Interspecific Hybridization Effect And Genetic Improvement Between Two Cupped Oyster, Crassostrea Hongkongensis And C. Gigas

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.