Mapping,Cloning And Functional Analysis Of ADL Gene In Asiatic Cotton

Cotton is an important economic crop.Plant architecture directly affects cotton’s mechanized picking and yield.At present,few target genes can be directly used in the molecular design of the ideal plant architecture of cotton.Apical dominance is one of the basic developmental phenomena in plant biology,which determines the overall architecture of the aboveground part of plants.Related research can provide theoretical support and genetic resources for the molecular design of cotton plant architecture,and is of great significance to the production practice of crops,fruit trees and flowers.The plants with missing apical dominance are dwarfed,which is easy to shape the ideal plant architecture,It is of great significance to study the regulation mechanism of cotton apical dominance loss plant architecture and improve cotton plant architecture.Previously,an apical dominance loss(adl)mutant with shorter plant height and longer lateral branches was obtained by ethyl methane sulfonate(EMS)mutagenesis of Asiatic cotton Shixiya No.1(SX1)in our group.In this study,the reference genetic map of Asiatic cotton was constructed,and the F 2 population was established by crossing M 2homozygous adl mutant with Asiatic cotton Changshulong(CL).Combined with the reference genetic map of Asiatic cotton,the ADL gene of Asiatic cotton was mapped and cloned,and then the function of the target gene was verified by upland cotton and Arabidopsis thaliana genetic materials.The main results are as follows:1.Construction of reference genetic map of Asiatic cottonAccording to the re-sequencing results of Asiatic cotton SX1 and CL,and their recombinant inbred lines,a large number of polymorphic In Del sites were found,and 633 pairs of primers for In Del loci on 13 c hromosomes were designed.Three hundred and one pairs of primers were screened by polyacrylamide gel electrophoresis(PAGE),which showed significant difference in the size of amplification products between SX1 and CL.Among them,248 polymorphic In Del mar kers evenly distributed on chromosomes were selected to construct the reference genetic map of Asiatic cotton,which laid the foundation for the mapping of EMS mutant genes.2.Phenotypic variation of adl mutantsPhenotypic observation showed that the apical dominance of the main stem was lost in the adl mutant at the late growth stage.Compared with wild type,the plant height of adl mutants was significantly lower,the internodes became shorter,the epidermal cells of internodes becam e shorter,the lateral branches became longer,and the angle between lateral branches and main stem became smaller,but there was no significant difference in boll number,fiber initiation number and mature fiber length.3.Genetic analysis of the adl mutantM2 homozygous adl mutant was crossed with Asiatic cotton CL.The phenotype of F1 generation was wild type,while that of F 2 population was segregated,including 1105 wild-type and 343 adl mutant phenotypic plants.The chi-square test indicated that the segregation ratio of wild type to mutant plants was consistent with the segregation ratio of 3:1(X~2=1.33<X~20.05=3.84),suggesting that the adl mutant trait was controlled by a pair of recessive nuclear genes in the Asiatic cotton.4.Identification of candidate genes for phenotypic control of adl mutantsAccording to the In Del marker loci on the reference genetic map of Asiatic cotton,the ADL gene was initially mapped between REF12 and REF17markers on chromosome 1.With single nucleotide polymorphism(SNP)markers detected with high resolution melting curve(HRM)analysis,this gene was finally mapped an interval of about 3Mb,between SNP1-41 and SNP1-46.In this region,there are 34 annotated genes.The re-sequencing results of SX1 and adl mutants showed that only one U-box type E3 ubiquitin ligase PUB protein-coding gene(Ga01G1670)had a single base mutation,resulting in early termination of protein translation.The comparative cloning results further proved that the Ga01G1670 gene in the adl mutant did have the mutation,so we took it as the candidate gene controlling the adl mutant.5.Study on the function of PUB63b gene in cottonPhylogenetic analysis showed that Ga01G1670 was similar to the atypical U-box domain protein AT3G19895(At PUB63b)in Arabidopsis thaliana,so the gene was named Ga PUB63b.The results of expression pattern analysis showed that Gh PUB63b was mainly expressed in root,stem,leaf and other vegetative organs in upland cotton.The results of transient expression in tobacco showed that the protein was located in the nucleus.Comp ared with the wild type,the plant height of Arabidopsis thaliana Atpub63b mutants decreased significantly,and the rosette branch numbers increased significantly.The CRISPR/Cas9 knockout mutants of Gh PUB63b phynocopied the adl mutant,and showed the phen otypes of shorter plant height,shorter internodes,increased lateral branch numbers and lengths.These results demonstrated that PUB63b gene played an important role in maintaining plant apical dominance,and Ga01G1670 was probably the phenotypic control gene of the adl mutant in Asiatic cotton.The results of transcriptome and plant hormone detection in stem apical tissues showed that,compared to the wild-type plants the expression of YACCA8(the key enzyme in auxin synthesis)and the auxin content decr eased significantly in the adl mutants.Therefore,we speculated that the decrease of auxin synthesis may be the cause of apical dominance loss and plant dwarfing in adl mutants.