Karyotype Analysis And Meiotic Chromosome Behavior Observation On Tree Peony Allotetraploid

Paeonia section Moutan is a traditional Chinese famous flower and has always been known as the“King of Flowers”.The inter-subsectional distant hybridization is a significant direction in modern tree peony breeding.The inter-subsectional hybrids generally have low fertility,while the inter-subsectional advanced generation hybrids show good cross compatibility in cross breeding.The cytological study of the inter-subsectional advanced generation hybrids can reveal the formation mechanism of their fertility differences.In this study,by optimizing the two aspects of pretreatment time and pretreatment solution,the pretreatment method of chromosome preparation was improved,and the quality of chromosome preparation was improved.On this basis,karyotype analysis was carried out on three tetraploid intersubsectional advanced generation hybrids of tree peony with significant differences in pistils and stamens.By studying the three advanced generation hybrids meiotic chromosome behavior and its performance in cross breeding,and then the mechanism of fertility differences of advanced generation hybrids was obtained from cytological level.The main results are as follows:(1)The chromosome preparation was optimized by using P.× lemoinei ‘High Noon’,and the most suitable pretreatment method for tree peony was selected as follows: pretreatment with pdichlorobenzene for 36 ～ 72 h or p-dichlorobenzene-α-bromonaphthalene for 24 ～ 48 h at 4 °C.The more accurate karyotype information of P.× lemoinei ‘High Noon’ was obtained,and the karyotype formula was 2n = 2x = 10 = 8m(2SAT)+ 2st(2SAT).Compared with the previous study,three satellite chromosomes were newly discovered.(2)Using the method of chromosome optimization,the accurate karyotype information of three advanced generation hybrids 07-LB-30,07-LB-22 and 07-LB-56 was obtained.The chromosome number of the three materials were 20,which were tetraploid tree peony,and the karyotypes were all 2A.The karyotype formula of 07-LB-30 was 2n = 4x = 20 = 16m(1SAT)+ 4st(1SAT);the karyotype formula07-LB-22 was 2n = 4x = 20 = 15m(1SAT)+ 1sm(1SAT)+ 4st(3SAT);the karyotype formula of 07-LB-56 was 2n = 4x = 20 = 16m(3SAT)+ 4st.Chromosome composition of the three materials was complex and showed high heterozygosity.(3)The rate of abnormal meiotic process of microspore mother cells of 07-LB-30,07-LB-22 and07-LB-56 respectively reached 40.67%,72.37% and 53.75%.Chromosomal abnormal behaviors were common in both the first meiotic division and the second meiotic division.In the first meiotic division,unequal chromosome segregation caused by the phenomenon of univalent,multivalent,lagging chromosome or early separation of sister chromatids,and chromosome fragment deletion due to the phenomenon of chromosome bridge and fragments may lead to reduce fertility of gametes and generation of aneuploid gametes.The dyad and triad due to abnormal spindle orientation such as parallel spindle,fused spindle,tripolar spindles of the second meiotic division,which may lead to the production of unreduced gametes,and the proportion of microspore mother cells producing dyad and triad in the three materials ranged from 8.61 to 12.72%.In addition to the above abnormal behaviors,other abnormal behaviors such as monads,empty spores and micronucleus or extra microspores were observed in meiotic products.(4)Three advanced generation hybrids showed different fertility in the hybridization experiment.The average seed setting rate of 07-LB-30 was as high as 5.83 seeds per flower for female and 3.96 seeds per flower for male,indicating that 07-LB-30 was a parent with good fertility performance.07-LB-22 was male sterile,and the average seed setting rate was as high as 5.78 seeds per flower,showing high fertility of female parent.The average seed setting rate of 07-LB-56 female parent was 0.30 seeds per flower,and the fertility of female parent was low.The average seed setting rate of male parent was 3.53 seeds per flower,showing high fertility of male parent.In this study,an optimized chromosome preparation method of tree peony was established,accurate karyotype information of three allotetraploid tree peony was obtained,and a high degree of heterozygosity of the karyotype was found.The cytological processes leading to reduced fertility,aneuploid gametes and unreduced gametes were revealed by observing the chromosome behavior during meiosis.The differences in fertility of different tetraploid tree peony when used as male and female parent were found through cross breeding experiments.The results of the study provided an important reference for exploring the origin of tetraploid tree peony and promoting its application in breeding.