Microsatellite-centromere Mapping And Integration Of Centromeric Regions On The Genetic Maps In Two Important Marine Mollusks And Sea Cucumber Apostichopus Japonicus

The marine shellfish has served as an important component of aquaculture in China. TheZhikong scallop (Chlamys farreri) and the Pacific abalone (Haliotis discus hannai) are twomajor economically important aquaculture species in China. In recent years, the artificialinduction of gynogenetic diploids and the cytological mechanism of gynogensis in thesespecies have been comprehensively investigated. In this study, we developed multiplex PCRsystems of microsatellite markers, evaluated the inbreeding efficiency through gynogenesis,performed half-tetrad analysis, and integrated centromeric regions on the microsatellitelinkage maps of the two molluscs. Meanwhile, we first reported the artificial induction ofgynogenetic diploids in the economically important sea cucumber Apostichopus japonicus,and studied the mapping of microsatellite loci in relation to the centromere by half-tetradanalysis using two gynogenetic diploid families of the A. japonicus. The main results of thesestudies are as follows:1. Development of four multiplex PCRs in the Zhikong scallop (Chlamys farreri) andtheir validation in parentage assignmentThis study reports four highly informative multiplex PCRs developed from12previouslydescribed microsatellites in Chlamys farreri. We evaluated and validated these multiplexPCRs in12full-sib families. The average polymorphism information content (PIC) was0.872.Inheritance analyses in the144tests showed that4.9%microsatellite loci deviated fromMendelian ratios. The frequency of null alleles was estimated as14.8%of all the allelessegregating based on a within-family analysis of Mendelian segregation patterns. Parentageanalysis of real offspring demonstrated that75%of all offspring were unambiguously allocated to a pair of parents based on only one multiplex PCR, and100%of the offspringwere correctly allocated to their parents when two multiplex PCRs were used.2. Microsatellite-centromere mapping in Zhikong scallop (Chlamys farreri) throughhalf-tetrad analysis in gynogenetic diploid familiesInheritance of27informative microsatellite loci was examined in three inducedmeiogynogenetic diploid families of zhikong scallop (Chlamys farreri) for centromeremapping using half-tetrad analysis. Mendelian inheritance was confirmed for the loci byexamining the genotypic segregation in three control crosses. Nine of the27microsatelliteloci showed the existence of null alleles in control crosses. All gynogenetic offspring onlypossessed the alleles of the mother, indicating100%success level for the three families. Thesecond division segregation frequency (y) of the microsatellite loci ranged from0.05to0.78with a mean of0.41, suggesting the existence of positive interference after a single chiasmaformation in some chromosomes in the scallop. Microsatellite-centromere (M-C) distancesranged from3cM to39cM under the assumption of complete interference. Information onthe positions of centromeres in relation to the microsatellite loci will represent a contributiontowards assembly of genetic maps in the commercially important scallop species.3. Genetic positioning of centromeres through half-tetrad analysis in gynogenetic diploidfamilies of the Zhikong scallop (Chlamys farreri)Centromere mapping is a powerful tool for improving linkage maps, investigatingcrossover events and understanding chiasma interference during meiosis. Ninetymicrosatellite markers selected across all linkage groups (LGs) from a previous Chlamysfarreri genetic map were studied in three artificially induced meiogynogenetic families forcentromere mapping by half-tetrad analysis. Inheritance analyses in the control crossesshowed that all90microsatellite loci were conform to Mendelian inheritance, while inmeiogynogenetic progeny,4.4%of the microsatellite loci showed segregation departures froman expected1:1ratio of two homozygote classes. The second division segregation frequency (y) of the microsatellites ranged from0.033to0.778with a mean of0.332, confirming theoccurrence of partial chiasma interference in this species. Heterogeneity of y is observed inone of42cases in which markers were typed in more than one family, suggesting variation ingene-centromere recombination among families. Centromere location was mostly inaccordance with the C. farreri karyotype, but differences in marker order between linkage andcentromere maps occurred. Overall, this study makes the genetic linkage map a morecomplete and informative tool for genomic studies and it will also facilitate future research ofthe structure and function of the scallop centromeres.4. Multiplex PCR panels of microsatellites for the Pacific abalone (Haliotis discushannai)Four multiplex PCRs were developed for the Pacific abalone Haliotis discus hannai, andthe results obtained indicate the usefulness of the four multiplexed microsatellite systems inparentage assignment. We evaluated and validated these multiplex PCRs in12full-sibfamilies. The average polymorphism information content (PIC) was0.82. Inheritance analysesin the144tests showed that4.9%microsatellite loci deviated from Mendelian ratios. Thefrequency of null alleles was estimated as10.6%of all the alleles segregating based on awithin-family analysis of Mendelian segregation patterns. Simulations and parentage analysisshowed that with the most informative multiplex set4, the assignment success could be86%and90%respectively, and100%of the offspring were correctly allocated to their parentswhen two or more multiplex PCRs were used.5. AFLP analysis to assess crossover interference and homozygosity in gynogeneticdiploid Pacific abalone (Haliotis discus hannai)Recombination analysis in gynogenetic diploids is a powerful tool for assess degree ofinbreeding, investigating crossover events and understanding chiasma interference duringmeiosis. To estimate marker-centromere (M-C) recombination rate, inheritance pattern of654amplified fragment length polymorphism (AFLP) markers was examined in the72-h veligerlarvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). Of the654genotypic ratios examined, all were compatible with3:1Mendelian segregationratio. The second division segregation frequency (y) of the AFLP loci ranged from0.00to0.96, with23.9%loci showing y values higher than0.67, evidencing the existence ofinterference. The average recombination frequency across the654AFLP loci was0.45,allowing estimation of fixation index of0.55, indicating that meiotic gynogenesis couldprovide an effective mean of rapid inbreeding in the Pacific abalone. The distribution ofy-value at AFLPs was uniform over the interval from zero to1.0, and no AFLP clusteringobserved. The AFLP loci have a small proportion (4.4%) of y values greater than0.90,suggesting that a relatively low or intermediate degree of chiasma interference occurred in theabalone chromosomes. This study demonstrated that meiotic gynogenesis is an efficientmethod for achieving a rapid accumulation of inbreeding in this commercially importantspecies.6. Centromere mapping in the Pacific abalone (Haliotis discus hannai) throughhalf-tetrad analysis in gynogenetic diploid familiesCentromere mapping is an essential prerequisite for our understanding of the compositionand structure of genomes. For centromere mapping in two meiogynogenetic families of thePacific abalone (Haliotis discus hannai), we screened97microsatellite markers that cover alllinkage groups from a currently available abalone linkage map. Microsatellite analysisshowed that no unique paternal allele was found in all gynogenetic progeny, which confirmed100%success of induction of gynogenesis. In the control crosses, all97microsatellite lociwere compatible with Mendelian inheritance, while in meiogynogenetic progeny,5.2%of themicrosatellite loci showed segregation distortions from an expected1:1ratio of twohomozygote classes. The second division segregation frequency of the microsatellites rangedfrom0.037to0.950with a mean of0.399, indicating the existence of interference.Heterogeneity among linkage groups in the crossover distribution was observed. Centromerelocation was mostly in accordance with the abalone karyotype, but differences in markerorder between linkage and centromere maps occurred. The half-tetrad genotypes in LG2(family A) were examined in detail to determine the possible presence of multiple crossoversbetween markers. In this case (108chromosomes in total),44chromosomes are non-crossover (NCO;40.7%),23are single crossover (SCO;21.3%),37are double crossover (DCO;34.3%),and four are triple crossover (TCO;3.7%). However, the same analysis performed in LG6(family A,180chromosomes; Afa172, Afa005, Awb002and Afa179) revealed a lower levelof recombination,111NCO (61.7%),54SCO (30%),12DCO (6.7%), and three TCO (1.6%),indicating that neither the frequency nor the pattern of crossover would be constant across thelinkage groups. Information on the positions of centromeres in relation to the microsatelliteloci will represent a contribution towards assembly of genetic maps in the commerciallyimportant abalone species.7. Artificial induction of gynogenetic diploids in sea cucumber Apostichopus japonicusGynogenetic diploids in sea cucumber (Apostichopus japonicus) were induced by meansof ultraviolet (UV) irradiation and cytochalasin B (CB) treatment for the inhibition of thesecond polar body release. The sperms irradiated by UV at an intensity of2561μw/(cm2s)for various durations were fertilized with the normal eggs. With increasing irradiation time,the rates of cleavage, survival at the early embryo stage，and development of early auricularialarva decreased, and none of auricularia larva occurred at30s. Thirty-five min afterfertilization,20~30%of eggs fertilized with sperm irradiated by UV for30s released the firstpolar body. Then the fertilized eggs were exposed to0.5μg/ml CB for20min. Comparedwith that in the control group, the developmental speed in the gynogenetic diploid group wasslower. Microsatellite analysis demonstrated that93.3%of gynogenetic diploids weresuccessfully induced.8. Microsatellite-centromere mapping in sea cucumber (Apostichopus japonicus) usinggynogenetic diploid familiesTwo families of gynogenetic sea cucumber (Apostichopus japonicu) were produced byfertilizing eggs with UV-irradiated sperm followed by inhibition of the second meioticdivision. Inheritance of43microsatellite markers were examined in the two meiogynogeneticdiploid families for centromere mapping using half-tetrad analysis. The ratio of gynogenesis was proven to be93.3%and100%in the two families, respectively. Inheritance analyses inthe control crosses showed that2.3%microsatellite loci deviated from Mendelian ratios. Thesecond division segregation frequency (y) of the microsatellites ranged from0.00to0.80,suggesting the existence of positive interference after a single chiasma formation. The meanrecombination frequency was0.21, corresponding to a fixation index of0.79in onegeneration. This is3.2times the expected increase in homozygosity after one generation ofsib mating (0.25), suggesting meiotic gynogenesis may be an effective means of rapidinbreeding in the sea cucumber. Microsatellite-centromere distances ranged from0.0cM to40.0cM under the assumption of complete interference. Forty two microsatellite loci weremapped in relation to the centromere by half-tetrad analysis, and two markers, AH028andAH112, were identified as tight linkage with their centromeres. Centromere position in maleLG3and LG20was estimated from distance of markers from the centromere. The informationabout the positions of centromeres in relation to the microsatellite loci will be useful for thefuture gene mapping in this commercially important species.In conclusion, the microsatellite-centromere mapping and integration of centromericregions on the genetic maps have been accomplished in two important marine mollusks andthe sea cucumber. This greatly improves the genetic linkage maps, and makes the genetic mapa more complete and informative tool for genomic studies, which provides the basis for futureintegration with the physical map. The interference phenomenon was found in this study, andjoint segregation analysis of markers in gynogenetic diploids reveals that neither thefrequency nor the pattern of crossover would be constant across the linkage groups. Theinformation obtained here has important theoretical and applied significance for fine geneticmapping and revealing the chromosome behavior during meiosis.