Evolution Of Anthocyanin Synthesis Genes In Cotton Domestication

Anthocyanins,belonging to the secondary metabolites of flavonoids,are widely found in plants.Besides being natural pigments to colorate plant organs such as flowers and fruits,with special antioxidant properties,anthocyanins also play important roles on defending plant species from the pests and diseases and protecting plant body against harmful UV radiation.In addition,the values of anthocyanins on the aspects of medicine,food and health care have been paid more and more attentions because of its important functions on anti-cancer,bacteriostasis and preventing people from cardiovascular disease.Although the anthocyanin synthesis pathway had been widely studied in many diploid model plants,very few were focused on the genetic changes of the pathway in domesticated allopolyploids.In this study,using upland cotton(Gossypium hirsutum L.),a species with AADD genome,as the experimental material,we investigated the morphological and genetic changes between the cultivated and wild varieties of this species.Seven structural genes(CHS,CHI,F3 H,F3 ? H,DFR,ANS and UFGT)in the anthocyanin synthesis pathway were identified.Then,sequence differences and expression divergences of every structural gene pairs of the cultivated and wild cottons were further compared and analyzed.The main contents and results were listed as follows:1.By morphological observing,it was found that cultivated cotton had dwarf and thick stems,dark green and hairy foliage,an early flowering time,larger and plentiful flower buds,unspotted petals,bigger bolls and longer and white fibers;whereas the wild cotton had tall and thin stems,light green and glossy foliage,a late flowering time,spotted petals,small bolls and short and brown fibers.2.Based on transcriptome data,seven structural genes(CHS?CHI?F3H?F3?H?DFR?ANS?UFGT)in the anthocyanin synthesis pathway were identified.Using the BLAST method,the coding sequences of these genes were then mapped to the published whole genome of upland cotton,and the structures and chromosome locations of these genes were further confirmed.In addition,by phylogenetically analyzing among G.hirsutum and AA or DD genome species,G.arboreum L.and G.raimondii Ulbr.,A and D genomic homoeologs of seven genes in G.hirsutum were further confirmed and distinguished.3.Using whole genome re-sequencing technology,sequence differences on the coding region of seven genes between wild and cultivated cotton were investigated.One to five SNP for homologous pairs of CHS,CHI and F3 H genes and no SNP for homologous pairs of F3?H,DFR and UFGT genes were found.This result indicated that the cultivated and wild cottonsshared a high level of sequence conservation for each homologous gene pairs.4.Using the method of Quantitative Real-time PCR,the expression levels of six structural genes in leaves between cultivated and wild cottons were compared.The result showed that all genes in cultivated cotton had a lower level of gene expression in contrast to their homologous copies in wild cotton.Among them,F3 ? H obtained the highest expression change,whose expression level decreased about five times in the cultivated cotton,suggesting more active biological synthesis of anthocyanins or flavonoids in wild cotton under normal conditions.In addition,under the conditions of aphid stress,the expression levels of multiple genes related the anthocynain synthesis were up-regulated at the third week in cultivated cotton,but similar situation would be occurred in the second week in wild cotton,indicating a more rapid reaction on the anthocyanin synthesis in wild cotton when needing to defend against insects.5.Due to every key enzyme in the anthocynain synthesis pathway was possibly encoded by more than one locus,and each locus usually possess both parental genomic homoeologs in an allopolyploid species,the evolution of the encoded genes of each key enzyme in upland cotton would be very complex.Here,using an important enzyme DFR as an example,expression statuses of the encode genes in leaf,petal,seed and fiber were studied.Two loci(DFR1 and DFR2)were found to encode the enzyme DFR in upland cotton,and each locus of DFR gene was confirmed to have both A and D genomic homoeologs,therefore,a total of four homoeologs occurred in upland cotton.By comparing our transcriptome data of different organs or tissues between cultivated and wild cotton,complicated temporal and spatial expression divergences of four homoeologs were revealed.6.In order to detect the possible genetic mechanism of those expression divergences between cultivated and wild cotton,the sequence changes of the promoter regions of seven structural genes in the anthocynain synthesis pathway were examined by using the next-generation DNA sequencing technology.In comparison with the homologous genes in cultivated cotton,CHS,ANS and DFR2 in wild cotton were found to have some indel mutations in their promoter regions.Due to any nucleotide change in the promoter region would probably produce different gene expression,the indels in the promoter regions of these genes should be paid more attentions.But,if the indels were the main causes for the divergent expression levels of these homologous genes between cultivated and wild cottons,more experimental verifications in the future were required.In summary,during the cotton domestication,the structural genes in the anthocynain synthesis pathway did not produce obvious sequence changes in their coding regions,but produce complicated gene expression divergences,suggesting that domestication had a greateffect on epigenetic changes rather than genetic mutations.The current results provide important molecular data for better understanding the effects of domestication on sequence and expression change of functional genes in allopolyploids and improving the genetic quality of cultivated cotton from the point view of the anthocynains biosynthesis.