Study On Transmitted Parental Heterozygosity Of 2n Gametes And Homologous Recombination Characteristics In Populus Tomentosa Carr.

Hybridization and polyploidization are effective ways to create variations,in which chromosome doubling of female gamete through physicochemical treatment is conductive to breed triploids.In the process of inducing sexual polyploidization,homologous recombination is closely related to the transmitted parental heterozygosity of 2n gametes.The transmitted parental heterozygosity varies by different origins of 2n gametes,resulting in different performance of triploid progenies.Therefore,studies on characterization of homologous recombination,and on the parental heterozygosity transmitted by 2n gametes induced by chromosomes doubling of gametes in plant have important theoretical significance and practical guiding value for plant genetic improvement,especially for triploid breeding.In this study,Populus tomentosa was used as the object,the technique of inducing chromosome doubling of megaspore to breed hybrid triploids was developed,and the parental heterozygosity transmitted by 2n gametes were analyzed using SSR markers.At the same time,triploid populations,established earlier in the laboratory,from artificiallyinduced chromosome doubling of embryo sac in P.tomentosa were served as material,based on the strategy that heteroduplex DNA(hDNA)could be detected through inhibiting postmeiosis of sister chromatids using the SSR markers,homologous recombination in P.tomentosa with different genotypes were characterized.Major results are as follows:(1)The hybrid triploids were obtained by inducing megaspore chromosome doubling with high temperature treatment for the first time in P.tomentosa.According to the correlation between megasporocytes and microsporocytes during meiosis,the meiotic stage of megaspore in female flowers could be determined by the meiotic stage of microspore in male flowers.When the microspores developed into the uninucleate eccentric stage,the corresponding meiotic stage of macrospore mother cell was at the stages from pachytene to metaphase I,which could be the optimal treating period for inducing megaspore chromosome doubling by 38? and 40? high temperature treatment for 4h.A total of 189 triploids were obtained in the two hybrid combinations,the average induction rate was around 50%.The growth speed of triploid hybrids was higher than that of the diploids,amid there were some differences between the different parental combinations,which suggested that some attentions should be paid to the parental selection.(2)It was proved that there was no significant difference in parental heterozygosity transmitted by 2n female gametes from the same origin in different female parents in P.tomentosa.A total of 110 hybrid triploids in Leuce poplar obtained by chromosome doubling of megaspore via high temperature treatment were served as materials,five pericentromeric SSR markers were selected to identify 30 individuals derived from the first division restitution(FDR),and 80 allotriploids from the second division restitution(SDR)in these two cross combinations.Twenty-six SSR loci were used to analyze the transmitted parental heterozygosity of 2n female gametes derived from two origins.The results showed that transmitted parental heterozygosity of FDR type 2n female gametes were 0.824 and 0.769,respectively,while that of SDR were 0.395 and 0.396,respectively.The transmitted parental heterozygosity of FDR type 2n female gametes was significantly higher than that of SDR in female parents.However,no significant difference was found in parental heterozygosity transmitted by 2n female gametes from the same origin in different female parents.(3)The characteristics of homologous recombination among poplar species and among individuals within species were preliminarily revealed.The two hybrid triploid populations from inhibited post-meiotic segregation in P.tomentosa were served as materials.111 pairs of SSR primers were used to study heteroduplex DNA(hDNA)on nine chromosomes in P.tomentosa with different genotypes,applying the strategy that hDNA were detected by constructing populations from inhibited post-meiotic segregation.The results showed that the frequency of hDNA between two female parents in P.tomentosa ranged 8.5%to 87.2%,and that the hDNA frequency was positively related to the distance from the centromere,but the average hDNA frequency of the same chromosome had no correlation with the chromosome length;1-3 times recombination events were detected on most chromosomes,four times recombination events were detected on a few,and five times recombination events were detected on few;the overall frequency of hDNA on the same chromosome in two genotypic individuals was roughly similar,while the hDNA frequency varied greatly at specific SSR loci;compared with Tacamahaca poplar,homologous recombination times detected and the frequency and location of hDNA were significantly different.(4)It was proved that more homologous recombination event times with high frequency would occur in the development of gametes in genome.With the increase of the number of SSR molecular markers for detection,the homologous recombination event times detected increased significantly.Using 87,62,and 79 pairs of SSR primer distributed on Chr01,Chr04,and Chr19,respectively,up to 12,8,18 recombination times were detected on three chromosomes investigated.The hDNA patterns on different chromosomes in P.tomentosa were little changed with increased pairs of SSR primer.Similarly,the highest frequency of hDNA occurred at the adjacent terminal on chromosomes,which was slightly higher than that at the terminal.Then,as the distance from the marker site to the centromere decreased,the frequency of hDNA decreased gradually.At the region with high methylation or the long terminal repeat(LTR)retrotransposon enrichment,the frequency of hDNA showed low frequency,and it appeared high frequency at the region with low complexity AT-sequence and high gene density.(5)The results of homologous recombination were of important instructive significance for tree breeding.Regular and high-frequency homologous recombination events occurred at high gene density regions largely affected the association between molecular markers and quantitative trait loci(QTLs),which was an important reason that it was difficult to achieve better results with molecular marker-assisted selection in tree breeding;similarly,it also led to particularly outstanding genotypes among triploid hybrid progeny populations originated from SDR and post-meiotic restitution(PMR)-type 2n gametes,indicating that SDR,PMRtype 2n gametes and FDR-type 2n gametes with higher parental heterozygosity also had significant breeding value.