Cloning The Drought Tolerance Gene Of Gossypium Anomalum Using A Single Segment Substitution Line

Drought is one of the key abiotic stresses limiting cotton production.The narrow genetic base of upland cotton,which is widely planted due to long-term artificial selection,is the main bottleneck for further improving drought resistance.Developing new germplasm resources of drought-resistant upland cotton and studying its physiological and molecular mechanisms are the basis for breeding new drought-resistant varieties.Gossypium anomalum,an Africa diploid wild species of the B₁ group,is distinctly characterized by its extreme drought tolerance and a valuable genetic resource for improving the drought tolerance of upland cotton.In previous studies,a set of introgression lines of G.anomalum were developed through techniques such as distant hybridization and whole genome selection.Drought tolerance of all CSSLs（chromosome segment substitution lines）was assessed using 20%polyethylene glycol（PEG）treatment at the seedling stage,and CSSL29 of chromosome 5 showed tolerance to PEG stress.In this study,we identified the drought resistance of 4 CSSL lines with overlapping segments of chromosome 5,and attempted to identify the candidate genes involved in drought tolerance by combining transcriptome analysis,qRT-PCR,sequence variation analysis and virus-induced gene silencing（VIGS）.We verified the function of candidate gene through extopic expression in Arabidopsis and elucidated its tolerance mechanism through phenotypic and physiological indicator measurements.The main results of this study are as follows:（1）Four overlapping CSSL lines of Chr.05（CSSL28,CSSL29,CSSL30,and CSSL31）and the recurrent parent G.hirsutum cv.Su8289were grown.The interval of CSSL28 is JAAS6365-JAAS0803,while CSSL29,CSSL30 and CSSL31 contain overlapping r intervals JAAS6365-JAAS5604.Su8289 and CSSL28 plants exhibited obvious wilting after 20%PEG6000 treatment,while CSSL29,CSSL30,and CSSL31 showed more tolerance to PEG stress than Su8289 seedling,indicating that loci involved in drought tolerance could be defined at JAAS0803-JAAS5604（1,415,831-2,878,211 bp）interval on Chr.05 of G.anomalum,which contained a total of 192 candidate genes.（2）Transcriptome sequencing analysis found 20 genes in JAAS0803-JAAS5604 interval were up-regulated in CSSL29 at least one time point after PEG stress.Of which,9 contained likely function-altering variation compared to their homologues in G.hirsutum L.acc.TM-1 and their expression levels were further verified by qRT-PCR.Candidate genes were screened through VIGS experiments.The Goano05G0268-silencing plants of CSSL29 were more sensitive than TRV:00 plants,while the silencing plants of the other eight genes showed no sensibility.Also,Goano05G0268-silencing plants had higher H₂O₂ and MDA content,but lower POD activity than TRV:00 plants under PEG stress at 72 hours.Goano05G0268 encodes a peroxidase,which belongs to the superfamily of antioxidant proteins.Therefore,it is supposed that Goano05G0268 is a key gene for drought tolerance in G.anomalum,which may regulate drought tolerance by increasing POD activity.Sequence variation analysis of Goano05G0268 in CSSL29 and its orthologous gene GH＿A05G0249 in Su8289 showed that GH＿A05G0249had 408 bp deletion,2549 bp,159 bp and 2 bp insertion in the second intron,and 11 SNPs in the CDS region.Goano05G0268 in G.anomalum was aligned with its orthologous gene in other allotetraploid cotton species,such as G.hirsutum L.acc.TM-1,G.barbadense L.cv.Hai7124,G.barbadense L.accession 3–79,G.tomentosum,G.mustelinum and G.darwinii.The result was similar with the alignment of G.anomalum and Su8289,that is,orthologous genes of Goano05G0268 in these allotetraploid cotton species had a～2500 bp insertion and other small deletions and insertions in the second intron,and SNPs variation with different size within exon 1,2,and 3.These results suggested that this gene likely became pseudogenized during Gossypium evolution.（3）We created ectopic expression lines of Goano05G0268 in Arabidopsis for further analysis of the function of Goano05G0268 under drought stress.The root length experiments of WT and T₁ generation EE lines indicate that the root length showed no significant difference between WT and EE lines on MS medium without mannitol.However,the EE lines on MS medium with 200 and 250 mM mannitol had longer roots than WT lines.Normal cultivation of WT and T₁ generation EE lines for drought treatment,25 days after drought,the whole WT plants wilted and lost vitality due to severe drought stress,but most EE plants kept alive.The relative content of H₂O₂and MDA increased significantly in WT lines compared with EE lines after 14 days drought stress treatment,while the POD activity was significantly higher in EE lines under drought stress than those in WT lines.These results were similar to those of drought tolerance identification for the homozygous T₃ EE lines seedlings.The phenotypic observation and identification of physiological indicators of Arabidopsis verified the drought resistance function of Goano05G0268.