩增且条带清晰，交叉扩增的成功率为40.00%，其中10对呈多态性，2对为单态性。10个多态性引物的等位基因数为2-8个，平均每个位点5.10个；观察杂合度（Ho）为0.0000-0.8667，平均0.5200；期望杂合度（He）为0.0655-0.8492，平均0.6053。10个位点的PIC值范围为0.062-0.815，平均0.555，其中6个呈高度多态性（PIC>0.5）；4个位点偏离哈代平衡。对大黄鱼和鮸鱼中相同引物的扩增序列进行比对，结果显示除MM007位点的相似度较低外（51.08%），其他位点相似度均较高（89.63%-99.36%）；除核心单元的重复次数不同外，微卫星侧翼序列在鮸鱼和大黄鱼间也存在差异，表现为个别碱基的转换/颠换或一段序列的插入/缺失。用4对交叉扩增成功的引物对鮸鱼精子诱导的雌核发育大黄鱼进行扩增分析，结果显示4个引物都可以在父本和母本间扩增出清晰的差异性条带，子代在4个微卫星位点均只出现母本条带而没有父本条带，表明子代是真正的雌核发育产物，同时多个位点的检测结果也证明精子中的遗传物质既没有以染色体形式也没有以DNA片段形式进入子代。
现有的核型分析显示大黄鱼没有异型分化的性染色体，目前尚无法鉴定大黄鱼的遗传性别。使用AFLP技术对雌、雄大黄鱼进行条带对比分析，结果显示64对选择性扩增引物组合中除E-ACC/M-CAT外均可得到清晰的条带，但仅引物组合E-ACC/M-CTG扩增到一条在雌、雄大黄鱼间显著差异的条带，表明大黄鱼雌性和雄性的基因组差异极小。将引物组合E-ACC/M-CTG扩增到的仅在雄鱼中出现差异条带进行回收测序，来自4个雄性个体的差异条带测序成功，结果显示虽然长度都是188bp，但为两段不同的序列，经BLAST搜索，两条序列在GenBank中均无同源序列。对两条序列分别设计引物进行PCR扩增检验，结果显示两对引物在雌性和雄性个体中都可扩增到条带，推测此AFLP条带可能是由DNA序列修饰（如甲基化等）和酶切识别等因素共同作用产生的性别差异性条带。
综上所述，本研究建立了异源（鮸鱼、黄姑鱼）精子诱导大黄鱼雌核发育的技术体系，除幼鱼外部形态观察和染色体分析外，还使用已发布的大黄鱼、黄姑鱼SSR引物对大黄鱼雌核发育子代进行了鉴定和比较分析，从个体、细胞和分子遗传等多个水平上确认了雌核发育的真实性，并对同源和异源精子诱导的雌核发育大黄鱼进行了胚胎发育时程的观察和比较；通过磁珠富集法筛选了30对鮸鱼SSR引物，并将这些引物在大黄鱼中进行交叉扩增，对交叉扩增成功的12对引物进行了鮸鱼、大黄鱼间的扩增序列比较并应用于鮸鱼精子诱导大黄鱼雌核发育子代的鉴定；使用AFLP技术对雌、雄大黄鱼基因组进行了扩增条带的比较分析，找到一条仅在雄鱼中出现的差异条带。这些结果为大黄鱼雌核发育、群体遗传结构分析、性别决定与性别分化、石首鱼科种系进化等理论研究提供资料，也为大黄鱼遗传育种、种质保护以及单性群体繁育等应用性研究奠定了基础，但雌核发育大黄鱼的生长发育特别是性腺发育情况以及遗传性别的鉴定等问题仍有待于更加深入的研究。
Large yellow croaker Pseudosciaena crocea is the largest artificial breeding and culture species ofmarine fish in China. In recent years, reared P. crocea have begun to degrade in germplasm and loss ofgenetic diversity. It is needed to improve the germplasm and breed superior variety. Gynogenesis is aspecial type of sexual reproduction whereby the chromosomes of offspring are exclusively inherited fromthe mother. It is a valuable tool both in genetic research and aquaculture, e.g., producing monosexpopulation and pure lines, revealing sex-determing mechanisms, constructing genetic maps. Althoughgynogenetic diploids of P. crocea have been induced by UV-irradiated homologous sperm, some researcheshave reported that paternal genetic material was revealed into some offspring, which may due to theinadequacy of UV-irradiation. To ensure that any surviving offspring are truly gynogenetic, using suitableheterologous sperm is an alternative. In this study, gynogenesis was induced in P. crocea using untreatedMiichthys miiuy sperm or UV-irradiated Nibea albiflora sperm, and cold-shock was performed to diploidizethe female chromosome. By altering the initial time, temperature and duration, cold shock at3-4℃for10-13min which started in2-3min after fertilization was found as the optimum protocol for retaining thesecond polar body and producing meiotic diploids P. crocea with heterologous sperm. Under the abovecondition, the morphologic of gynogenetic P. crocea induced by unirradiated M. miiuy and UV-irradiated N.albiflora sperm were all similar compared to normal P. crocea, and cytogenetic analysis showed all thegynogenetic larveas were diploid with~48chromosomes. The results of microsatellite DNA analysisshowed that gynogenetic progeny were exclusive maternal inheritance.
The embryonic development and SSR patterns were compared between the two groups of gynogenetic P.crocea which were induced by UV-irradiated homologous sperm and untreated heterologous (M. miiuy)sperm, respectively. The fertilization and hatching rate of heter-gynogenesis was41.33±0.58%and36.67±0.58%, respectively, and both higher than the rate of homo-gynogenesis, which was20.67±1.52%and24.67±0.58%, respectively. But the survival rate was opposite, which was3.28±0.09%forheter-gynogenesis and6.93±0.15%for homo-gynogenesis. In the two gynogenesis groups, the embryoswhich were failed to inhibit the extrusion of the polar body show obvious haploid syndrome; the embryoswhich were successful diploidized had the same process with the normal diploid embryos. However, thedeveloping speed of gynogenetic diploid was slower than control group: the homo-and heter-gynogenesisembryos delayed3min and2min at blastodisc stage,35min and45min at low blastula stage,55min and1h at late gastrula stage,50min and1h45min at heartbeat stage, and1h and1h30min at hatching stage,respectively. The delay may be due to the disturbance of cell division cycle caused by cold shock. Inhomo-gynogenesis, the analysis of SSR primes KPC9and KPC45showed in24tested offsprings,83.3%(20/24) had maternal bands only;12.5%(3/24) individuals had paternal bands in the two loci, suggestingthey derived from normal fertilization; and1individual showed paternal band in locus KPC9but absent inlocus KPC45, suggesting there were genetic leakage of the paternal fish. In heter-gynogenesis, the analysisof SSR prime KPC43showed18tested offsping all have maternal specific band only. The results showedheterologous sperm can avoid paternal gene inflowing to offspring. In gynogenesis induction, the selectionof homo-or heterologous sperm should accord to their relative merits.
In order to analyze gynogenetic P. crocea induced by sperm of M. miiuy, as well as manage and protect M.miiuy and study the phylogeny in Sciaenidae, SSR markers were isolated for M. miiuy using genomiclibrary enriched by magnetic beads, and subsequently these markers were cross-amplified in P. crocea andused to analyze the gynogenetic P. crocea induced by sperm of M. miiuy. In total,247positive clones weresequenced and got138unique sequences that contained microsatellite motifs. Thirty-seven primer pairswere designed for35sequences and amplified in30M. miiuy individuals. Thirty primer pairs wereeffective, and within the tested population, twenty-four were polymorphic with an average of4.33allelesper locus (range from2to8). The observed (Ho) and expected (He) heterozygosity ranged from0.0667to 1.0000(mean0.6361) and0.3828to0.8139(mean0.6443), respectively. The mean PIC value was0.576,ranging from0.348to0.823. Seventeen loci showed high polymorphism (PIC>0.5). Eight loci weresignificant deviations from HWE, which may be due to the small sample size. Twelve out of the30primerpairs showed amplification in the different genus fish P. crocea and ten showed polymorphic with anaverage of5.10alleles per locus (range from2to8). The observed (Ho) and expected (He) heterozygosityranged from0.0000to0.8667(mean0.5200) and0.0655to0.8492(mean0.6053), respectively. The meanPIC value was0.555, ranging from0.062to0.815. Six loci showed high polymorphism (PIC>0.5). Fourloci were significant deviations from HWE. Except MM007(the homologous was51.08%between the twospecies), the sequence alignment of microsatellite flanking regions of other11loci showed highlyhomologous (range from89.63%to99.36%) between the two species. The gynogenetic P. crocea inducedby sperm of M. miiuy were analyzed through four cross-amplified primer pairs, and no specific paternalbands were found in the offspring. The results of four SSR markers indicate the genetic material of M.miiuy sperm did not revealed to the gynogenetic P. crocea as neither chromosome nor DNA fragment.The karyotype showed there was no identifiable sex chromosome in P. crocea. The inability to determinethe genotypic sex of P. crocea is a problem in aquaculture. In this study, AFLP technique was used toanalyse the bands between female and male P. crocea. Sixty-three out of64primer combinations couldamplify clear bands, but only the primer combination E-ACC/M-CTG got a band wihch was present in allmales and absent in all females. The result indicates there were only a few genomic differences betweenfemale and male P. crocea. Alignment showed this male-specific candidate band was two differentnucleotide sequences which were both188bp, and BLAST showed there was no homologous sequence inGenBank. Two PCR primers were designed, but amplification showed bands were appeared both in femaleand male P. crocea. We speculate that this sex-difference AFLP band came from the interaction ofrestriction enzyme recognition sites, DNA sequence modification (e.g, methylation) and other factors.
In conclusion, this study established a technique that inducing gyonogenetic P. crocea by heterologoussperm (M. miiuy and N. albiflora) and using cold-shock to diploidize the female chromosome, verifiedgynogenesis through morphology, cytogenetic and SSR markers analysis, compared the embryonicdevelopment and SSR patterns between two group of gynogenetic P. crocea which were induced byhomologous sperm and heterologous (M. miiuy) sperm, isolated and characterized SSR markers for M.miiuy, verified the gynogenetic P. crocea using M. miiuy SSR markers which were cross-amplifed in P.crocea besides P. crocea SSR markers, screened a male-specific ALFP band. The result of this study wasuseful both for theoretical research and application research. Nevertheless, the growth, development(especially gonadal development) of gynogenetic offspring and identification of genotypic sex in P. croceaare needed to be further investigated.
引文
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