Fine Mapping Of The Major-effect QTL Fnl3.1 For Fruit Neck Length In Cucumber And Functional Verification Of The Candidate Gene CsFnl3.1

Cucumber（Cucumis sativus L.）is one of the main vegetable crops that widely cultivated in China.The fruit neck length（the length of the non-seed cavity-developed part near the stalk）is one of the most important characters affecting the commercial and edibe quality of cucumber.The hard texture and low sugar content in the fruit neck result in poor taste of this part.Therefore,the shorter the fruit neck,the higher its commercial value and the better the taste.In order to find the key genes controlling the fruit neck length in cucumber,we selected the cucumber inbred line KP2 with long fruit and short fruit neck（average fruit neck length is 4.86 cm）and the cucumber inbred line Jin5-508 with long fruit and long fruit neck（average fruit length neck is 9.65 cm）as parents to construct F2 populations.QTL-seq method was used to identify the major-effect QTL controlling fruit neck length in cucumber.RNA-seq analysis was used to conduct transcriptomic profiling of fruit necks at different developmental stages after pollination.The main results are listed as follows:1.The average fruit neck length of KP2 and Jin5-508 was 4.86cm and 9.65cm,respectively.The average fruit neck length of KP2×Jin5-508 F1 hybrid was 7.36 cm.The F2 population showed a normal distribution trend,indicating that the fruit neck length of cucumber is quantitatively inherited.2.The F2 population was planted in three consecutive seasons and extreme individual plants were selected for QTL-seq identification,and a major locus was identified on Chr3.By enlarging F2 population,adding molecular markers and screening recombinant individual plants,the interval was finally mapped to a-757.4kb（Chr3:34,723,012-35,480,427）region.By comparing resequencing data and gene cloning results,CsaV3₃G042060（CsFnl3.1）,which encodes GA regulatory protein,was preliminously identified as the candidate gene.3.RNA-seq analysis identified a total of 3410 differentilly expressed genes between KP2 and Jin5-508 fruit necks harvested at 0,3,6,and 9 days after pollination.Functional enrichment analysis showed that DEGs were significantly enriched in the plant hormone signal transduction pathway,phenylpropanol biosynthesis pathway,photosynthesis-antenna proteins pathway and MAPK signal transduction pathway.The DEGs were aligned to the previous localization results,and 12 DEGs were found to be located in the candidate interval,including CsFnl3.1 The results of qPCR analysis showed that the expression level of CsFnl3.1 was significantly down-regulated with the elongation of fruit neck,and the expression level of KP2 was significantly higher than that of Jin5-508,indicating that CsFnl3.1 had a negative regulatory effect on the length of fruit handle.4.The result of liquid chromatography-tandem mass spectrometry showed that the total GA content in fruit neck of Jin5-508 was significantly higher than that of KP2.The fruit neck length of the parents increased upon exogenous gibberellin GA3（100 mg/L）treatment,but decreased upon inhibitor DPC（100 mg/L）treatment.After exogenous GA3 induction,the expression of CsFnl3.1 in fruit neck was down-regulated,and after DPC induction,the expression of CsFnl3.1 in fruit neck was up-regulated.The result of subcellular localization indicates that CsFnl3.1-GFP is localized in the nucleus.