Research On Verticillium Wilt Resistance And Fiber Quality Of Cotton Chromosome Segment Introgression Lines By RNA-seq

Cotton(Gossypium)is not only the most widely cultivated fiber crops in the world,but also the significant resource of plant protein and edible oil,which plays an important role in the human economic and social development.Faced with the increasing human population while diminishing arable land,the demand for higher fiber production and better quality becomes increasing along with the rapid development of mordern cotton textile industry.There are many threats causing the output reduction of cotton fiber,however,the so-called “Cotton cancer” Verticillium wilt has already become one of the main severe obstacles for affecting yield and fiber quality of cotton in our country,even around the world.Therefore,it has been such a huge challenge for us to work out how to develop new cotton varieties possessing high yield,superior fiber quality and VW resistance at the present stage.The chromosome segment substitution lines(CSSLs)were constructed from a cross between the donor parent Hai 1,G.barbadense with excellent fiber performance and high VW resistance,and the receptor parent CCRI36,G.hirsutum with high yield and extensive adaptability.After five generations of backcrossing with CCRI36 as the recurrent parent and three generations of consecutive selfing,the CSSLs were successfully developted in virtue of modern marker-assisted selection(MAS)techniques.Based on the trait investigations for years in different environments,we separately selected the cotton varieties harboring high VW resistance and superior fiber performance from our CSSLs population,and RNA-seq was performed to identify the key genes or significant signal pathways during the processes of cotton resistance to VW and fiber development.The results are as followed:1.Analysis on the cotton VW resistance(1)Seven CSSLs with high VW resistance were selected from the cotton VW investigations of natural field in 2014,namely as MBI8232,MBI8255,MBI8983,MBI9066,MBI9626,MBI9743 and MBI9758.Subsequently,the greenhouse,natural field and artifical disease nursery were separately employed to investigate the VW resistance of the seven CSSLs,CCRI36 and two controls(Zhongzhimian2 and Jimian11),which showed that only MBI8255 has the stable VW resistance,while its recurrent parent CCRI36 belongs to one of the stable lines susceptible to VW.Combined with the results of fiber quality test and genetic background identification of the seven CSSLs,MBI8255 and CCRI36 were separately chosen as the resistant and susceptible cotton varities to VW for further RNA-seq and biochemical tests.(2)To explore the relationships between the specific signaling pathways or biochemical substances and cotton VW,biochemical tests of MBI8255 and CCRI36 in response to V991 infection were conducted in root at 0,1 and 2 DAI,including five enzyme activities(CAT,SOD,POD,PAL and PPO)and 4 biochemical substance contents(MDA,Proline,Soluble sugar and Soluble protein).In combination with the results of three biological repetitions per sample,we found that POD and PPO activities were positively correlated with VW resistance,while MDA content was negatively correlated with VW reaistance,which is consistent with previous results.(3)In virtue of RNA-seq technique,the root tissues of MBI8255 and CCRI36 infected by V991 were separately sampled at 0DAI,1DAI and 2DAI,and three biological repetitions per sample were designed to conduct the transcriptome sequencing and analysis,which obtained a total of 77412 expressed genes(including 6934 novel predictive genes)and 23180 differentially expressed genes(DEGs)after multiple comparisons between samples.Through the temporal expression pattern analysis on the up-regulated and down-regulated DEGs,plenty of pathways involved in VW resistance were identified,such as the significantly enriched phenylpropanoid biosynthesis,plant hormone signal transduction and flavonoid biosynthesis.And the expression profiling of DEGs related with phenylopropanoid metabolic pathway,oxidation-reduction process,and plant resistance were carried out to identify the key candidate genes or pathways,which provides plenty of useful information to reveal the differences of resistant and susceptible lines to response V991 infection and lays a soild foundation of understanding the mechanism of VW resistance.2.Transcriptome analysis of cotton fiber qualityIn order to reveal the mechanism of fiber development,two CSSLs(MBI9915 and MBI9749)with superior fiber performance,together with the recurrent parent CCRI36 with the relatively ordinary fiber quality were chosen to conduct transcriptome analysis during the fiber development(10,15,20,25 and 28DPA).After library constructions and sequencing on the fifteen samples,a total of 10198 expressed genes were identified,resulting in 1801 DEGs through the Gfold algorithm and multiple comparisons between samples.After GO enrichment and KEGG annotation,the 903 up-regulated DEGs were mainly involved in cell wall organization and response to oxidative stress and auxin,while the 898 down-regulated ones participated in translation,regulation of transcription,DNA-templated and cytoplasmic translation.Based on the analysis of temporal expression patterns,29 DEGs related with oxidation-reduction process were identified in the profile with the most abundant DEGs,which turned out the significant roles of ROS-ralated genes in fiber development in combination with previous results.Furthermore,the expression profiling of POD genes in oxidation-reduction process and COBL gene in growth process were performed to explore potential contribution of chromosome substitution segments from Sea Island cotton to fiber quality of Upland cotton.At last,the obtained transcriptome data was reassembled with on reference genome,and then comparisons of sequence difference site were conducted between the assembly results of the two CSSLs and CCRI36.Through the filtration with the genome information of Sea Island cotton,we finally identified 1457 and 1585 candidate introgressive genes in MBI9915 and MBI9749,respectively,which doubtlessly lays a soild foundation for further identification of key genes in combination with QTL(Quantitative trait loci)mapping results.