Analysis Of Homologous Recombination Characteristics And 2n Gamete Heterozygosity In Populus

Sexual polyploidization, based on unreduced gametes (2n gametes), is a major pathway for the production of plant polyploid. Homologous recombination is an important reason for the heterozygosity variation among different types of 2n gametes. Homologous recombination and formation pathways of 2n gametes jointly affect the heterozygosity of 2n gametes, and subsequently allopolyploid. Studies including analysis of homologous recombination characteristics and 2n gamete heterozygosity in plant, effective assessment of the source of triploids, and analysis of transmission of heterozygosity by different types of 2n gametes, are significant for promoting the basic theory research of tree polyploid breeding and the effective utilization of 2n gametes. In this study, based on the production of triploid population origined from three different types of 2n gametes and diploid control population with the cross combination between Populus pseudo-simonii×P. nigra ’Zheyin3#’ and P. × beijingensis, a strategy was firstly proposed to characterize the reombination production (heteroduplex DNA; hDNA) in higher plants. And on the basis of this strategy, homologous recombination characteristics of the female parent P. pseudo-simonii × P. nigra ’Zheyin3#’ were analyzed. According to homologous recombination characteristics of the detected loci, suitable marker loci were selected to assess the formation pathways of 2n gametes and heterozygosity of 2n gametes with different origins. Major results as follows,(1) A full sib triploid hybrid population with different 2n gamete origins in Populus was established by megaspore chromosome doubling and embryo sac chromosome doubling. According to morphological characteristics of female flower buds, when the bract scales dehisced and the catkin came out a little, at which stage the megaspore mother cells were at the period from diplotene to diakinesis, high temperature treating with 41℃ for 4 h was applied to induce 2n gametes through megaspore chromosome doubling. One hundred and sixty four allopolyploid were produced via first-division restitution (FDR) and second-division restitution (SDR). Additionally, according to the timing from the optimal pollination period of P. pseudo-simonii×P. nigra ’Zheyin3#’, when 54-84 h after pollination, high temperature treating with 41 ℃ for 4 h was applied to induce 2n gametes through embryo sac chromosome doubling. Ninety four allopolyploid were produced via postmeiotic restitution (PMR). It provides the material basis for the studies on the source identification and the heterozygosity evaluation of 2n gametes.(2) A new strategy was reported to characterization hDNA through the inhibition of post-meiotic segreation (PMS). Based on inhibiting chromosome segragation at the process of embryo sac development (which is equal to PMS) after meiosis by physical or chemical mutagens, chromosome with hDNAs would form two sister chromatids during this mitosis and then be captured within the same unreduced 2n female gamete cell without segregation. Hybridization with a sperm cell could fix hDNA information into a triploid zygote. Finally, hDNAs could be detected through the allelic configurations analysis of triploid hybrids, using co-dominant simple sequence repeat (SSR) markers. According to this strategy, it was proved that hDNA was indeed produced during the process of homologous recombination in Populus. With the advantages of low cost, no need for complex technology, and being suitable for the non-sequencing species, this strategy has realized the direct detection of recombination product hDNA in angiosperms at the molecular level and opened up a new way for the study of homologous recombination mechanism in poplar and other angiosperms.(3) Based on the new strategy for characterization of hDNA in higher plants, homologous recombination characteristics were uncovered firstly in Populus. It was shown that hDNAs frequencies, which were positive correlated with locus-centromere distance, were varied from 5.3% to 76.6% among different SSR loci. It was also reported that distribution patterns of recombination rates, the number variation of MeDIP reads, the proportion of bases in LTR Gypsy retrotransposons and low-complexity sequences, gene density were covaried with hDNAs frequencies. However, no correlation was found between physical chromosome length and average frequency of hDNA in this study. According to formation status of hDNA for two loci which were adjacent to each other at the same chromosome, the number of recombination events per meiosis was counted for each chromosome. It was shown that 89.89% of chromosomes displayed 1-3 recombination events per meiosis. Additionally,4-5 recombination events per meiosis were detected in 5.57% of chromosomes, and one chromosome displayed 6 recombination events per meiosis, which were different with previous studies. The large number of recombination events detected in this study might be attributed to the high heterogeneity of the female parent P. pseudo-simonii × P. nigra ’Zheyin3#’.(4) A strategy was proposed that using SSR markers with low recombination frequencies to identify the formation pathways of 2n gametes in plants. Facing the two problems including FDR and SDR 2n gametes could not be distinguished due to asynchronism of megasporogenesis and homologous recombination effect the assessment of 2n gametogenesis, it was proposed that based on homologous recombination characteristics, using SSR markers with low recombination frequencies could accurately identify the formation pathways of 2n gametes of triploid hybrids. One hundred and sixty four allopolyploid origined from megaspore chromosome doubling were analyzed by using six SSR markers with low recombination frequencies, and it proved that forty triploids originated from FDR 2n eggs, whereas one hundred and twenty four triploids originated from SDR 2n eggs. Accurate assessment of formation pathways of 2n gametes laid a foundation for the heterozygosity evaluation of different types of 2n gametes and their efficient utilization.(5) The maternal heterozygosity transmitted by different types of 2n gametes was analyzed on the population level. And then based on the homologous recombination characteristics, compositive SSR primers selection strategy was proposed for heterozygosity analysis. It was shown that heterozygosity transmitted by different chromosomes were dramatically various, FDR 2n gametes showed the smallest variation of heterozygosity transmitted by chromosomes (CV=13.50%), whereas SDR and PMR 2n gametes showed a greater variation of heterozygosity transmitted by chromosomes (CV=51.92% and CV=63.14% for SDR and PMR 2n eggs, respectively). This was probably because not all, but only half, of the heterozygous parental loci will become homozygous in FDR 2n gametes. FDR, SDR, and PMR 2n eggs transmitted different maternal heterozygosities (74.80%,39.58%, and 35.90%, respectively). The maternal heterozygosity transmitted by FDR 2n eggs was significantly higher than that of SDR and PMR 2n eggs, both with P<0.05. Additionally, on the basis of studies on homologous recombination characteristics, a compositive primers selection strategy, which was relied mainly on suitable primers selection strategy while random primers selection strategy subsidiary, was proposed to provide the basis for the effective screening of polymorphism markers loci in heterozygosity analysis. Related studies has great significance to the theoretical research and efficient application of 2n gametes in Populus and other species.(6) Based on the general understanding that there was more utility value of breeding in FDR type 2n gamete with high parents heterozygous, a different viewpoint was proposed. According to the homologous recombination characteristics in poplar, while the average heterozygosity of SDR and PMR types 2n gamete were approximately half of that of FDR type 2n gamete, hDNAs frequencies were covaried with gene density on chromosome and then there was more opportunity to generate chromosome crossover in these regions, thus specific target genes should be heterozygous in SDR and PMR types 2n gametes and then transmit more parental heterozygosity in these regions. It was showed that breeding value of SDR and PMR types 2n gametes is not necessarily lower than that of FDR type 2n gamete. All of three different types of 2n gametes should have certain utility value in breeding.