Screening And Cloning Of The Key Genes Involved In Self-incompatibility Of Citrus Reticulata Blanco Cv.‘Wuzishatangju’

Citrus reticulata Blanco cv.’Wuzishatangju’,which is seedless and self-incompatibility,is evolved from youzishatangju.Studies showed that the seedless of wuzishatangju was caused by the self-incompatibility of gametophyte,and the self-incompatibility response occurred in the ovary.Self-incompatibility is a complex physiological process.There are a lot of genes involved in self-incompatibility,such as: F-box,S-RNase,the ubiquitin gene and so on.Using wuzishatangju and youzishatangju as materials,the F-box in the pollen,S-RNase in the pistil,and some differential genes were screaned out,combined with the transcriptome sequencing technology.The main results are as follows:1.According to the annotation of unigenes,308 unigenes annotated as F-box were screaned out.Fourteen protein sequences of SLF or SFB were obtained based on the NCBI database.Phylogenetic tree were constructed to analyze the relationship between those F-box candidate genes and genes downloaded from NCBI database.A total of 13 unigenes F-box genes were achieved.2.In the transcriptome,22 unigenes are annotated as RNase,and 121 genes are annotated as Ribonuclease.Then the clustering analysis with 24 reported RNaseshowed that there were 5 Unigenes,which were Unigene0019191,Unigene0040115,Unigene0001013,Unigene0036542 and Unigene0017501.Among them,Unigene0019191 and Unigene0040115 were two transcriptions of the same gene,which was identical with the reported S-RNase gene(GU220071).3.The RPKM≥10,either in ’youzishatangju’ or ’wuzishatangju’,and|Log2 Ratio|≥5 work as the Screening criteria to define the significantly different expressed gene.Acrroding to the standard,14 and 30 unigenes were picked out from pollen and stigma,respectively.4.Among the 13 screened F-box genes,there were seven genes specifically expressed in pollen.They were F-box4(Unigene0050323),F-box5(Unigene0001060),F-box6(Unigene0004230),F-box7(Unigene0004222),F-box8(Unigene0012037),F-box9(Unigene0004272)and F-box11(Unigene0048889),respectively.In the mature pollen of ’youzishatangju’ and ’wuzishatangju’,4 F-box genes(F-box6,F-box7,F-box9 and F-box11)showed different expression levels.5.The sequence analysis of cDNA,DNA and promoter in those 4 F-box genes was finished.Then the sequencing results showed that there were some same heterozygous loci in cDNA of F-box7 and F-box9,and the cDNA sequence of F-box6 and F-box11 was exactly identical.In addition,there were one,four and five identical heterozygous loci in the DNA sequence of F-box6,F-box9 and F-box11,respectively.Two same sequences of F-box9 were found in ’wuzishatangju’ and tetraploid ’youzishatangju’,which was different compared with one single sequence in ’youzishatangju’.Finally,there was no significant difference between the promoter sequences of those four F-box genes.6.Several S-RNase screened out from the phylogenetic tree were used for analysis of tissue specificity,the results showed that S-Like RNase(Unigene0001013)was expressed in pollen,stigma,style and ovary,and there were significant difference between ’wuzishatangju’ and ’youzishatangju’.While the RNase-P(Unigene0017501)showed no obvious tissue specificity.Sequencing analysis of S-Like RNase showed that the O RF of S-Like RNase is 677 bp,and there is no difference in cDN A sequence between ’wuzishatangju’ and ’youzishatangju’.7.The experiment to prove the 44 significantly different expressed genes was conducted.The results showed that,24 unigenes matched with the data of transcriptomes.Then,the expression levels of 24 unigenes in ovary among 3d-4d after self and crossing pollination were observed: the most unigenes expressed differently,in addition to Unigene0018095 and Unigene0031979.8.8 unigenes were selected out from the 24 different unigenes to analyse their tissue specificity and sequence.The results showed that,copper transporter2 and Pectate_lyase specifically expressed in ovary and pollen,respectively,while the other 6 unigenes showed no tissue specificity.Copper transporter2 performs significantly different between ’youzishatangju’ and ’wuzishatangju’,however,the Pectate_lyase behaves the same expression level.Sequencing results showed that,Copper transport-like and Fe(III)-chelate reductase have some different loci between the cDNA of ’youzishatangju’ and ’wuzishatangju’,the other genes share the same cDNA sequence.The b HLH101 promoter sequence contains 1 heterozygous loci in ’youzishatangju’ and ’wuzishatangju’,but not found in tetraploid ’youzishatangju’;Copper Transporter1 promoter contains 7 heterozygous loci only in ’wuzishatangju’;pectate_lyase promoter also contains a number of heterozygous loci,but there were no different among ’youzishatangju’,’wuzishatangju’ and tetraploid ’youzishatangju’.