Genetics Research And Candidate Gene Identification For Fuzz Trait In Gossypium Arboreum

Cotton fiber fibers are commonly divided into lint（long fibers）and fuzz（short fibers）according to their length.Currently,the researches on cotton fiber were mainly focused on the lint development,the mechanism of fuzz development was rarely reported.Asiatic cotton（G.arboreum）was a diploid species which was an ideal model species to study fiber development.The genetic diversity study of Asiatic cotton fuzzless germplasm was essential to understand the mechanism of cotton fiber development.In the present study,the Asiatic cotton natural population（n=215）,fuzzless mutant（GA0149）and wildtype（GA0146）were selected as experimental materials to conduct Mendel’s genetic study,genome-wide association studies（GWAS）and various molecular biological analysis.One candidate gene Ga FZ for fuzz trait was identified,and its function and molecular mechnism were investigated in details.The main results were as follows:（1）Genetic Analysis of fuzzless trait in Gossypium arboreum germplasmFifty-five F₁ populations were constructed by crossing 55 fuzzless mutant accessions of Gossypium arboreum（female parents）with G.arboreum Shixiya 1 Hao（male parent）for the analysis of the dominant and recessive inheritance of fuzzless trait in cotton.Fifteen F₂ populations were obtained for further analysis inheritance of fuzzless trait pattern.The results showed that the inheritance of fuzzless trait was complex,with 37.5%of dominant inheritance and 62.5%of recessive inheritance.The fuzzless trait was controlled by a single dominant gene in acc.GA0149 and acc.Hengfeng Tiezi,while a single recessive gene was responsible for the fuzzless mutation in Changzi 1 Hao.However,in most of the accessions,the fuzzless trait was controlled by two pairs of genes with dominant and epistatic effects.Particularly,eight accessions carried two dominant epistatic inhibition genes and 4 accessions had a pair of genes with complementary effect responsible for fuzzy trait.The correlation analysis showed that the fuzzless trait was positively correlated with leaf hair but some cross combinations negatively corrected with lint percentage.In some cross combinations,it was positively correlated with the leaf area but negatively with the gossypol number.（2）Genome wide association studies for the seed fuzz and leaf hair in G.arboreumIn our previous study,we found that the fuzzless trait had a significant negatively correlation with leaf hair.In order to identify the causal genes of fuzzless trait,the phenotypes for seed fuzz（presence/absence）and leaf hair（number）,along with SNP,In Dels,large insertions/deletions,and copy number variations genotypes,in 215 G.arboreum accessions were used to perform GWAS.A total of 19 and 39 SNPs were found to be significantly associated with leaf hair and seed fuzz,respectively.A non-SNP marker(large deletion,designated as lar INDEL_FZ)was found to be associated with the above two traits.Further analysis showed that the lar INDEL_FZ(-log₁₀P=33.60,Chr08:～885,000 bp)co-located with SNP signals with stronger intensity(SNP_FZ,-log₁₀P=18.95,Chr08:862,509 bp)in the common genomic location（～600 kb GWAS region）at the end of Chromosome 8,indicting that this region was related to the fuzzless and leaf hair trait.This region contained a total of eight genes.SNP_FZwas located on the intronic region of a gene encoding a Casparian-strip membrane protein（CASP）（Ga08G0117）and lar INDEL_FZ was located approximately on the upstream 18 kb region of an unannotated gene（Ga08G0121）,respectively.Population genotyping result revealed that lar INDEL_FZwas more relevant to the fuzzless phenotype.（3）The sequence characteristic of lar INDEL_FZ and its linkage relationship with traitsTo determine the exact location and sequence characteristic of lar INDEL_FZ,we amplified and assembled the whole sequence across this region（ranging from～880 kb to～903 kb on chromosome 8）in fuzzy GA0146(lar INDEL_FZ absent)and fuzzless GA0149(lar INDEL_FZ present)accessions with 19 pairs primers,respectively.However,only Primer-6F6R could amplify different bands between the two accessions.Sequencing analysis revealed a 6,226 bp fragment inserted in the GA0149 genome.Bioinformatic analysis implied that this insertion may be caused by the amplification of nearby repeated sequences.To further assess the linkage relationship between this fragment and traits,an F₂segregated population generated by GA0146 crossing with GA0149 was used to identify the presence and absence of lar INDEL_FZ.By integrating seed fuzz and leaf hair traits with electrophoresis band,the result showed that the segregation ratio of homozygous fuzzy:heterozygous fuzzless:homozygous fuzzless was consistent with a 1:2:1（Chi-square test,P=0.192）,which indicated that the fuzzless trait of GA0149 was controlled by a typical single dominant gene.And lar INDEL_FZ was found to be tightly linked fuzzless trait.Meanwhile,the accessions carrying lar INDEL_FZ showed significantly fewer leaf hairs（P<0.0001）.Namely,leaf hair and seed fuzz traits were conferred by lar INDEL_FZ in G.arboreum.（4）The analysis of lar INDEL_FZ regulating Ga FZFor identifying the causal gene which controlled fuzz fiber development,the transcriptome data and RT-PCR analysis found that Ga FZ（Ga08G0121）specifically expressed in GA0149（fuzzless）at the critical development stage of fuzz fiber（from+3DPA to+5 DPA）,comparing to that in GA0146.There was no significant difference for remaining genes in the candidate region between these two accessions.These results indicated that Ga FZ might be the key gene to control the target traits.Moreover,except lar INDEL_FZ,another four variations were found at Ga FZ gene and its flanking genomic region.For deciphering the connecting between genomic variations and expression of Ga FZ,we conducted the experiments to analyze the promoter activity of Ga FZ gene,and luciferase activity of various-length lar INDEL_FZ.The results showed that the expression of Ga FZ gene in GA0149 was activated by lar INDEL_FZ.Additionally,cis-acting element analysis demonstrated that lar INDEL_FZ might act as an enhancer to regulate Ga FZ gene.（5）Phenotype analysis of Ga FZ transgenic plantsGa FZ（Ga08G0121）was a single exon gene without intron and functional annotation.The subcellular localization result showed that Ga FZ expressed in both nucleus and cell membrane,which suggested that Ga FZ might be a shuttling protein.T.In order to verify the function of Ga FZ gene,the Ga FZ overexpression vector was constructed and transformed into Arabidopsis and cotton（G.hirsutum）,respectively.Transgenic phenotype investigation results showed that the development of leaf hair in Arabidopsis and stem hair,leaf hair and seed fuzz in cotton was inhibited.In conclusion,Ga FZ negatively regulates the development of leaf hair and seed fuzz in cotton.（6）Regulatory relationship between Ga FZ and R2R3-MYB/b HLH/WD40-GL2（MBW-GL2）system,as well as Very Long Fatty Acid Extension（VLCFAE）PathwayThe transcriptome of ovules and fibers between GA0149 and GA0146 at four critical fiber developmental stages（0 DPA,+3 DPA,+5 DPA,+8 DPA）were compared.Differential expression analysis exhibited that the homologous genes of MBW-GL2system in Asiatic cotton,including Ga WER,Ga HOX1,Ga HOX2,Ga HOX3,Ga DEL65,and Ga WD40 showed no significant difference between two accessions.Additionally,the expression levels of At GL1,At GL3,and At TTG1 between the Arabidopsis wild-type and Ga FZ overexpressing lines also showed no significantly difference.Besides,the yeast two-hybrid experiment demonstrated that no interaction occurred between Ga FZ and MBW-GL2 systems at the protein level.GUS enzyme activity and luciferase experiments further proved that Ga FZ did not regulate the expression of Ga GL2.The above results implied that Ga FZ might affect the development of leaf trichome and fuzz independently of the MBW-GL2 system in G.arboreum.We further analyzed the gene expression of downstream pathways in both cotton and Arabidopsis,and found that 3-ketoacyl-Co A synthetase（KCS）involved in the very-long-chain fatty acid elongation（VLCFAE）pathway（ko00062）,and several genes involved in cutin,suberine and wax biosynthetic pathways（ko00500）were significantly decreased in GA0149 and Ga FZ transgenic lines.These results indicated that Ga FZ might negatively regulate the development of trichome（leaf hair）and seed fuzz by inhibiting VLCFAE pathway,and reducing the content of cutin,suberine and wax in plants.