Map-based Cloning And Transcriptome Analysis Of Gl Gene In Melon(Cucumis Melo.L)

Melon(Cucumis melo L.)is an annual viningcrop of cucurbitaceae family,with high genetic diversity and phenotypic diversity.China has the largest cultivation area and the highest yield in the world,and China is also one of the countries that have the longest history of melon planting.Trichome is the derived structure of epidermal cells which is the best model to study plant cell differentiation,so exploring the mechanism for the formation of trichome in melon can help us to enrich the theory of plant cell differentiation.With the rapid development of next generation sequencing(NGS)technology and the crossover between molecular biology and bioinformatics,it greatly accelerates map-based cloning and functional analysis of important traits in different crops.In this study,we developed a F2 population of the glabrous material M68 and the normal trichome material M465 for map-based cloning of glabrous gene in melon.The gl gene was first primarily mapped using the bulked segregant analysis(BSA).Then the two parental lines were resequenced and compared in the candidate region,and new markers were developed for fine mapping based on the sequence difference.The ORFs in the candidate region was predicted and compared between two parental lines,and the candidate gl gene was finally cloned.Furthermore,the RNA-seq was used to investigate the regulatory network controlling trichome development in melon.The results will provide a solid foundation for the further exploring the function and molecular mechanism of the gl gene.The results identified in our study are as follows:1.A 1536 F2 population was developed between the parental line of M68 and M465 which included 1156 normal plants and 380 glabrous plants.The segregation data for trichome and glabrous was conformed with 3:1 ratio(χ2=0.5556<χ20.05=3.84),suggesting that the glabrous phenotype in melon was controlled by a single recessive gene which was named gl.2.A BSA method was used for screening polymorphic markers with 10 plants randomly selected for glabrous pool and trichome pool,respectively.480 SSR markers were screened and eight ones showed polymorphic markers between two pools.Then a 256 F2 mapping population was used for primarily mapping,and the gl gene was mapped between Cm SSR19480 and Cm SSR19495 on chromosome 8 which had a genetic distance of 0.9 c M and 2.8 c M with gl gene,respectively.3.The two parental lines M465 and M68 were resequenced and compared in thecandidate region.6 Indel and one dCAPS markers were developed based on the sequence difference,and four Indel markers showed good polymorphic between two parental lines.Then we randomly selected 438 F2 populations for fine mapping,and the gl gene was mapped between Indel1 and Indel6 with a genetic distance was 0.3 c M and 0.1 c M,respectively.Furthermore,we enlarged the F2 population to 1536 plants and the gl gene was further narrowed down between Indel2 and Indel5 which was physically 11.58 kb for this two markers.In this candidate region,there was only ORF predicted which encoded a CDS of 2166 bp.BLASTP for the function analysis for this gene revealed that it encoded a HD-ZIP IV protein showing 99% identity with a cucumber gl gene Csa6M514870 which had been cloned in cucumber.4.Based on the resequence mapping between two parental lines for gl gene,10 SNPs were detected in the genomic region.Of them,six SNPs were located in the introns and four SNPs were located in the exons.The fourth SNP on the exons resulted in a premature termination for this gene in the mutant.5.A BSR-seq strategy was used to investigate the regulation network for gl gene.20 plants were randomly selected for glabrous pool and trichome pool for RNA-seq analysis.Totally 2368 differentially expressed genes(DEGs)were identified with 1195 genes up-regulated and 1173 genes down-regulated,respectively.Function analysis of these DEGs revealed that there were eight enrichment pathways involved including the aromatic tyrosine and tryptophan in phenylalanine metabolism pathway.GO function analysis showed that there were312 functional annotation of biological processaccounting for 63.3%.Among them,the maximum number of annotation genes was related to metabolism process.6.Based on SNPs detected between two bulks by mapping the RNA-seq to the reference,we performed an ED association analysis with gl gene between them.Finally,the candidate region were predicated on chromosome 8 between 1.98 Mb and 3.72 Mb,and it was consistent with the fine mapping result where the gl gene was located around 2.45 Mb on chromosome 8.