Causes Of Seedless Forming And Identifiction Of Seedless Related Genes From ’Xiangshui’ Lemon

’Xiangshui’lemon (Citrus limon (L.) Burm. F.) is a seedless cultivar with a strong sweet smell and flowers throughout the year. Genetic relationship of Xiangshui lemon and other cultivar was analysed by ISSR and SCoT markers, furthermore new SSR primer pairs were designed base on lemon gene database. To investigate the mechanisms that may contribute to the seedlessness of’Xiangshui’, we conducted studies addressing pollen and embryo sac fertility, embryo development and compatibility of self-and cross-pollination. In this study, RNA-seq was used to analyze the transcriptomes of stigmas with styles of the ’Xiangshui’lemon24h after no-, self-and cross-pollination. Candidate genes associated with seedlessness were identified, and three of them were cloned. Gene expression patterns were analyed by real time quantitative PCR and the three genes were transformed into tobacco plants for factional study. The main results were summarized as follows:1. The genetic relationships and genetic diversity of39lemon (Citrus limon (L.) Burm. F.), lime (C.aurantifolia (Christm.) Swingle), and Rangpur (C. limonia Osb.) germplasm resources were examined by SCoT and ISSR markers in this study. Both SCoT markers and ISSR markers could discriminate the lime and lemon. All the varieties of Citrus limonia’Guangxi’clustered in one group, while the introduced cultivars from Europe and America clustered in another group.’Meyer’lemon was clustered in a separate group, which was a good agreement with previous reports since’Meyer’lemon is reported to be as a hybrid between lemon and sweet orange or mandarin. There was some difference between the results given by the SCoT and ISSR markers. The’Tahiti’was clustered in the same group with the European and American lemons by SCoT markers, while it was in the same group with’Xiao lime’by ISSR markers. This result may imply that the parents of ’Tahiti’belong to lime and lemon. The lemon varieties’Lisbon’,’Eureka’,’Femininello’ and’Fino’could be distinguished by SCoT markers but not by ISSR markers. The results showed that’Xiangshui’lemon has the nearest genetic relationship with’Baihua’lemon. 2. Microsporogenesis and development of gametophytes of’Xiangshui’lemon was observed by fuchsin dye squash technique. No chromosome bridges, lagging chromosomes or other types of variation were observed in the stages of microsporocyte meiosis of ’Xiangshui’. At the microsporocyte stage, cytokinesis was simultaneous type. Tetrads were mainly tetrahedral, several were isobilateral. Mature pollen grain was2-cell type. Mature pollen grains from’Xiangshui’were spherical, similar to that of’Baihua’. Number per anther, viability and germination of’Xiangshui’and’Baihua’pollen was compared. The results showed no significant difference between them, indicating that the male gametes of ’Xiangshui’lemon are normal..3. Megasporogenesis and embryo sac development of’Xiangshui’lemon was observed by paraffin section technique. The ovule was anatropous, bitegmic and crassinucellate and the development of embryo sac belonged to Polygonum type. The female gametophyte of ’Xiangshui’was normal, as shown by the presence of one egg cell, two synergids, three antipodal cells and a big central cell containing two polar nuclei within the mature embryo sac.4. Hand pollination experiment was carried out. Cross-pollination of’Xiangshui’, cross-pollination of’Baihua’and self-pollination of’Baihua’produced seeded fruits. Fruits of self-pollination and fruits from emasculated, unpollinated flowers of’Xiangshui’were seedless. The results indicated that both the male gamete and embryo sac of’Xiangshui’ are in fact fertile. Furthermore,’Xiangshui’has the ability to participate in parthenocarpy. We observed typical embryonic initiation and development in cross-pollinated’Xiangshui’. But no zygotes and embryonic development was abserved of self-pollinated’Xiangshui’5. To compare the kinetics of pollen tube growth, self-and cross-pollinated pistils were sequentially evaluated. In both self-and cross-pollination, most of pollen grains germinated and pollen tubes elongated in stigma. At the base of stigma, self-pollen tubes became twisted, or the tube tips enlarged and ruptured in the course of extension with appearance of exudant, while cross-pollen tube growth was normal. Cross-pollen tubes elongated and reached the style base48h after pollination, but in self-pollinated pistils, no pollen tube was observed reaching the top of the style and ovary, having been rejected at the bottom of stigma. In cross-pollinated pistils, pollen tubes reached the ovary in72-96h and entered the ovule through the micropyle to achieve fertilization.6. In this study, candidate genes associated with SI were identified using high-throughput lllumina RNA sequencing (RNA-seq). A total of61,224unigenes were produced, with an average length of948bp and an N50of1457bp. A total of47,260unigenes were annotated by comparison to six public databases (Nr, Nt, Swiss-Prot, KEGG, COG and GO). Differentially expressed genes were identified by comparing the transcriptome of no-, self-and cross-pollinated stigma with style in the’Xiangshui’lemon. Several differentially expressed genes that may be associated with SI were obtained.1356unigenes were up-regulated in the self-pollinated transcriptome than others, including F-box gene CL2009contigl.757unigenes were up-regulated and1452unigenes down-regulated in the cross-pollinated transcriptome than others, the ribonuclease genes unigenl4715and unigene17211were among the down-regulated genes. These genes are involved in pollen tube growth, cell death, localization, productive and other processes.7. Ribonuclease genes unigen14715, unigene17211and F-box gene CL2009contigl were cloned, named CIS1, CIS2and ClF-box, respectively. CIS1gene was differently spliced in Xiangshui and Baihua lemon.177amino acids were encoded in Xiangshui, while only118amino acids were encoded in Baihua. This may result fuction changing of C1S1protein, which maybe the reason of compatibility between Xiangshui pollen and Baihua pistls. The results of homology alignment showed that CIS1and ClS2were close to self-incompatibility ribonuclease genes of Nicotiana sylvestris, Petunia and Medicago truncatula. ClF-box gene was close to S-locus F-box gene of Rosaceae fruit trees.8. Gene expression patterns was analyed by real time quantitative PCR. CIS1and CIS2were expressed in pistls specially, and expression was highest in stigma, followed by style. ClF-box gene was expressed in anther specially. All of them were up-regulated in self-pollinated pistls than cross-pollinated ones. CIS1and CIS2genes were highest’ expressed in stigmas at4hours after self-pollination, ClF-box gene was highest expressed in stigmas at3days after self-pollination.9. The construction of plant expression vectors was carried out by In-Fusion kit. CIS1, CIS2and ClF-box genes were transformed into tobacco plants mediated by Agrobacterium tumefaciens.10. SSRs (simple sequence repeats) detection was done with software MicroSAtellite of the61,224unigenes. Total13,318SSRs were identified in10,628unigene with frequency of21.75%. Except mono-nucleotide repeats, trinucleotide repeats were the most abundant repeat units, followed by dinucleotide repeats. Finally1717primer pairs were designed with the production of80-500bp.15of them were selected for genetic relationship analysis in16lemon cultivars. The results were simily with ISSR markers and SCoT markers. It is useful for the identification of closely related lemon cultivars, such as Verna, Villafranca and Kutdiken.