QTL Identification And Candidate Genes Analysis For Central Flesh Hardness Of Watermelon(Citrullus Lanatus)

Watermelon(Citrullus lanatus L.)is an important horticultural crop around the world.The hardness of watermelon flesh is a primary factor of shelf life indication which also affects the edible preferences of consumers.The flesh texture,cell wall structure and composition,hormones accumulation and regulation,are main factors which triggers the overall hardness of watermelon fruit by exhibiting a positive or negative associations.Hence,the exploration of comprehensive inter-correlation of fruit hardness and its related traits is a great interest of research in the commercial industry of watermelon,by selection and breeding of desired watermelon varieties for producing the new varieties having suitable desired characteristics.In the current research,two contrasted watermelon lines(W1-1,as female parent with soft flesh and PI 186490,as male parent with hard flesh)were selected for fine genetic mapping and analysis of main-effect genes controlling the center flesh hardness of watermelon.The plant population of six generations(P₁,P₂,F₁,F₂,BC₁P₁and BC₁P₂)were derived and analyzed for genetic inheritance of center flesh hardness trait.The candidate genes controlling the center flesh hardness were primarily mapped through whole genome BSA-seq and genetic linkage map construction.The parameters of center flesh hardness,cell wall composition content,hormonal content,cell size and their arrangement,and genes expression level of the parental lines were observed at different developmental stages simultaneously.The histochemical analysis and primary genetic mapping results were combined to identify the candidate genes in targeted region of controlling the center flesh hardness of watermelon through molecular marker-assisted selection(MAS).This molecular and genetic breeding method laid out a strong theoretical foundation for speedy breeding process.The main research contents and results are as followed;1.Analysis of genetic inheritance of watermelon center flesh hardness.A normal distribution of center flesh hardness was noticed in the contrasted parental lines"W1-1 and PI 186490"and their derived F₂,BC₁P₁,and BC₁P₂populations.The combined genetic inheritance analysis of six generations exhibited that watermelon center flesh hardness is polygenic in nature and conforms to the E-3(MX2-A-AD)model,which is 2 pairs of additive-major genes in addition with additive-dominant polygenes.Overall,the additive effect of polygenes was observed positive and negative,and the dominant effect was noticed positive.The first pair of major genes and the second pair of major genes showed negative additive effect.In the derived F₂population,a total of89.19%was observed in which the heritability of major genes was about 88.26%and 0.92%,respectively.2.Analysis of QTLs and candidate genes controlling center flesh hardness of watermelon.An F₂segregation population of"W1-1 and PI 186490"was derived,used for whole gnome BSA-seq analysis,and total 153 CAPS markers were developed for genetic linkage map construction.All the genotypic and phenotypic data was combined and total of 3 QTLs(CH2.1,CH6.1 and CH8.1)were identified for watermelon center flesh hardness.QTL“CH2.1”was located on chromosome 2between the two adjacent markers“W2-1811213 and W2-23770587”,with physical distance of5.66 Mb,PVE contribution rate of 17.41%,and the additive effect of-1.0409.QTL“CH6.1”was located on chromosome 6 between the two adjacent markers“W6-12805511 and W6-13817233”,with physical distance of about 1.01 Mb,PVE contribution rate of 33.39%,LOD value of 9.82,and additive effect QTL“CH8.1”was positioned on chromosome 8 between the two CAPS markers“W8-207553 and W8-403137”.The physical distance was about 195 kb,PVE contribution rate was24.44%,additive effect was 0.01,and the total contribution rate of these 3 QTLs was 75.25%.The candidate gene Cla004102 in the targeted region of chromosome 6 was an auxin-responsive protein gene,which was differentially expressed in the two contrasted parental materials;the candidate gene Cla012507 in the targeted region of chromosome 8 was a MADS-box transcription factor.This transcription factor had been reported in many crops,mainly responsible for fruit ripening and softening.So,it is speculated that Cla004102 and Cla012507 were the major genes that controlled the central flesh hardness of watermelon.3.TPA analysis of the center flesh of"W1-1"and"PI 186490"at each developmental stage.The characteristics of hardness,chewiness,cohesiveness,springiness and resilience at each developmental stage were higher for"PI 186490"line than that of"W1-1"line.The change trend of hardness and chewiness was almost similar which increased at the beginning of fruit development and continues to reach the maximum value at 21 days after pollination,and gradually decreased and then slightly increased.The change trend of cohesiveness and resilience was similar,and both parent showed overall continuous decrease trend.Correlation analysis depicted that hardness was significant and positive correlated with chewiness,springiness and resilience,and the correlation coefficients were 0.90,0.81,and 0.411,respectively.It was significantly observed that watermelon fruit texture,hardness,chewiness,springiness and resilience can be used as important assessment indicators.4.The content analysis of propectin,cellulose and hemicellulose in the center flesh of"W1-1"and"PI 186490".The content of propectin,cellulose and hemicellulose in"PI 186490"were all higher than that of"W1-1"at different developmental stages.Correlation analysis showed that the watermelon center flesh hardness was significantly positively correlated with the content of propectin,cellulose and hemicellulose,and the correlation coefficients were 0.67,0.64,and0.81,respectively.The difference in cell wall substance content between the two watermelon lines may be the main reason that affected the hardness of the watermelon center flesh.5.The endogenous content analysis of IAA and ABA in the center flesh of"W1-1"and"PI186490".The content of IAA was higher at early stage of"W1-1",and as the fruit matured,the IAA content continued to decrease,but the IAA content of"PI 186490"had a similar lower trend which was lower than"W1-1"at each developmental stage.The content of ABA was low at early development stage of"W1-1",and as the fruit continued to mature,the content of ABA also continued to arise,while the content of ABA remained at low level during the development stage of"PI 186490".Correlation analysis showed that the center flesh hardness of watermelon was significant but negatively correlated with the content of IAA and ABA,having coefficient values of-0.55 and-0.47,respectively.We concluded that the hormonal content difference between two watermelon lines might be the main reason that affects the hardness of the watermelon center flesh.6.Observation of the central flesh cells of"W1-1"and"PI 186490"at five developmental stages.In the early stage of development,the cell numbers of"PI 186490"were significantly more than that of"W1-1",and the cell sizes were small and tightly arranged.However,the cells continued to expand during the development of two watermelon lines but"PI 186490"cells at any developmental stage were comparable with"W1-1",and both lines showed smaller cell sizes,more number of cells,tighter cell arrangement,and clearer cell edges.We concluded that the change of watermelon fruit texture might be related to the size and arrangement of flesh cells.7.RNA-seq analysis.The routine analysis and WGCNA analysis of central flesh transcriptome data of"W1-1"and"PI 186490"at different developmental stages exhibited few genes related to cell wall enzymes,and fruit softening related enzymes,e.g.,polygalacturonase(PG),pectin methyl esterase(PME),auxin response protein,abscisic acid receptor,ethylene response transcription factor,glycosyltransferase and expandin,etc.These genes were seemed to be differentially expressed in two parental line at each developmental stages which may control the ripening and softening phenomenon of watermelon fruits traits by regulating the cell wall metabolism and hormone metabolism.