A Comparative Genome-wide Analysis Of The R2R3-MYB Gene Family:Sequence Variation And Association With Fiber Quality And Yield In An Interspecific G.hirsutum × G.barbadense Population

Cotton(Gossypium spp.)is the most important natural fiber crop in the world.Two tetraploid cultivated species,G.hirsutum and G.barbadense,are grown commercially around the world for natural textiles.G.hirsutum has a high yield potential that accounts for >95 % of the total world fiber production.However,G.barbadense is renowned for having superior fiber qualities(superior fiber strength,length,and fineness).The R2R3-MYB transcription factors have been demonstrated to contribute to cotton fiber quality,however,natural sequence variations in these genes have been largely unexplored for their potential roles in fiber development.In this study,a comprehensive genome-wide comparative analysis was performed among the four Gossypium species with genome sequences.To determine whether any of the Upland cotton R2R3-MYBs are genetically associated with fiber quality and yield,a genome-wide co-localization analysis was first performed for all genes coding for R2R3-MYB transcription factors in the sequenced genome TM-1(G.hirsutum,Texas Marker-1).The experimental results were showed as follows:1.The whole genome sequence scaffolds from the ancestral diploids G.raimondii(D5)and G.arboreum(A2)as well as their decedent tetraploids G.hirsutum(AD1)and G.barbadense(AD2)were used for a genome-wide search for R2R3-MYB genes in Gossypium.As a result,216 R2R3-MYBs genes based on the sequence information of D5,213 R2R3-MYBs in the draft A2 genome,406 R2R3-MYBs in the draft AD1 genome and 393 R2R3-MYBs in the draft AD2 genome were identified.Seven specific models of gene duplication or loss event were found in the present study.Extensive close orthologous relatives of R2R3-MYBs were identified in the four Gossypium species.Interestingly,R2R3-MYB gene family in the A sub-genome might have evolved faster than in the D sub-genome both in Upland and G.barbadense,suggesting that asymmetric evolution occurred in R2R3-MYB gene family for the two sub-genomes.Furthermore,GbR2R3-MYBs might have evolved faster than GhR2R3-MYBs both A sub-genome and D sub-genome compared with their corresponding progenitor genomes,indicating R2R3-MYB gene family had a faster evolution rate in G.barbadense.To compared the features of R2R3-MYB domain sequences among the four Gossypium genomes,the homologous domain amino acid sequences of 1228 and 126 R2R3-MYBs from four Gossypium species and A.thaliana were aligned to produce the sequence logos.The distribution of conserved amino acids of the R2 and R3 MYB domains of the four Gossypium species(including A sub-genome and D sub-genome)were very similar to those of A.thaliana.A series of regularly spaced and highly conserved tryptophan(Trp,W)residues were observed in R2 and R3 MYB repeats of all four Gossypium species.Several highly conserved amino acid residues are mainly distributed in the turn and the third helix of the helix-turn-helix(HTH)motif,which is in good consistent with A.thaliana.Overall,there is an evolutionary conservation of R2R3-MYBs among the four Gossypium species.In this part,the phylogenetic relationships involved in gene duplication and loss and codon substitution rate distribution of R2R3-MYB family members were evaluated in in four Gossypium species.The results offered a useful framework for future research to understand the evolution of R2R3-MYB gene family.2.In the present study,a comprehensive genome-wide comparative analysis was performed among the four Gossypium species with genome sequences,i.e.,tetraploid G.hirsutum(AD1)and G.barbadense(AD2)as well as their likely ancestral diploid exants G.raimondii(D5)and G.arboreum(A2).To elucidate whether the R2R3-MYB genes are genetically associated with fiber quality and yield,86 R2R3-MYB genes were co-localized with quantitative trait loci(QTL)hotspots for fiber quality and yield,including 42 genes localized within the fiber length QTL hotspots,in interspecific G.hirsutum × G.barbadense populations.There were 20 interspecific nonsynonymous single-nucleotide polymorphism(SNP)sites between the two tetraploid cultivated species,16 of which developed from 11 R2R3-MYB genes were significantly correlated with fiber quality and yield in a backcross inbred population(BIL)of G.hirsutum × G.barbadense at least in one of the four field tests.Taken together,these results indicate that the sequence variation in these 11 R2R3-MYB genes is associated with the natural variation(i.e.,QTL)in fiber quality and yield.Moreover,the functional SNPs of five R2R3-MYB allele pairs from the AD1 and AD2 genomes were significantly correlate with fiber quality related to gene expression in fiber development.The present finding will be useful to further elucidate the role of the R2R3-MYB genes during fiber development.3.To reveal if a genetic difference in GhMYB5_A12 was associated with fiber traits,this study first showed that GhMYB5_A12 on chromosome A12 was co-localized with a fiber trait quantitative trait locus(QTL)cluster including a fiber-length QTL hotspot.A nonsynonymous nucleotide polymorphism in the GhMYB5_A12 gene was associated with the natural variation in fiber length and strength and lint percentage in a backcross inbred population(BIL)of G.hirsutum × G.barbadense.The expression of the gene was up-regulated at the early fiber developmental stage in three BILs with longer fibers than in three BILs with shorter fibers,while other genes within the QTL region did not show significant differences at both the RNA expression level and the DNA sequence level.4.Suppressing the expression of GhMYB5_A12 in cotton using virus-induced gene silencing(VIGS)inhibited fiber initials and decreased lint percentage.GhMYB5_A12 in overexpressed transgenic Arabidopsis increased fiber-like seed hair production from 0.47% to 7.08-8.52%.The overexpression of GhMYB5_A12 in transgenic cotton increased fiber initials,lint percentage and fiber length.5.Furthermore,GhMYB5_A12 was found to interact with three fiber-related proteins,i.e.,GhHOX3 encoding a homeodomain leucine-zipper(HD-ZIP)protein,GhEGL3 encoding a basic helix-loop-helix(bHLH)protein.Taken together,these results suggest that GhMYB5_A12 is genetically involved in regulating fiber initiation and elongation through its interactions with GhEGL3 and GhHOX3,providing a useful gene for genetically improving both lint percentage and fiber length.Overall,the present provide an important lead for further analysis to identify the valuable putative genes from fiber development advantageous gene family and their involvement in the natural variation of fiber quality and yield.Moreover,our results demonstrated a potential strategy in genetic engineering for the improvement of quality and yield in cotton.Further more,the present study implied that the GhMYB5_A12 regulatory network in cotton is similar to that in Arabidopsis and plays an important role in developmental pathways via various combinations of factors required for the full development of cotton fibers.