| Chinese chestnut(Castanea mollissima BL.),belongs to the Fagaceae family,is known as the "king of dried fruits" because of rich nutrients in its seed kernel.However,the yield per unit area of Chinese chestnut is relatively low,which is closely related to the imbalance of male-female flower ratio,poor pollination and fertilization,and embryo abortion.Fertilization and embryonic development are the key links of ensuring higher yield of Chinese chestnut,but abortion at any stage of these processes will reduce its seed setting rate and thus seriously affecting the yield.In this study,using the chestnut cultivar ‘Zunhuaduanci’ as test materialthe,and then the key stages of fertilization and embryo development were determined by cytological observation of ovaries and ovules after pollination,and the key genes related to these processes were selected by transcriptome sequencing of ovule at the critical stages of fertilization and seed development,which will provide a reference for the study of gene function.The main results are as follows:(1)From the anatomical structure,in the 7th day after pollination,chestnut had significantly enlarged ovaries,and ovules had been formed.In the 15 th day after pollination,the embryo sac was mature,but fertilization had not been completed.In the pollinated ovule of 18 th day after pollination,the nucellus tissue gradually disintegrated and the endosperm nucleus began to divide,indicating the completion of double fertilization.Therefore,the pollinated ovules completed double fertilization in 16-17 th day after pollination,however,the embryo sac of the unpollinated ovule was immature.In the same ovary of 21 st day after pollination,there were significant differences in morphology between developed and abortive ovules,and the massive internal endosperm nuclei were divided.In the 27 th day after pollination,the globular embryo and a large number of endosperm cells had been formed in the developed ovules,while the vacuolar embryo sac was observed in abortive ovules.(2)Transcriptome analysis of the unpollinated ovules and pollinated ovules of 18 th day after pollination yielded 7023 differentially expressed genes,including 2915 up-regulated genes and 4108 down-regulated genes.The key genes related to fertilization mainly include aldehyde dehydrogenase family 2 member B7,sugar carrier protein C,phospholipase D alpha 4,transcription factor WRKY40,wall related receptor kinase 1,glycine-rich DOT1 protein,and MYB108,etc.These results indicate that the key genes expressed in the pollinated ovules are beneficial to the improvement of disease resistance,the supply of energy for the completion of fertilization,and the rapid growth of female flower clusters.(3)Transcriptome analysis of the developed ovules and abortive ovules of 21 st day after pollination yielded 1615 differentially expressed genes,including 530 up-regulated genes and 1085 down-regulated genes.The selected key genes related to embryo development mainly include B3 domain-containing transcription factor LEC2,transcription factor MYB118,glutelin type-B 2,sodium/calcium exchanger NCL1,nucleolin,eukaryotic translation initiation factor,polygalacturonidase,glycine-rich cell wall structural protein 1,expansin-A23,and histidine-containing phosphotransfer protein 2,etc.These results indicate that the key genes expressed in the developed ovules regulate embryogenesis,cell wall plasticity,cell proliferation and growth to promote the enlargement of female flower clusters.(4)Transcriptome analysis of the developed ovules and abortive ovules of 27 th day after pollination yielded 4370 differentially expressed genes,including 2151 up-regulated genes and 2219 down-regulated genes.The key genes related to embryo development were pectinesterase 2,expansin-A23,AGL61,MYB115,AHP2,two-component response regulator ARR21,SAUR21,cell number regulator 6,and sucrose synthase 2,etc.These results indicated that the key genes expressed in developed ovules regulated embryo and endosperm development,cell division and expansion,and cell wall plasticity,promoting the further enlargement and hardening of female flower clusters.This study reveals the ovule development process,double fertilization period and embryo development process after female flower cluster pollinated,and provids a large amount of transcriptome data on fertilization and embryonic development,which could offer a theoretical basis for improving seed setting rate of Chinese chestnut and further research on molecular mechanism of embryonic development. |
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