The Role And Mechanism Of Allotriploid In Polyploid Formation Of Brassica

Genome duplication has been a significant feature in plant evolution and speciation.The triploid bridge pathway and triploid block play an important role in the formation and evolution of polyploids.However,the detailed process and mechanism of allopolyploid formation via allotriploid remain poorly understood.The ease of ploidy manipulation and interspecific hybridization,the existing suite of molecular cytogenetic techniques,and the availability of reference genome sequences make Brassica an ideal model species for investigating the consequences and mechanisms of de novo allopolyploidization.In this study,we obtained four types allotriploids（F₁）by crossing between different Brassica parents,and then analyzed their chromosome pairing and segregation behavior during meiosis using chromosome accurate identification technique.We continuously tracked the chromosome constitution of selfing F₂-F₆progenies of allotriploids,combining with the data of pollen viability and seed yield,we discovered the role and mechanism of allotriploids in allopolyploid formation.In addition,introgression analyses were performed in the newly formed allotetraploids from allotriploid self-fertilization progeny using bioinformatics,demonstrated the role of allotriploid in unidirectional gene flow between diploid and allotetraploid.Our findings provide a mechanism of polyploid formation and persistence.The main results are as follows:1.Karyotype verification of the numerical and structural chromosome changes for all hybrid strains to select the true allotriploid AAC BrassicaAllotriploids（AAC,2n=3x=29）were produced by different crossing methods using diploid Brassica rapa（AA,2n=2x=20）,as well as natural and resynthesized allotetraploid Brassica napus（AACC,2n=4x=38）as parents.Karyotyping results showed that only 25 out of 41 candidate allotriploids were true allotriploids,which possess two complete sets of A genome chromosomes and one complete set of C genome chromosomes.The remaining plants were aneuploids containing incomplete genomes of AAC allotriploids.2.Allotriploid AAC Brassica undergoes regular meiosis and produces viable aneuploid gameteIn this study,we demonstrated that allotriploids exhibit basically regular chromosome pairing and segregation during meiosis.We found that the main factors impacting chromosome abnormal pairing and mis-segregation,abnormal pairing included homoeologous pairing and 45S r DNA association,mis-segregation were homologous chromosome nondisjunction and premature sister chromatid separation,which were induced by homoeologous associations and univalents at diakinesis,respectively.We demonstrated that pre-existing homoeologous arrangements in heterozygous plants lead to high rates of meiotic abnormalities.In addition,our results showed that allotriploids AAC Brassica are semi-fertile,and the gametes produced by allotriploid are predominantly viable aneuploid gametes.These results demonstrated that the chromosomal inheritance of allotriploid Brassica has its own rules and undergoes regular meiosis.3.Triploidy is a mediator of the origins of different ploidy cytotypesWe demonstrated that the immediate progeny of allotriploid AAC Brassica were predominantly aneuploids with a wide range of genome contents,including near-triploid,near-pentaploid,and near-hexaploid.Our results further demonstrated that allotriploid Brassica can generate offspring with different ploidal levels from near-triploidy to near-hexaploidy,but do not support the hypothesis that allotetraploid formation via triploids occurs directly through the fusion of euploid gametes in Brassica.4.The pathway from triploid to tetraploidTo investigate the fate of the self-fertilized allotriploids,we comparatively analyzed the karyotypes and fertility of the progeny from the F₂to F₄generations.Our studies demonstrated that within three generations of self-fertilization,allotriploids mainly formed near or complete allotetraploids similar to B.napus via gradually increasing chromosome numbers and fertility,suggesting that allotriploids could act as a bridge in polyploid formation,with aneuploids as intermediates.5.The underlying mechanism of polyploid formation:genome balance and a dosage effect drive the transition from triploid to tetraploidOur results demonstrated that AAC allotriploids readily formed allopolyploids after 2-3 generations of self-pollination by gradually increasing their number of C chromosomes,and there was selection in favor of individuals containing at least one complete set of chromosomes from the C genome in the progeny of AAC allotriploids.These results suggested that disrupting the genome balance of both the A and C genomes had a detrimental effect on fertility,and aneuploid individuals containing fewer imbalanced chromosomes had higher viability and fertility.To explain why selection is likely driving increased chromosome number toward tetraploidy,we propose that genome balance and dosage effects drive allopolyploid formation by constant selection in favor of aneuploid individuals with fewer alterations to the balance of chromosomes among the progeny of allotriploids.6.Reproductive barrier and gene flow between diploids and allopolyploids in BrassicaCombined the introgression analysis of self-fertilized progeny from natural A_nA_rC_n-M allotriploids,we found the pathway from triploid to tetraploid is very feasible,and genome sequences from the diploid B.rapa were transferred to the newly formed allotetraploids.These results demonstrated the process of recurrent triploid-derived allotetraploid formation in Brassica not only help allopolyploids remain isolated from their diploid progenitors but also promote gene flow from diploids to allopolyploids.