Analysis Of Physiological Characteristics And Cloning Of Gene V-2 Of A Virescent Leaf Mutant In Cucumber(Cucumis Sativus L.)

Leaf is the important photosynthetic organ of plants and algaes.Leaf color changes often affect the photosynthetic efficiency of plants and thus affect the yield.Leaf color mutants are ideal materials for studying the mechanism of leaf color variation.They have been widely used in researches such as plant photosynthesis,photomorphogenesis,chloroplast structure function and genetic development regulation mechanism.In addition,leaf color mutants can also be used as marker traits for assisted breeding and production of hybrid generation seeds.In this study,a new cucumber virescent leaf mutant“C777”and its wild type CCMC were used as materials.Photosynthetic pigment content,photosynthetic characteristics,chlorophyll fluorescence parameters,and leaf microstructure were measured and observed respectively.The characteristics were analyzed;at the same time,the morphological observation and genetic analysis of the mutant revealed that the mutant trait was controlled by a recessive single gene.The genetic populations were generated from a cross between the C777 muatnt and the North China type cucumber“9930”and North American cucumber“GY14”.Based on the F₂ population,the virescent leaf gene（v-2）was mapped using molecular markers,and the v-2 gene was cloned.The above work lays the foundation for the next step in the study of the function of the v-2.The main findings are as follows:1.By observing the phenotypic characteristics of C777,it was found that the C777 was yellow when the cotyledons expansion,as the first true leaf unfold it turns yellow green slowly.The first true leaf was yellow when it unfold.With the second true leaf expansion,the first true leaf slowly turns green and finally turns into a yellow-green leaf.The mutant showed this color change during the whole growth stage.2.The results of photosynthetic pigment content of C777 in seedling showed that before the leaves turned green chlorophyll a and chlorophyll b and carotenoid content were significantly lower than wild type,which were respectively 27.27%、23.53%、40.00%of wild type.After the leaves turned green,the content of chlorophyll a,b and carotenoids of the mutant were significantly increased.Especially the total chlorophyll content was 3.5 times higher before the leaf color changed to green,but still lower than the wild type.The contents of chlorophyll a,chlorophyll b,total chlorophyll,and carotenoid were respectively 82.95%,75.00%,81.11%,and 83.33% of the wild type.The chlorophyll a/chlorophyll b is higher than wild type before and after the change of the leaf color of the mutant.The carotenoid/total chlorophyll was significantly higher than that in the wild type before the change of leaf color of the mutant,but no significant difference was found after the change of the leaf color of the mutant.3.Photosynthetic characteristics analysis showed that the net photosynthetic rate was 5.51μmol·m^-2·s^-1,which was significantly lower than wild type before the leaves turned green.The net photosynthetic rate increased significantly at the after the leaves turned green,but still lower than wild type.The intercellular CO₂ concentration and stomatal conductance were higher than wild type at the two stages,but there was no significant difference in transpiration rate.4.Chlorophyll fluorescence parameters analysis showed that the initial fluorescence F_o、maximum fluorescence yield Fm、maximum light energy conversion efficiency Fv/Fm、and initial light energy capture efficiency Fv’/Fm’of the mutant were significantly lower than that of the wild type,which were respectively 49.41%、24.94%、78.48% and 58.82% of wild type.The photochemical quenching coefficient of mutant was significantly lower than that of wild type,which was 78.31%of wild type.The non photochemical quenching coefficient of NPQ was significantly higher than that of the wild type,0.24 higher than the wild type.The photosynthetic electron transport rate（ETR）of mutant was significantly lower than that of wild type,which was 45.44%of the wild type.The chlorophyll fluorescence imaging showed that the QYmax（Fv/Fm）of mutant was lower than wild type.5.For leaf tissue microstructure observation of mutant,the results showed leaf palisade tissue and spongy tissue structure were significantly looser than the wild type,indicating that the v-2 gene not only controlled the leaf color change of the C777,but also affected the leaf tissue structure,Which in turn affects the photosynthesis of the C777.6.Genetic analysis of the C777 showed that the yellow-green leaf trait was a quality trait controlled by a recessive single gene.The v-2 gene was mapped using two separate populations:C777x9930F₂ and C777xGY14F₂.Finally,the gene was mapped to the genetic distance of 0.2cM on the chromosome3 of the cucumber and flanked by CAPs777-1 and Indel777-hll-1,the physical distance is 45.3kb.There wre eight candidate genes in this region.These eight genes were sequenced and only one G-A non-synonymous mutation was found in the CDS region of the CsHD gene.One pair of dCAPs marker dCAPS777-13 was designed based on the mutation site,and the linkage analysis in the localized F₂ population revealed that the marker was co-segregated with the target gene.Using this dCAPs marker,a site uniqueness test was performed in 115 cucumber natural germplasms,confirming that the mutation site is not present in natural germplasm.The qPCR results showed that there was no significant difference in the expression of CsHD gene between the mutant and the wild type before the mutant turned green,indicating that the point mutation did not affect the normal transcription of the mutant gene.Therefore,all the evidences indicated that the CsHD gene was the most likely candidate gene for the v-2.