Genetics Studies On Sex Identification In Seabuckthorn

Sea buckthorn(Hippophae L.)is a dioecious shrub that exhibits sexual dimorphism with male and female flowers present on separate plants.Its sex differentiation is noticeable but only discernible during the flowering and fruiting period.Early-stage sex determination in sea buckthorn is critical for genetic improvement and commercial cultivation.In this study,highthroughput RNA sequencing data from leaves of two Seabuckthorn species,Hippophae rhamnoides L.subsp.sinensis and H.neurocarpa subsp.neurocarpa,were utilized to identify differentially expressed genes between male and female plants.Subsequently,molecular tools were developed for sex identification of the two Seabuckthorn species.Finally,chromosome karyotypes of male and female sea buckthorn materials were obtained,providing data for clarifying the presence of sex chromosomes in sea buckthorn.This study enhances our understanding of the sex determination mechanisms in dioecious plants and the evolution of sex chromosomes.The experimental results are as follows:(1)Transcriptome sequencing is a powerful tool for identifying novel genes and understanding complex biological processes.In this study,the leaves of Seabuckthorn subspecies were subjected to transcriptome sequencing using Illumina HiSeq high-throughput sequencing technology.A total of 40 GB of usable data with an average error rate of 0.015%was generated,which was assembled using Trinity software into 214,928 unigenes and 281,671 transcripts.The unigenes had a maximum length of 17,356 bp,and the average lengths of the unigenes and transcripts were 588 bp and 781 bp,respectively.BLASTx analysis against public databases revealed that 99,421 unigenes were annotated in at least one database,accounting for 46%of the total unigenes.Furthermore,a total of 44,072 potential Simple Sequence Repeat(SSR)sites were detected in the transcriptome data.Gene expression analysis between the two transcriptomes showed that 101 genes were significantly upregulated in female Seabuckthorn subspecies,while 12 genes were downregulated.Additionally,two genes were found to be expressed only in males,and one gene was expressed only in females providing insights into the sex determination mechanism of dioecious Seabuckthorn subspecies in H.rhamnoides L.subsp.sinensis.(2)In this study,the transcriptome of male and female plants of H.neurocarpa subsp.neurocarpa were generated using Illumina HiSeq high-throughput paired-end sequencing technology.Using Trinity software resulted in a total of 151,066 unigenes and 210,961 transcripts,with a maximum length of 17,886 bp and average lengths of 588 bp and 808 bp,respectively.BLASTx analysis against public databases revealed that 59,075 unigenes were annotated in at least one database,accounting for 39.1%of the total unigenes.Additionally,a total of 31,016 potential SSR sites were detected in the transcriptome data.Differential gene expression analysis using DESeq2 identified 64 genes with significant expression differences between male and female plants,including 14 downregulated genes and 44 upregulated genes in female plants.These findings provide valuable insights into the molecular mechanisms underlying sex determination in the dioecious H.neurocarpa subsp.neurocarpa.The annotated unigenes and potential SSR sites identified in this study provide a valuable resource for future studies aimed at investigating the functional roles of genes involved in the development and growth of Seabuckthorn.(3)Based on previous research on sex identification in Seabuckthorn using molecular markers such as SCAR and RAPD,two male-related molecular markers,ZHAD1(5’A********C-3’)and ADZH2 F(G************************CA)&ADZH2 R(GA******************CA),were identified.These markers exhibited polymorphism between male and female genotypes of H.rhamnoides L.subsp.sinensis and H.neurocarpa subsp.neurocarpa,and successfully identified the genotypes of male and female plants.Therefore,they can serve as genetic markers for sex identification and accelerate the commercial application of this species,ultimately improving its economic value.(4)Study conducted a detailed karyotype analysis of H.rhamnoides L.subsp.sinensis and H.neurocarpa subsp.neurocarpa,revealing that both species have 2n=24 chromosomes,indicating the presence of sex chromosomes.Comparison of their karyotype characteristics and symmetry degree confirmed the existence of sex chromosomes in both species.The study also analyzed the similarities and differences in chromosome karyotypes between HNN-1(♀)and HNN-2(♂),and HRS-1(♀)and HRS-2(♂)The karyotype formula for HNN-1(♀)was 2n=24=14M+10m,while that for HNN-2(♂)was 2n=24=10M+14m.The karyotype formula for HRS-1(♀)was 2n=24=12M+12M,and that for HRS-2(♂)was 2n=24=10M+14m,with a karyotype asymmetry coefficient of 52.4%,51.8%,52.6%,and 51.6%,respectively.These results suggest that the sex chromosomes of male plants are heteromorphic,while those of female plants are homomorphic.The Y chromosome was found to be larger than the X chromosome.These findings have important implications for the understanding of the genetic basis of sex determination in sea buckthorn and lay a foundation for further studies on the molecular mechanisms underlying sex determination.The results also provide valuable information for the breeding of improved cultivars of this important plant species.