| Chinese jujube（Ziziphus jujuba Mill）,a member of the genus of Ziziphus in Rhamnaceae is one of the most traditional important dried fruit and its annual output was ranking first in china.But Chinese jujube with very low kernel content caused by self-incompatibility has restricted the development of the jujube industry.Therefore,the study of the self-incompatibility in China jujube has supplied significant guidance for the cross breeding and effective production.This experiment is intended to study the self-incompatibility of jujube from both the physiological and molecular levels.In the aspect of physiological experiment,through the designed different self-and cross-pollination combinations,used fluorescence microscopy and paraffin sectioning techniques to explore the pollination compatibility among different jujube cultivars;in the aspect of molecular experiment,the forward and reverse specific primers were designed by using the known Jujube S-RNase gene,and through the PCR specific amplification,target fragment recovery,cloning and sequence of PCR production,homologous detection,sequence alignment analysis,and phylogenetic tree construction using MEGA to identify the S-RNase genes of 32 jujube cultivars and 11 wild jujubes and their genotypes.The main results were as follows（1）Using specific primers to clone genes.4 S-RNase alleles,JZS11-RNase（S11）,JZS12-RNase（S12）,JZS13-RNase（S13）,JZS14-RNase（S14）were identified from 32 jujube cultivars and 11 wild jujubes,and had strong homology with the similarity with 100%,85%,86% and 86% compared to XP015875313（termed S-F11-like）,respectively.S11 reported previously was the same gene as S-F11-like,while the remaining 3 new alleles were deposited in GenBank with the accession numbers of MG553632,MG53633 and MG553634.S-RNase protein sequence of the jujube showed less similarity with that involved in Rosaceae,Solanaceae and Rutaceae,no homology among them,but they all belonged to T2-RNase family.S-RNase genotypes were classified into three categories based on amino acid sequence alignment and cluster analysis,6 individuals as heterozygote S13S14 were Dunhuangdazao,Lingwuchangzao,Jinzao,Qiyuexian,Linyilizao,Yongjihamazao;a single one was heterozygote S12S14 was Qingjiansuanzao;remaining 36 individuals as homozygote S11S11.（2）Fengmiguan,Xiangfenyuanzao,Huizao,Jinzao,Yanchuanlaoyasuanzao,Dongzao and Yongjihamazao were selected as materials and designed self-and cross-pollination combination.Fluorescence microscopy was used to observe pollen germination and pollen tube growth after pollination.The results indicated that Some pollen grains germinated in stigma,3 h AFP（after pollination）;pollen tube penetrated mastoid cell into the stigma,8 h AFP;pollen tube penetrated at 1/3 of style,24 h AFP;pollen tube penetrated at middle of style,48 h AFP;pollen tube that stopped growing while penetrated at middle of style,96 and 129 h AFP,as was observed in self-pollination combination（Dongzao × Dongzao,Huizao × Huizao）;In cross-pollination combination of Fengmiguan × Xiangfenyuanzao,Fengmiguan × Yanchuanlaoyasuanzao,Huizao × Jinzao,Xiangfenyuanzao × Yongjihamazao,some pollen grains germinated into stigma,3 h AFP;pollen tube penetrated mastoid cell into the stigma,8 h AFP;pollen tube penetrated at 1/2 of style,24 h AFP;pollen tube grew to the lowest part of the style,48 h AFP（Fig.4-D,G,P）;pollen tube penetrated the stylar base,72 h AFP.Self-pollinators exhibited self-incompatibility and cross-pollinators showed compatibility.（3）13 days after pollination,paraffin section and microscopic observation showed that in ovary of pollinator Fengmiguan × Xiangfenyuanzao,one ovule degenerated and the other kept on developing,embryonic development was at the stage of the small globular gradually formed after the transverse fissure of zygote;endosperm was at the free nuclear stage,the division of the free nucleus was extremely rapid.embryo abortion happened in Huizao × Huizao.In the unfertilized ovary,the outer integument was shrunk,and the egg apparatus and embryo sac disintegrated.Overall,two types,cross-compatibility and self-incompatibility,were involved in Chinese jujube,and the self-incompatibility belongs to prezygotic mechanism.Moreover,the compatible phenomenon we found in some pollination combinations indicated that both the pistil S-RNase gene and the pollen SFB gene were required to be comprehensively considered in design of pollination combinations.This current study revealed that the jujube with very low kernel content is caused firstly by self-incompatibility,secondly hypoplasia of embryo result in abortion.This finding also provides insight into crossbreeding and parental choice for the jujube. |
