| Six leaf- wrinkled and dwarf plants(8~#,10~#,14~#,15~#,28~#,42~#) derived from the hybridization of Pingyi Tiancha[M.hupehensis(Pamp.) Rehd.var.pingyiensis Jiang]and Zha'ai Shandingzi[M.baccata (L.) Borkh.]were selected as the experimental materials and the two parents as the contrasts to make the overall observation and systematic investigation about the characteristics of their florescence and fruit setting and comparison of their apomictic capabilities,as well as their embryogenesis development by the help of laser scanning confocal microscope(LSCM) and paraffin-section method.For definition ploidy and karyotype characteristics of the offspring derived from the hybridization of Pingyi Tiancha,using four leaf-wrinkled and dwarf plants(10~#,14~#,15~#,28~#) derived from the hybridization of Pingyi Tiancha and Zha'ai Shandingzi,one smooth-leaf plant and four seedlings of two groups derived from di-embryonic seeds of Pingyi Tiancha as the experimental materials,the two parents and normal M.baccata as the contrasts,the chromosome number was observed and the karyotype was analysed by the methods of Squash and flow cytometer.The possible evolution trend and the genetic relationship among the experimental materials were discussed.Based on the observation of embryogenesis development,the occurrence time of apomixis in Pingyi Tiancha was determined,the suppresson subtractive cDNA library of Pingyi Tiancha ovaries was constructed in florescence and the sequence and homology of the differential genes acquired from suppresson subtractive cDNA library was analysed through differential screening. This research will establish a foundation for cloning the related apomictic genes of Pingyi Tiancha and genetic engineering of fruit trees,as well as this research will enrich the utilization and breeding of apomictic rootstock resources in Malus and afford embryological,cytological and molecular biological reference for revealing the genetic mechanism of apomixis in Pingyi Tiancha.The experimental results as follows:1.All the leaf-wrinkled and dwarf plants(8~#,10~#,14~#,15~#,28~#,42~#) derived from the hybridization of Pingyi Tiancha and Zha'ai Shandingzi had no pollen.Their fruit setting percentage and average seed number per fruit were lower than their maternal parent Pingyi Tiancha,but there was relative diversification among the six plants.The average weight per fruit in order of precedence was Zha'ai Shandingzi＜the leaf-wrinkled and dwarf plants＜Pingyi Tiancha.After the treatment of pistil-cut,all the six plants' fruit setting percentage declined obviously,all their fruit-producing percentage were lower than 20%and were lower than that of Pingyi Tiancha(84.1%) significantly.As well as their average seed numbers per fruit were lower than that of natural pollination.All above indicated that these leaf-wrinkled and dwarf plants had some apomictic capabilities,but the degrees of the apomictic capability were greatly lower than Pingyi Tiancha.2.The six leaf-wrinkled and dwarf plants could carry on the normal process of the meiosis and form the functional megaspores from which the sexual embryo sacs derived.From the full blooming day to 2 days later,the eight-nucleate embryo sac began to develop and the sperm cells or the pollen inclusion emerged and the fertilized eggs were partially observed in the embryo sacs of the six leaf-wrinkled and dwarf plants.The proembryos formed about 10 days after the full blooming day.There were some differences about the distributing way of the nucleolus in the specific time during the development of the sacs,and few poly-embryo sacs were observed in some of the leaf-wrinkled and dwarf plants.So that the embryogenesis characteristics of the leaf-wrinkled plants were basically consistent with Zha'ai Shandingzi whose characteristics of the sexual reproduction were distinct.Compared with Pingyi Tiancha,they did not have the complex phenomena during the process of the embryogenesis development.Generally speaking, the sexual reproduction was the main reproductive mode of the leaf-wrinkled and dwarf plants which had the weak capability of apomixis.3.The results of the ploidy determination indicated that the ploidy of the two groups of seedlings derived from Pingyi Tiancha di-embryonic seeds(8-1,8-2;12-1,12-2 ) were 3X,four leaf-wrinkled plants (10~#,14~#,15~#,28~#) derived from the hybridization of Pingyi Tiancha and Zha'ai Shandingzi were 4X,the chromosome number of the leaf-smooth plant(aneuploid plant) was 2n = 43.The karyotypes of these materials were all small chromosomes whose morphological variations and relative length ratios were not obviously different.The karyotypes of these materials were composed of metacentric chromosomes(m) and submetacentric chromosomes(sm) and different karyotypes presented different degrees of differences, which related to four karyotypes of 1A,2A,1B and 2B respectively based on the characteristics of the karyotypes.4.The suppression subtractive cDNA library of Pingyi Tiancha ovaries in florescene was constructed by suppression subtractive hybridization technology using Pingyi Tiancha ovaries at the full blooming day (tester) and 1d before the full blooming day(driver) as the initial matierials.Key points in the course of construction,including qualities of total RNA and starting mRNA,adaptor-ligated efficency and hybridization efficiency were controlled for the better library quality.768 white clones were selected by blue-white spot screening whose inserted cDNA fragments were distributed between 250 and 1200bp which was in accord with expectation of cDNA library after PCR amplification.All of the above were the basis of screening of differentially expressed genes with high efficiency.5.Dot-blotting was performed to screen subtractive cDNA library and 304 positive clones were acquired.After sequencing the positive clones,114 effective expressed sequence tags(EST) were obtained. BLAST analysis,functional annotation and category in Genebank showed that 85 ESTs encode for functionally known proteins and other 29 unique sequences had no match proteins and non-redundancy database which probably represented new genes or high variant non-coding regions of cDNAs.Biological functions of the 84 ESTs were related to basal metabolism,protein metabolism,metal transport,membrane protein,stress response,transcription factor and so on.All above indicate that the representative genes of these ESTs in Pingyi Tiancha during this period participated in many aspects of plant cellular activities.6.EST sequencing and analysis indicated that there were some related apomixis genes in the subtractive cDNA library.Ubiquitin 4(Contig13),cysteine protease(B20) and senescence-associated protein(A21) were found that they were associated to the related genes of programmed cell death(PCD) in ovaries which was further confirmed the important phenomenon that the sexual embryo sac aborted and the asexual embryo sac developed during the course of 1d before and 1d after the blossom day in Pingyi Tiancha,and which also further verified the characteristics of facultative apomixis in Pingyi Tiancha. Similar to S-adenosylmethionine decarboxylase(B26),S-adenosylmethionine methyl-transferase(B70), allene oxide synthase(B38),ethylene receptor(B96),cytochrome P450(B58) and methionine synthase (B77) were found in ovaries of Pingyi Tiancha at 1d before the blossom day,which related to plant hormone and could participate in metabolic pathways of ethylene,polyamine,jasmonate acid and gibberellin,et al.,as well as had close relationship with maturity and senescence of plants.All of these indicated that the occurrence of the asexual embryo sac development and the sexual embryo sac abortion might be a omplicated,interaxtive system involving multiple approaches and multiple genes.
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