Fine Mapping And Candidate Genes Analysis Of Two Virescent Genes(v₁ And Vsp)in Upland Cotton

Cotton industry occupies a pivotal position in the national economy in China.Utilization of heterosis is one of the most effective methods to increase cotton yield by a big margin,improve fiber quality and enhance resistance to biotic and abiotic stress.However,often cross-pollination and self-fertilization of sterile line affected by temperature or light cause impure cotton hybrid,consequently low cotton yield and quality.The young leaf of virescent mutant in cotton is yellowish.Virescent mutation in cotton can be used as marker trait for removing off-type plants in sterile line and cotton hybrid,which may improve hybrid purity.In addition,virescent mutant can be used to research chlorophyll(Chl)biosynthesis,choloroplast development and photosynthesis mechanism.Therefore,elucidating the genetic basis of virescent mutation has great theoretical and applied value in cotton heterosis breeding.In this study,G.hirsutum acc.(TM-1,Zhongmiansuo60,Zhongmiansuo58 and its mutant vsp,v₁ mutant)and G.barbadense acc.(Hai7124)were selected as experimental materials.Based on SNP,SSR and In Del markers and using F₂ populations,v₁ and vsp genes were finely mapped,and the candidate genes were cloned and analyzed.As a result,we preliminarilily clarified the genetic bases of virescent traits of the v₁ and vsp mutants.The major research results are as follows:I.Fine mapping and candidate gene analysis of the virescent gene v₁ in Upland cotton(i)The true leaves of the v₁ mutant are yellowish and gradually turn light green as they reach maturity,accompanied by an increase in Chl conent.F₂ populations(TM-1×v₁)were constructed,in which the ratio of plants with virescent leaves to plants with green leaves was 1:3.This result showed that the virescent phenotype in the v₁ mutant was caused by a pair of recessive nuclear genes.Using SNP,SSR and In Del markers and the F₂ population,the v₁gene was narrowed down to an 84.1-kb region.(ii)Gene annotation of this region revealed that the Gh Chl I gene encoding the cotton Mg-chelatase I subunit(CHLI)is the candidate gene related to the virescent mutation.BLAST analysis showed that the Gh Chl I has two homologous genes(Gh?A10G0282 and Gh?D10G0283)in Upland cotton genome.Sequence analysis indicated that the length of coding region of the Gh Chl I is 1269 bp,with three predicted exons and one non-synonymous nucleotide mutation(G1082A)in the third exon of Gh?D10G0283,with an amino acid(AA)substitution of arginine(R)to lysine(K).Phylogenetic analysis revealed that the AA sequence encoded by the third exon of the Gh Chl I is highly conserved across diverse plant species,in which AA substitutions frequently cause changes in leaf color in various species.Gh Chl I-silenced TM-1 plants exhibited virescent phenotype and significant reduction in Chl content.Based on the above-mentioned results,we assumed that the Gh Chl I is a key gene in regulating virescent leaves in the v₁ mutant.(iii)Promoter analysis showed that there is no variation among Gh?D10G0283promoters from the TM-1,Zhongmiansuo60,and v₁ lines,whereas variation sites in the promoters were identified between Gh?A10G0282 and Gh?D10G0283 in the v₁mutant.Specific primers based on the SNP sites between Gh?A10G0282 and Gh?D10G0283 were designed to determine the expression levels of the Gh Chl I by q RT-PCR analysis.The results showed that in the v₁ mutant,the transcript levels of Gh Chl I in the true leaves were significantly higher than those in Zhongmiansuo60,but the transcript level of the Gh Chl I between the v₁mutant and TM-1 has no significant difference.In the phytotron,at the 12th day stage of the mutant v₁,the transcript level between the Gh?D10G0282 and Gh D10G0283 did not differ,but at the 18-day stage,the expression level of the Gh?D10G0282 was significantly higher than that of Gh?D10G0283.Based on these results,we assumed that the transcript level of the Gh?D10G0283 may be normal,and the mutation(G1082A)within Gh?D10G0283 causes the virescent phenotype,and the increase in the Gh?A10G0282 dosage may partially make up for the deficiency of Gh?D10G0283 in the v₁ mutant.II.Fine mapping and candidate gene analysis of the virescent gene vsp in Upland cotton(i)Using the parents(TM-1 and vsp),F₂ populations were constructed,in which the ratio of plants with virescent leaves to plants with green leaves was 1:3.The results indicated that the virescent phenotype was controlled by vsp which is a recessive nuclear gene.A high-density genetic map,including 4472 Bin markers,was constructed by sequencing the parents(TM-1 and vsp)and 100 F₂individuals using genotyping by sequencing(GBS).The genetic map included 26 linkage groups with total length 5384.33c M and average distance between 4472 Bin markers was about 1.2 c M.Based on this map,the vsp gene was mapped to a 38.32-c M region on chromosome D04 between the markers Bin3262 and Bin3268.Using the molecular markers developed by resequencing the TM-1and vsp,the vsp gene was mapped to a 23.11-c M region.A total of 484 virescent individuals of F₂ population from the cross vsp×Hai7124 were used to map the vsp gene which was narrowed down to a 353.7-kb region.(ii)Sequence analysis of genes in this region showed that the Gh PUR4 gene encoding formylglycinamidine ribonucleotide synthetase from Zhongmiansuo58 was 4468 bp in length with two predicted exons and one intron;the first and the second exon were 42 bp and 4197 bp long respectively,whereas the intron was 229 bp.The 201 bp deletion(from960 to 1160)was found in the second exon of the Gh?D04G1108 gene in the vsp mutant.Three homologous genes of Gh PUR4(Gh?D04G1108,Gh?A04G0646 and Gh?A06G0069)have been identified through BLAST in the G.hirsutum genome.The Gh PUR4 gene encodes a polypeptide with 1412 AA with an estimated molecular mass of154.15 k Da.The 201-bp deletion of Gh?D04G1108 gene caused the deletion of 67 AA(244 to 310)in Gh PUR4 protein.Sequence alignment results of PUR4 proteins from 20species found that there were 33 highly conserved AA and 13 completely conserved AA among the 67 AA.