Discovery Of Genes Associated With Dichogamy In Walnut(Juglans Regia L.)Based On Transcriptomic Analysis

Walnut is a dichogamy plant which ensures self-fertilization does not occur.It is divided into two main types,Protogynous?female flowers mature early?and Protandrous?male flowers mature early?.There is difference in maturing time between male and female flowers in same species,leading to low pollination rate resulting into the low fruit set.This has resulted to the decline of the quality and yield of walnut fruits.Therefore,the experiment was aimed to study the significance of flowering mechanism in walnut.In this study,flower buds of protandrous cultivar‘Xin Zaofeng'?‘XZF'?and protogynous cultivar‘Ji Zaofeng'?‘JZF'?were used as experimental materials.The genes responsible for regulating the difference in time for the opening of the flowers in the walnut were studied during the experiment with the main contents leading to the following conclusions:1.The phenotypic character of male and female flower buds were observed during the whole development periods of protandrous cultivar‘XZF'and protogynous cultivar‘JZF'in Henan.The growth pattern and morphological development of the fruits of protandrous and protogynous cultivars were recorded during the study.The results indicated that after entering the spring season,the external morphology of female and male flower buds of cultivars‘XZF'and‘JZF'gradually showed significant differences at?P<0.05?.The germination stage in spring season,in late February to early March could be selected as a critical period for male and female flower buds differentiation time between protandrous and protogynous cultivars in study area.2.In the experiment area Henan,walnut flower buds entered to the stage of morphological differentiation during March.The female flower buds of protogynous cultivar‘JZF'differentiated faster than those of‘XZF'.While,in protandrous cultivar,male flower buds of‘XZF'differentiated faster than those of‘JZF'.By using the method of paraffin section,we found that on the dates of Feb 6^th,Mar 21^stt and Apr 7^thh in 2018,the female flower buds of‘JZF'were in the perianth primordium differentiation stage,pistil primordium differentiation stage and female flower maturation stage and the female flower buds of‘XZF'were in the sepal primordium differentiation stage,perianth primordium differentiation stage,and pistil primordium differentiation stage,respectively.Similarly,the male flower buds of‘XZF'were in the stamen primordium differentiation stage,anther differentiation stage and male flowers maturation stage during Feb 6^th,Mar 21^stt and Apr 7^thh in 2018 and the male flower buds of‘JZF'were in the bract primordium differentiation stage,stamen primordium differentiation stage and anther differentiation stage,respectively.The development and maturation of male and female flower buds of‘XZF'and‘JZF'developed in advance for one development period,respectively.3.Sequencing analysis of transcriptome revealed 11 genes related to the flowering mechanism of dichogamy in walnut,including 10 genes related to the flower development and one gene related to gibberellin response signal.They were as:one FT-like gene,one FUL-like gene,one SOC1-like gene,one SPL-like gene,one SVP-like gene,one AGL24-like gene,one AP1-like gene,one LEAFY-like gene,one FLC-like gene,and one FD-like gene,and one gibberellins response gene DELLA-like.The results of transcriptome sequencing were verified by qRT-PCR.The analysis results of structure and expression showed that these genes played a key role in the flowering process of walnut.4.Two candidate genes were selected for homology comparison,cloning and functional analysis.They were FT-like gene?WALNUT₀0002262?and FUL-like gene?WALNUT₀0024135?.The ORF length of FT-like was 525bp,coding 174 amino acids,and contained a PEBP domain structure.Phylogenetic analysis showed that FT-like coding amino acid sequence had the highest similarity with FT and TSF in Arabidopsis thaliana,up to 94%.The ORF length of FUL-like was 747bp,coding 248amino acids,and contained a MADS-box domain structure and a K-box domain structure.Phylogenetic analysis showed that FUL-like coding amino acid sequence had the highest similarity with FUL inArabidopsis thaliana,up to 72%.It showed that these two genes may be key genes regulating the flowering of walnut.We also constructed the gene over expression vectors of these two genes for further function research.