Development Of G.hirsutum-g.Australe Disomic Addition Lines And Study On Alien Gene Expression

Gossypium australe,one of diploid wild cotton species(2n=26,G2G2)native to Australia,belonging to the tertial gene pool of cotton,possesses valuable characteristics unavailable in the cultivated cotton gene pool,such as resistance to Verticillium wilt and certain plant pests,tolerance to cold and drought,and glandless-seed and glanded-plant.In this study,using a set of G.hirsutum-G.australe monosomic alien addition lines,the G.hirsutum-G.australe disomic alien addition lines were obtained.Meanwhile,the full-length transcriptome sequencing technology was used to analyze the G.australe alternative splicing and gene model,and RNA-Seq technology was used to analyze gene dosage effect on G.australe gene expression in alien addition lines.The main results were as follows:1.Development of G.hirsutum-G.australe disomic alien addition lines Combined with the genomic in situ hybridization(GISH)technique and molecular marker,disomic alien addition lines(DAs)could be identified from the selfed progenies of monosomic alien addition lines(MAs).A total of 819 selfed progenies from 13 MAs were tested by chromosome-specific markers first and then it was found that 298 seedlings contained G.australe chromosomes.Based on their GISH patterns,progenies 15-02-20,15-03-9,15-0478 and 15-07-29 had 54 chromosomes,of which one pair of chromosomes displayed brightgreen hybridization signals,demonstrating that it came from G.australe.A total of 52 primer pairs(SSR markers)located on 13 homeologous chromosome groups in cotton were employed to analyze the chromosomal composition of the four progenies.The results indicated that four SSR markers(STV030,NAU6692,cgr5029,cgr5675)located on chromosome 2G,four SSR markers(TMK08,NAU805,NAU3292,dPL0245)located on chromosome 3G,three SSR markers(NAU2120,NAU3437,NAU3791)located on chromosome 4G and five SSR markers(NAU2620,NAU2733,NAU2680,NAU2862,cgr5181)located on chromosome 7G generated clear G.australe-specific bands in 15-02-20,15-03-9,15-04-78 and 15-07-29,respectively.Using the primer pairs distributed on the other 12 chromosomes,G.australe-specific bands appeared in neither these ALs nor TM-1,reconfirming that the two added chromosomes in these putative DAs are a pair of homologous chromosomes.In conclusion,based on the results of GISH and the distribution of SSR markers on the homoeologous chromosomes of the Dt-subgenome in tetraploid cotton,15-02-20,15-03-9,15-04-78 and 15-07-29 are confirmed as G.hirsutum-G.australe 2G,3G,4G and 7G disomic alien addition lines(DAs),denote as DA2G,DA3G,DA4G and DA7G,respectively,which are the first report about the disomic alien addition lines obtanined in cotton.Among them,DA2G seemed normal in fertility and alien chromosome transmission,DA3G produced several seeds but unfurturnately,the 3G chromosome transmited at a very low rate,and DA4G and DA7G showed completely sterile,which hindered the further studies on DLs.The anther numbers significantly decreased in most ALs.Moreover,DAs showed fewer anthers than that of the corresponding MAs.The flower size decreased gradually from TM-1,MA2G to DA2G,indicating that the gene controlling the flower size is located on G.australe Chr.2G and its expression may be regulated by dosage.2.Full-length transcriptome analysis of G.australe Here we sequenced the Australian wild cotton species,G.australe,full-length transcriptome using Pacific Biosciences singlemolecule long-read isoform sequencing(Iso-Seq)from ten tissues pooled cDNA to identify transcript loci and splice isoforms.As a result,we reconstructed the G.australe full-length transcriptome and identified 25,010 genes,74 pre-miRNA and 1,502 lncRNA.In addition,most genes(16,761,67.08%)exhibited two or more isoforms,which suggests a high degree of transcriptome complexity in G.australe.A total of 34,637 alternative splicing(AS)events that involved in five major types were found among the 10,734 gene loci.Among five major types,intron retention were the most frequent,accounting for 66.37%of AS events.30,427 polyadenylation sites were detected from 14,658 genes,8,020 of which have alternative polyadenylation sites(APA).Using the full-length transcriptome as a reference,analysis of root RNA-seq data from hpi24,hpi48,and hpi72 showed that 285,846,and 1535 genes and 299,349,and 361 alternative splice events were differentially expressed,respectively.In addition,based on our AS events annotations,RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination.Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds.RT-PCR indicated that the gel banding pattern and the size of the fragments were consistent with the AS events identified from the Iso-Seq data.We further recharacterized and identified 38 potential genes involved in gossypol biosynthesis,of which 30 contained multiple isoforms.Moreover,intron retention level of gene PB.13680 coding for MK(Mevalonate kinase).gradually decreased during seed germination in G.australe.In conclusion,the reconstructed gene sequences and their AS in G.australe provides a useful information for exploring beneficial characteristics in G.australe.3.Gene dosage effect on expression change Compared to MAs,DAs contain a pair of homologous chromosomes from G.australe,which can be regarded as chromosomeduplication to mimic genome duplication.Here,we developed several G.hirsutum-G.australe alien chromosome addition lines(ALs)that contain a single(MA)or a pair(DA)of G.australe chromosomes in the G.hirsutum background,which can be regarded as G.australe chromosome-duplication to mimic genome duplication and to elucidate the shortterm effects of their gene duplication on gene expression changes.We then investigated the profiling of G.australe gene expression in three pairs of MAs and DAs(2G,3G,and 7G).The results indicated that majority of genes on alien chromosomes displayed dosagedependent expression and maintained their original transcription level,being involved in organelles and primary metabolic activities.Genes displaying dosage-independent were involved in stress responses,cell signaling and endomembrane system.In addition,a significant positive correlation between orthologues by pairwise comparisons was observed,implying trans-regulation played pivotal roles for dosage-independent expression.Thus,dosage-dependent genes are predominantly regulated by cis elements to maintain metabolism and growth stability while dosage-independent genes are mainly regulated by trans-regulator to response environmental changes.The results will provide new insights into mechanisms of gene dosage effect on expression changes induced by chromosome duplications.