Identification Of Short-juvenility Germplasm And Screening Of Associated Genes Of Walnuts In Xinjiang

Xinjiang walnut（Juglans regia L.）can be divided or classified into two types:early-flowering（EF）type and late-flowering（LF）type.Among them,EF walnut seedlings may can complete the transition from juvenility phase to adult phase in the duration of 1-3 years（also known as“short-juvenility”）.However,the LF walnut seedlings usually take 5-8 years to complete the floral transition.Study on the regulation mechanism of short-juvenility has important applications for mining excellent traits and accelerating the breeding of walnut varieties.Thus,this study aims to screen the EF germplasm of Xinjiang and to discover the regulatory genes of short-juvenility traits,to pave the way for the basic research and breeding applications in walnut.In this study,the segregated F₁ materials were obtained from the crossing between EF walnut cultivar‘Xinwen 81’and the local LF walnut germplasm.Transcriptome（RNA-seq）and small RNA-seq of flower buds undifferentiated leaf buds from EF and LF in the population.Furthermore,the transcriptome and small RNA sequencing data were compared with the published walnut reference genome information to identify the differentially expressed genes and differentially expressed mi RNAs related to the flowering transition of walnut,and screened out the genes that regulate the flowering transition of early fruiting walnut.And then,these candidate genes were verified by heterologous expression in Arabidopsis thaliana.The main results were listed as follows:1.A total of 167 hybrid seeds was obtained by crossing between‘Xinwen 81’（EF,female parent）and a late-bearing walnut（LF,male parent）with good resistance and high yield.After seeding,84 F₁ individuals were obtained.In the F₁ population,the single plant that completed the flower transformation in the same year after sowing was H16;the traits survey in the second year found that 7 plants（including H16）had bloomed;the survey in the third year found that a total of 36 F₁ plants had completed flower bud differentiation,the early-flowering rate was 42.86%;until the seventh year,all 84 F₁ plants completed the transition into flower.2.We have used the BSA analysis strategy,RNA-seq analysis was performed on the above-mentioned early-flowering and late-flowering F₁ stage in individual plants,where3,009 differentially expressed genes（DEGs）were identified.Among them,933 genes were up-regulated（31%）,and 2,076 were down-regulated（69%）.Part of these DEGs have been verified by q RT-PCR,and the homologous genes Jr FLC,Jr FT,Jr SOC1 and Jr LFY related to Arabidopsis during flowering stage,were further identified in early-flowering and late-flowering stage in individual plants.Among them,Jr FLC was down-regulated in early flowering stage plants,while Jr FT,Jr SOC1 and Jr LFY were up-regulated.In addition,small RNA-seq analysis was also performed in this study,and 45 differentially expressed mi RNAs related to the flowering transition of early fruiting stage in walnut were found and verified by q RT-PCR.3.The flower-inhibiting gene Jr FLC and the flower-promoting gene Jr LFY were cloned in walnut,and functionally verified by heterologous overexpression transformation model plant A.thaliana.Statistical finding of flowering schedule patterns in transgenic plants and wild type（WT）revealed that 35S:Jr FLC inhibited flowering,while 35S:Jr LFY promoted flowering.This indicates that walnut Jr FLC and Jr LFY may have similar functions to Arabidopsis At FLC and At LFY,and further homologous transformation of walnut ontology is required to study.In conclusion,this research investigated the juvenility traits of walnut resources in Xinjiang,and acquired a lot of number of DEGs and mi RNAs by combining BSA with RNA-seq and small RNA-seq,providing important genetic resources and data for the study of walnut juvenility traits.In particular,the walnut flowering-related genes Jr FLC and Jr LFY verified by heterologous transformation are expected to further explore the flower transition of Xinjiang EF in EF walnut.In addition,the F₁ individual plant population obtained by crossing the EF walnut‘Xinwen 81’with excellent comprehensive quality and the LF walnut with good resistance and high yield is also expected to breed new EF walnut for cultivars.In conclusion,the results of this study can provide clues for the short-juvenility genetic characteristics of Xinjiang EF walnut,and provide the materials and candidate functional genes for genetic improvement and breeding application in walnut.