香港巨牡蛎Crassostrea hongkongensis与长牡蛎C.gigas种间杂交效应及遗传改良研究
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摘要
种间杂交是动植物基本育种手段之一，它可以显著的扩大动植物的基因库，促进种间交流，引入异种有利基因，创造出前所未有的变异类型，甚至会合成新的物种。香港巨牡蛎是我国南方主要经济种，喜好高温低盐环境，主要分布在广东、广西，年产量在120万吨左右；而长牡蛎是我国北方最重要的经济种，喜好低温高盐环境，主要分布在我国的辽宁、山东，年产量近100万吨。本文以这两种在生态类型及地理分布上互补的牡蛎为材料，在突破生殖隔离的基础上，成功的获得了大量种间杂交子，构建了牡蛎远缘杂交育种技术体系；并且通过建立杂交家系对其杂交效应进行了剖析；还采用诱导异源多倍体及种间回交等手段对其表型性状进行了遗传改良。此外，对所有的杂交子均进行了遗传鉴定，还对其多态性的不育格局进行了分析。
（1）首先，评估了香港巨牡蛎和长牡蛎在我国北方沿海的表型性状。结果表明：在温度和盐度相同下，长牡蛎卵径、受精率、孵化率及D形幼虫均大于香港巨牡蛎；香港巨牡蛎幼虫浮游前期生长较慢，而后快于长牡蛎。温度是影响幼虫生长、存活、变态的最主要因素，其次为盐度，交互作用几乎尚未起到作用。中间育成阶段，室外比室内培育效果更好，环境是影响稚贝生长的最主要因素；无论室内还是室外两种牡蛎稚贝的存活率均在90%以上。幼贝阶段，长牡蛎表型性状显著优于香港巨牡蛎；且香港巨牡蛎稚贝在北方越冬期间出现了大量致死现象。
（2）在比较了两种牡蛎表型性状的基础上，开展了两种牡蛎的人工种间杂交。结果表明：香港巨牡蛎的卵子可以与长牡蛎的精子受精，相反方向不能受精；种间配子兼容性受到温度、盐度及其精子浓度的影响，而且其兼容性存在着较大的个体间差异。杂交子在不同的环境条件下，存活能力不同，在适宜的条件下，表现出较高的变态率、存活率，具有明显的存活优势；在不利条件下，幼虫存活能力低，能或者不能获得少量稚贝。杂交子生长能力较差，具有明显的生长劣势，表现出远交衰退。对其所有的杂交子均进行了遗传鉴定，表明其为真正意义上的两性融合杂交子。杂交子性腺颜色与性别相关联，表现出伴性遗传；大部分杂交子是不育的，仅有8.64%的雌性杂交子完全可育；还有一些杂交子部分可育的；经流仪分析发现杂交子均产生单倍体配子。
（3）为了进一步分析杂交子到底能否产生生长优势，建立了9个香港巨牡蛎、9个长牡蛎及45个杂交家系，并引入杂种潜力、配合力及杂种遗传力来剖析杂交效应。从杂种潜力上看，杂交子产生了明显的生长劣势，稚贝阶段具有明显的存活优势，幼虫具有显著的变态优势。从配合力效应上分析：对于杂交子生长性状而言，长牡蛎一般配合力具有正向效应，香港巨牡蛎几乎尚未表现出一般配合力，杂交家系平均水平上为负向特殊配合力，这种效应主要来自于双亲基因互作的非加性效应；对杂交存活性状而言，长牡蛎一般配合力具有正向效应，香港巨牡蛎为负向效应，杂交家系平均水平上为正向配合力，即出现正向存活优势；对于变态率而言，长牡蛎与香港巨牡蛎均表现出负向一般配合力，而杂交家系表现出正向特殊配合力，其效应主要来自于双亲基因的显性与上位效应。从其杂种遗传力上看，父系半同胞的生长性状遗传力为0.48~0.67，存活性状为0.19~0.41，幼贝产量为0.50。
（4）为了对杂交子生长性状进行改良，采用低渗的方法诱导了香港巨牡蛎与长牡蛎的异源多倍体。通过检测，发现3个家系的异源三倍体家系倍化率均为100%，而且在稚贝阶段出现了个别嵌合体。杂交三倍体幼虫具有明显的生长、存活劣势；但是一旦转成稚贝以后，就具有明显的生长及存活优势。遗憾的是，虽然相对杂交二倍体而言具有优势，但是其表型性状仍不及长牡蛎，但优于香港巨牡蛎了，也就是说，杂交三倍体使得其生长性状得到了部分改良。
（5）虽然大部分杂交子具有高度不育现象，但仍有少部分个体含有少量的可育雌性配子。筛选出部分可育个体，进行了与亲本种的种间回交育种研究。结果表明：杂交子与长牡蛎的回交中，回交子出现了明显的生长及存活优势，其生长性状更是出现了超级杂种优势。杂交子与香港巨牡蛎的回交中，以杂交子为母本的杂交组合出现了回交优势，而对应的杂交组没有出现显著的杂种优势。采用复合COI及ITS2对回交子及杂交子F_2进行了遗传鉴定，结果表明：在回交的过程中回交子出现了不同程度的同型选配现象；雌雄异体间杂交子F_2的ITS2出现了1:2:1式种间分离，但雌雄同体间的杂交子F_2仍为杂交子，其母本均为香港巨牡蛎，其少量个体表现为香港巨牡蛎与长牡蛎的双单亲遗传。
（6）香港巨牡蛎与长牡蛎的种间杂交生殖隔离可以由以下3种机制来解释：①种间配子的单向受精：可以使得种间配对概率减少50%；②杂交子的多态性不育格局：仅有8.64%雌性个体完全可育，总体上表现为二态性的雄性不育，这与霍尔丹定律一致，使得生殖隔离的隔离率减小90%；③回交中的同型选配：这样一来通过回交，实现了物种复原，即可以部分的回避生殖隔离。这三大机制保证了这两种牡蛎在自然群体中很难实现天然杂交，从而有效的被隔离开来。
总之，通过对香港巨牡蛎与长牡蛎远缘杂交及遗传改良研究。获得了具有高度不育，抗逆性较强的种间杂交子，这可以大大的扩大了牡蛎养殖面积，从而获得较高的产量；获得的具有较好表型性状的杂交三倍体，这将会进一步抑制其性腺发育，实现完全不育的目的；最后，获得了具有超级杂种优势的种间回交子。这些远缘杂交新品系对于目前牡蛎的种质改良及其产业品种提升指明了方向，奠定了坚实的理论基础，具有良好的开发应用前景。
Interspecific hybridization can generate transgressive hybrid phenotypes as acommon breeding method. These hybrid phenotypes have extreme trait valuesexceeding the combined range of the parental species. Such genetic variation not onlycan enlarge the working surface for natural selection, but also may facilitate theevolution of novel adaptations where ecological opportunity exists, even to newspeciation. Crassostrea hongkongensis is one of most important oyster speciescultured due to its high market value in South China. It is distributed from Fujian toGuangxi provinces, with populations centered in Guangdong province. Crassostreagigas is the most commonly used species in attempts at interspecific hybridization,owing to its worldwide distribution, rapid growth, and dominant position incommercial oyster culture. In North China, they are mainly farmed in Liaoning andShandong Province. Although both are Crassostrea oysters with similar karyotypesand are well known for high yields, C. gigas is distinct from the C. hongkongensis inbreeding season, preferred temperature, and salinity tolerance. In this text, a lager ofhybrids was obtained on the base of breaking through the reproductive isolation. Thedistant hybrid technology was established, and cross effect was examined by thehybrid families. To improve the phenotypic character, allotriploid induction andinterspecific backcrosses were carried in this study. In addition, the geneticconfirmation of hybrids was analyzed by the molecular measurement, while the sterilepatterns of hybrids were investigated by the histological method.
(1) To determine the possibility of transplantation of C. hongkongensis fromSouthern to Northern China, the early phenotypic traits of both larval and juvenile C.hongkongensis and C. gigas were determined under identical environments. Theresults showed that the shell width of C. hongkongensis was significantly larger than that of C. gigas (P<0.05), but the shell height of C. hongkongensis was significantlysmaller (P<0.05). No significant differences in terms of shell length and fecundity(P>0.05) were found between the species. The egg diameter, fertilization rate,hatchery success, and the larval size of C. gigas were all larger than those of C.hongkongensis. The larval growth of C. hongkongensis was smaller than that of C.gigas during the early planktonic stage, but it was larger during the later planktonicstage under the same temperature and salinity. The order of two larval survival abilitywas high temperature group>middle temperature group. The middle salinity group ofC. hongkongensis larval size was larger than that of the high salinity group, and thehigh salinity group of C. gigas larval size was larger than that of the middle salinitygroup at the same temperature. Larval metamorphosis was not only delayed, it alsodeclined with decreasing temperature; the larval metamorphic size increased duringthe larval metamorphic stage under the same condition. Larval phenotypiccharacteristics were mainly affected by temperature, and the secondary factor wassalinity, but mutual effects showed no positive action. The environment was the majorfactor for growth spurts during the juvenile stage and the phenotypic characteristics ofspurts outdoors were better than those indoors. The shell height of C. hongkongensiswas significantly larger than that of C. gigas on Day60(P<0.05). The survival ratesof the two oyster species were over90%during the juvenile stage, and no significantdifference between the experimental groups (P>0.05) was found. Our analysis clearlydemonstrates that seeds of C. hongkongensis were successfully achieved, and theirtransplantation to Northern China may yield considerable benefits by the artificialbreeding. As to two youths, the phenotypic traits of C. gigas was superior to C.hongkongensis. However, the higher mortality of C. hongkongensis the youth wasobserved during the overwinter stage. It is promising to supply the resource of C.hongkongensis, and also provide the scientific base on the interspecific hybridizationbetween two oyster species in China.
(2) The artificial hybridization between two species was introduced on the base ofcomparison on the phenotypic characters. C. hongkongensis eggs was fertilized to C.gigas sperms, and the reverse direction was no happened. The interspecific gametal compatibility was affected by temperature, salinity, sperm concentration and theindividual variation. Hybrid weakness of growth was observed at all times, whereassurvival heterosis was fluctuant under diverse culture sites due to the adaptivedifference of hybrids. Genetic analysis confirmed that the HG spat contained DNAfrom both species and thus were true hybrids. When hybrids grew to sex mature byone year culture, we observed that most of hybrids were sterile, but with a smallproportion being fertile. A certain extent of color polymorphism for gonad part ofhybrids was observed, including milk white, duff, saffron yellow, sandy beige,grayish and grayness six major colors, and these of corresponding with completelyfertile female, partially fertile female, partially fertile hermaphrodic, partially fertilemale, completely sterile male and asexual types, respectively. That is, the gonadalcolor and sex was linked. The patterns of hybrid sterility according to thegametogenesis were classified as below three modes:(1) Complete sterile: themajority of male hybrids (58.06%) produced spermatocytes without sperms, and theothers (7.27%) were asexual without gametes.(2) Partial fertile: several femalehybrids (19.65%) were fertile with0.21%normal oogenesis, a bit of hermaphroditic(1.40%) were fertile both0.06%normal oogenesis and0.04%normalspermatogenesis, while a few of male hybrids (5.06%) were fertile with0.55%normalspermatogenesis.(3) Complete fertile: only a small of female hybrids (8.64%) wascompletely fertile with33.72%normal oogenesis. Histological and flow cytometryanalyses of the hybrid gonads revealed that the sterile mode was defined as“dimorphism hybrid male sterility”, with some gonads but abnormal spermatogensisor few sperms. To our knowledge, the present finding is a novel example of hybridsterility in the Crassostrea species. Our analysis explicitly demonstrates thatinterspecific hybridization between C. hongkongensis and C. gigas is possible in onedirection and their hybrids are generally sterile, resulting in partial reproductiveisolation in postmating and postzygotic isolation.
(3) To further analysis the growth heterosis,9familes of C. hongkongensis,9families of C. gigas, and45hybrid families were established, and heterosis potence,cross-heritability and combining ability were introduced to ascertain the heterosis effect. Considering heterosis potence, growth weakness, spat survival heterosis andmetamorphosis advantage were observation in this study. Considering combiningability, positive effect of C. gigas, hardly nil of C. hongkonensis, and negative effectof hybrids occurred on the growth traits. Thus, this effect was come from the twoparental gene interactions with additive allelic effect. The positive effect of C. gigas,negative of C. hongkonensis, and positive effect of hybrids occurred on the survivaltraits. Moreover, this effect produced the survival heterosis. The negative effect of C.gigas and C. hongkonensis, and positive effect of hybrids occurred on themetamorphosis traits with the metamorphosis heterosis. Considering cross-heritability,the heritability of paternal half-sibs was0.48~0.67for growth trait, was0.19~0.41forsurvival trait and was0.50for youth yield.
(4) To further improve performance of their hybrids, allotriploid introduction byinhibiting the second polar body of eggs from C. hongkongensis using the hypotonictreatment method was conducted at Dalian in North China on July2011. Three realreplicates were successful introduced, and each one was consist of the twointraspecific families GGand HH, an interspecific hybridization family HG, and anallotriploid family HHG. Heterosis and allotriploid advantage of experimental groupswere evaluated for traits such as fertilization and hatching success, survival,metamorphosis, larval and juvenile growth. The egg cleavage rate of HHG group issimilar to HG group, and they were significantly smaller than the two intraspeicifccrosses. The D larval rate of HHG group was the smallest in these experimentalgroups. Survival heterosis of diploid hybrids was always positive, but no growthheterosis was observed. Allotriploid advantage of survival and growth were bothnegative in larval stage and positive in juvenile stage. Genetic analysis confirmed thatthe HG (HHG) spat were true hybrids by complex COI and ITS2. D larvae of threeHHG families were all triploid hybrids in D larval stage, whereas one diploid hybridspat and two diploid-triploid mosaics of HHG families were observed during spatstage. Our finding suggested that the phenotypic character of the allotriploid spatswith obvious alltriploid advantage was superior to HH and HG, but inferior to GG.
(5) Although most of hybrids were sterile, a small number of these was able to produce fertile gametes. Thus, interspecific backcross was introduced by the partialfertile hybrids and parental species progenies. The superior heterosis of growth andsurvival was observed on the backcrosses between hybrids and C. gigas. However, thebackcross between hybrid eggs and C. hongkongensis sperms occurred superiorheterosis, in contrast, the other backcross was no obvious heterosis. The backcrosseswere confirmed by the complex COI and ITS genes. The partial assortative matingphenomena was observed in the backcross. The ITS2gene was separated to1:2:1phenomena during the hybrids F2by the cross families, but no separation in thehybrids F2by the self-fertilized families. Interestingly, the maternal gene COI wasalways found in the hybrids F2, and a few of them occurred the doubly uniparentalinheritance.
(6) The reproductive isolation between C. hongkongensis and C. gigas wasdemonstrated by the three mechanisms:①Asymmetry fertilization: The probabilityof interspecific hybridization was reduced50%.②The polymorphism sterile pattern:Only8.64%females was completely fertile, that is, was summarized dimorphic malesterility, and it accorded with the Haldane Rule. Thus, above90%individuals wasisolated to avoid the reproductive isolation.③Assortative mating: the species wasrecover by the assortative mating in backcross, that is, it partially avoid thereproductive isolation. Above three mechanisms ensured the great lower probabilityof hybridization in the natural populations.
In conclusion, hybrids between two oyster species with the higher sterile wereable to increase environmental tolerances, increase harvest ability, and to increaseoverall hardiness in culture conditions. The gonad of allotiploids were great sterilewith the excellent phenotypic character. Finally, the superior heterosis of backcrosseswere obtained by the cross between hybrids and two parental species. These hybridstrains were propitious to oyster genetic improvement, and which offered the newdirection for oyster breeding with the prospect industry.
（1）首先，评估了香港巨牡蛎和长牡蛎在我国北方沿海的表型性状。结果表明：在温度和盐度相同下，长牡蛎卵径、受精率、孵化率及D形幼虫均大于香港巨牡蛎；香港巨牡蛎幼虫浮游前期生长较慢，而后快于长牡蛎。温度是影响幼虫生长、存活、变态的最主要因素，其次为盐度，交互作用几乎尚未起到作用。中间育成阶段，室外比室内培育效果更好，环境是影响稚贝生长的最主要因素；无论室内还是室外两种牡蛎稚贝的存活率均在90%以上。幼贝阶段，长牡蛎表型性状显著优于香港巨牡蛎；且香港巨牡蛎稚贝在北方越冬期间出现了大量致死现象。
（2）在比较了两种牡蛎表型性状的基础上，开展了两种牡蛎的人工种间杂交。结果表明：香港巨牡蛎的卵子可以与长牡蛎的精子受精，相反方向不能受精；种间配子兼容性受到温度、盐度及其精子浓度的影响，而且其兼容性存在着较大的个体间差异。杂交子在不同的环境条件下，存活能力不同，在适宜的条件下，表现出较高的变态率、存活率，具有明显的存活优势；在不利条件下，幼虫存活能力低，能或者不能获得少量稚贝。杂交子生长能力较差，具有明显的生长劣势，表现出远交衰退。对其所有的杂交子均进行了遗传鉴定，表明其为真正意义上的两性融合杂交子。杂交子性腺颜色与性别相关联，表现出伴性遗传；大部分杂交子是不育的，仅有8.64%的雌性杂交子完全可育；还有一些杂交子部分可育的；经流仪分析发现杂交子均产生单倍体配子。
（3）为了进一步分析杂交子到底能否产生生长优势，建立了9个香港巨牡蛎、9个长牡蛎及45个杂交家系，并引入杂种潜力、配合力及杂种遗传力来剖析杂交效应。从杂种潜力上看，杂交子产生了明显的生长劣势，稚贝阶段具有明显的存活优势，幼虫具有显著的变态优势。从配合力效应上分析：对于杂交子生长性状而言，长牡蛎一般配合力具有正向效应，香港巨牡蛎几乎尚未表现出一般配合力，杂交家系平均水平上为负向特殊配合力，这种效应主要来自于双亲基因互作的非加性效应；对杂交存活性状而言，长牡蛎一般配合力具有正向效应，香港巨牡蛎为负向效应，杂交家系平均水平上为正向配合力，即出现正向存活优势；对于变态率而言，长牡蛎与香港巨牡蛎均表现出负向一般配合力，而杂交家系表现出正向特殊配合力，其效应主要来自于双亲基因的显性与上位效应。从其杂种遗传力上看，父系半同胞的生长性状遗传力为0.48~0.67，存活性状为0.19~0.41，幼贝产量为0.50。
（4）为了对杂交子生长性状进行改良，采用低渗的方法诱导了香港巨牡蛎与长牡蛎的异源多倍体。通过检测，发现3个家系的异源三倍体家系倍化率均为100%，而且在稚贝阶段出现了个别嵌合体。杂交三倍体幼虫具有明显的生长、存活劣势；但是一旦转成稚贝以后，就具有明显的生长及存活优势。遗憾的是，虽然相对杂交二倍体而言具有优势，但是其表型性状仍不及长牡蛎，但优于香港巨牡蛎了，也就是说，杂交三倍体使得其生长性状得到了部分改良。
（5）虽然大部分杂交子具有高度不育现象，但仍有少部分个体含有少量的可育雌性配子。筛选出部分可育个体，进行了与亲本种的种间回交育种研究。结果表明：杂交子与长牡蛎的回交中，回交子出现了明显的生长及存活优势，其生长性状更是出现了超级杂种优势。杂交子与香港巨牡蛎的回交中，以杂交子为母本的杂交组合出现了回交优势，而对应的杂交组没有出现显著的杂种优势。采用复合COI及ITS2对回交子及杂交子F_2进行了遗传鉴定，结果表明：在回交的过程中回交子出现了不同程度的同型选配现象；雌雄异体间杂交子F_2的ITS2出现了1:2:1式种间分离，但雌雄同体间的杂交子F_2仍为杂交子，其母本均为香港巨牡蛎，其少量个体表现为香港巨牡蛎与长牡蛎的双单亲遗传。
（6）香港巨牡蛎与长牡蛎的种间杂交生殖隔离可以由以下3种机制来解释：①种间配子的单向受精：可以使得种间配对概率减少50%；②杂交子的多态性不育格局：仅有8.64%雌性个体完全可育，总体上表现为二态性的雄性不育，这与霍尔丹定律一致，使得生殖隔离的隔离率减小90%；③回交中的同型选配：这样一来通过回交，实现了物种复原，即可以部分的回避生殖隔离。这三大机制保证了这两种牡蛎在自然群体中很难实现天然杂交，从而有效的被隔离开来。
总之，通过对香港巨牡蛎与长牡蛎远缘杂交及遗传改良研究。获得了具有高度不育，抗逆性较强的种间杂交子，这可以大大的扩大了牡蛎养殖面积，从而获得较高的产量；获得的具有较好表型性状的杂交三倍体，这将会进一步抑制其性腺发育，实现完全不育的目的；最后，获得了具有超级杂种优势的种间回交子。这些远缘杂交新品系对于目前牡蛎的种质改良及其产业品种提升指明了方向，奠定了坚实的理论基础，具有良好的开发应用前景。
Interspecific hybridization can generate transgressive hybrid phenotypes as acommon breeding method. These hybrid phenotypes have extreme trait valuesexceeding the combined range of the parental species. Such genetic variation not onlycan enlarge the working surface for natural selection, but also may facilitate theevolution of novel adaptations where ecological opportunity exists, even to newspeciation. Crassostrea hongkongensis is one of most important oyster speciescultured due to its high market value in South China. It is distributed from Fujian toGuangxi provinces, with populations centered in Guangdong province. Crassostreagigas is the most commonly used species in attempts at interspecific hybridization,owing to its worldwide distribution, rapid growth, and dominant position incommercial oyster culture. In North China, they are mainly farmed in Liaoning andShandong Province. Although both are Crassostrea oysters with similar karyotypesand are well known for high yields, C. gigas is distinct from the C. hongkongensis inbreeding season, preferred temperature, and salinity tolerance. In this text, a lager ofhybrids was obtained on the base of breaking through the reproductive isolation. Thedistant hybrid technology was established, and cross effect was examined by thehybrid families. To improve the phenotypic character, allotriploid induction andinterspecific backcrosses were carried in this study. In addition, the geneticconfirmation of hybrids was analyzed by the molecular measurement, while the sterilepatterns of hybrids were investigated by the histological method.
(1) To determine the possibility of transplantation of C. hongkongensis fromSouthern to Northern China, the early phenotypic traits of both larval and juvenile C.hongkongensis and C. gigas were determined under identical environments. Theresults showed that the shell width of C. hongkongensis was significantly larger than that of C. gigas (P<0.05), but the shell height of C. hongkongensis was significantlysmaller (P<0.05). No significant differences in terms of shell length and fecundity(P>0.05) were found between the species. The egg diameter, fertilization rate,hatchery success, and the larval size of C. gigas were all larger than those of C.hongkongensis. The larval growth of C. hongkongensis was smaller than that of C.gigas during the early planktonic stage, but it was larger during the later planktonicstage under the same temperature and salinity. The order of two larval survival abilitywas high temperature group>middle temperature group. The middle salinity group ofC. hongkongensis larval size was larger than that of the high salinity group, and thehigh salinity group of C. gigas larval size was larger than that of the middle salinitygroup at the same temperature. Larval metamorphosis was not only delayed, it alsodeclined with decreasing temperature; the larval metamorphic size increased duringthe larval metamorphic stage under the same condition. Larval phenotypiccharacteristics were mainly affected by temperature, and the secondary factor wassalinity, but mutual effects showed no positive action. The environment was the majorfactor for growth spurts during the juvenile stage and the phenotypic characteristics ofspurts outdoors were better than those indoors. The shell height of C. hongkongensiswas significantly larger than that of C. gigas on Day60(P<0.05). The survival ratesof the two oyster species were over90%during the juvenile stage, and no significantdifference between the experimental groups (P>0.05) was found. Our analysis clearlydemonstrates that seeds of C. hongkongensis were successfully achieved, and theirtransplantation to Northern China may yield considerable benefits by the artificialbreeding. As to two youths, the phenotypic traits of C. gigas was superior to C.hongkongensis. However, the higher mortality of C. hongkongensis the youth wasobserved during the overwinter stage. It is promising to supply the resource of C.hongkongensis, and also provide the scientific base on the interspecific hybridizationbetween two oyster species in China.
(2) The artificial hybridization between two species was introduced on the base ofcomparison on the phenotypic characters. C. hongkongensis eggs was fertilized to C.gigas sperms, and the reverse direction was no happened. The interspecific gametal compatibility was affected by temperature, salinity, sperm concentration and theindividual variation. Hybrid weakness of growth was observed at all times, whereassurvival heterosis was fluctuant under diverse culture sites due to the adaptivedifference of hybrids. Genetic analysis confirmed that the HG spat contained DNAfrom both species and thus were true hybrids. When hybrids grew to sex mature byone year culture, we observed that most of hybrids were sterile, but with a smallproportion being fertile. A certain extent of color polymorphism for gonad part ofhybrids was observed, including milk white, duff, saffron yellow, sandy beige,grayish and grayness six major colors, and these of corresponding with completelyfertile female, partially fertile female, partially fertile hermaphrodic, partially fertilemale, completely sterile male and asexual types, respectively. That is, the gonadalcolor and sex was linked. The patterns of hybrid sterility according to thegametogenesis were classified as below three modes:(1) Complete sterile: themajority of male hybrids (58.06%) produced spermatocytes without sperms, and theothers (7.27%) were asexual without gametes.(2) Partial fertile: several femalehybrids (19.65%) were fertile with0.21%normal oogenesis, a bit of hermaphroditic(1.40%) were fertile both0.06%normal oogenesis and0.04%normalspermatogenesis, while a few of male hybrids (5.06%) were fertile with0.55%normalspermatogenesis.(3) Complete fertile: only a small of female hybrids (8.64%) wascompletely fertile with33.72%normal oogenesis. Histological and flow cytometryanalyses of the hybrid gonads revealed that the sterile mode was defined as“dimorphism hybrid male sterility”, with some gonads but abnormal spermatogensisor few sperms. To our knowledge, the present finding is a novel example of hybridsterility in the Crassostrea species. Our analysis explicitly demonstrates thatinterspecific hybridization between C. hongkongensis and C. gigas is possible in onedirection and their hybrids are generally sterile, resulting in partial reproductiveisolation in postmating and postzygotic isolation.
(3) To further analysis the growth heterosis,9familes of C. hongkongensis,9families of C. gigas, and45hybrid families were established, and heterosis potence,cross-heritability and combining ability were introduced to ascertain the heterosis effect. Considering heterosis potence, growth weakness, spat survival heterosis andmetamorphosis advantage were observation in this study. Considering combiningability, positive effect of C. gigas, hardly nil of C. hongkonensis, and negative effectof hybrids occurred on the growth traits. Thus, this effect was come from the twoparental gene interactions with additive allelic effect. The positive effect of C. gigas,negative of C. hongkonensis, and positive effect of hybrids occurred on the survivaltraits. Moreover, this effect produced the survival heterosis. The negative effect of C.gigas and C. hongkonensis, and positive effect of hybrids occurred on themetamorphosis traits with the metamorphosis heterosis. Considering cross-heritability,the heritability of paternal half-sibs was0.48~0.67for growth trait, was0.19~0.41forsurvival trait and was0.50for youth yield.
(4) To further improve performance of their hybrids, allotriploid introduction byinhibiting the second polar body of eggs from C. hongkongensis using the hypotonictreatment method was conducted at Dalian in North China on July2011. Three realreplicates were successful introduced, and each one was consist of the twointraspecific families GGand HH, an interspecific hybridization family HG, and anallotriploid family HHG. Heterosis and allotriploid advantage of experimental groupswere evaluated for traits such as fertilization and hatching success, survival,metamorphosis, larval and juvenile growth. The egg cleavage rate of HHG group issimilar to HG group, and they were significantly smaller than the two intraspeicifccrosses. The D larval rate of HHG group was the smallest in these experimentalgroups. Survival heterosis of diploid hybrids was always positive, but no growthheterosis was observed. Allotriploid advantage of survival and growth were bothnegative in larval stage and positive in juvenile stage. Genetic analysis confirmed thatthe HG (HHG) spat were true hybrids by complex COI and ITS2. D larvae of threeHHG families were all triploid hybrids in D larval stage, whereas one diploid hybridspat and two diploid-triploid mosaics of HHG families were observed during spatstage. Our finding suggested that the phenotypic character of the allotriploid spatswith obvious alltriploid advantage was superior to HH and HG, but inferior to GG.
(5) Although most of hybrids were sterile, a small number of these was able to produce fertile gametes. Thus, interspecific backcross was introduced by the partialfertile hybrids and parental species progenies. The superior heterosis of growth andsurvival was observed on the backcrosses between hybrids and C. gigas. However, thebackcross between hybrid eggs and C. hongkongensis sperms occurred superiorheterosis, in contrast, the other backcross was no obvious heterosis. The backcrosseswere confirmed by the complex COI and ITS genes. The partial assortative matingphenomena was observed in the backcross. The ITS2gene was separated to1:2:1phenomena during the hybrids F2by the cross families, but no separation in thehybrids F2by the self-fertilized families. Interestingly, the maternal gene COI wasalways found in the hybrids F2, and a few of them occurred the doubly uniparentalinheritance.
(6) The reproductive isolation between C. hongkongensis and C. gigas wasdemonstrated by the three mechanisms:①Asymmetry fertilization: The probabilityof interspecific hybridization was reduced50%.②The polymorphism sterile pattern:Only8.64%females was completely fertile, that is, was summarized dimorphic malesterility, and it accorded with the Haldane Rule. Thus, above90%individuals wasisolated to avoid the reproductive isolation.③Assortative mating: the species wasrecover by the assortative mating in backcross, that is, it partially avoid thereproductive isolation. Above three mechanisms ensured the great lower probabilityof hybridization in the natural populations.
In conclusion, hybrids between two oyster species with the higher sterile wereable to increase environmental tolerances, increase harvest ability, and to increaseoverall hardiness in culture conditions. The gonad of allotiploids were great sterilewith the excellent phenotypic character. Finally, the superior heterosis of backcrosseswere obtained by the cross between hybrids and two parental species. These hybridstrains were propitious to oyster genetic improvement, and which offered the newdirection for oyster breeding with the prospect industry.
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