| Cucumber,（Cucumis sativus L.）（2n=2x=14）,is very popular in the world and according to the World Food and Agriculture Organization（FAO）,cucumber ranked sixth in sown area and third in production among all vegetables in 2020.Leaf color mutants are important for understanding chloroplast development,chlorophyll synthesis and catabolism in leaves.‘SC311’ originated from USDA（PI 164465）,and the virescent leaf mutant isolated was named‘SC311Y’ after its progeny was found to have virescent leaf separation during the daily management of cucumber germplasm resources.The virescent leaf mutant was isolated by mapbased cloning combined with BSA-Seq,RNA-Seq,the virescent leaf（v-3）was cloned,and the mechanism of virescent leaf formation was initially explored.The main findings of this study are as follows:1.The phenotypical characteristics of the ‘SC311Y’ mutant and the response to temperature and light were analyzed.The results showed:（1）The cotyledons and true leaves of the mutant were yellow and flat in the early stage of growth,and the leaf color turned green gradually as the leaves grew.Leaf folds with white stripes appear from the fifth true leaf.（2）Compared with wild type,the chlorophyll and carotenoid content of mutant plant leaves decreased significantly,but the difference in pigment composition was not significant;The pn of mutant plants decreased significantly,the photosaturation point decreased significantly,and the chlorophyll fluorescence parameters differed significantly.Ultrastructural observations show that the chloroplast structure is abnormal.（3）Based on temperature 28℃ and light 25000 lx,set 3 temperature treatments（21℃,28℃ and 35℃）and 3 light intensity treatments（10000lx,25000 lx and 50,000 lx）,it was found that when the light intensity increased from 10000 lx to 25000 lx,the pigment content in the mutant leaves increased significantly,indicating that‘SC311Y’ was a light-sensitive mutant;When the light intensity is certain and the temperature increases from 21℃ to 28℃,the pigment content in the mutant leaves increases significantly,indicating that the mutant is also a thermo-sensitive mutant.（4）Mutant plants show male sterility and female flower dysplasia.2.In order to further identify the biological processes in which mutants participate in regulation,a transcriptome sequencing of the newly flattened cotyledons of wild and mutants was carried out.A total of 1104 DEGs were obtained.DEGs are mainly enriched in UDP formation,ribosome composition,transcription factor complex.KEGG enrichment analysis shows that both up-regulation and down-regulation genes are significantly enriched in ribosome pathways.The GO and KEGG analysis suggests that the mutant may have influenced the process of RNA metabolism.In addition,the down-regulation of Csa V31G014830 encoding ferredoxin may affect the photosynthetic efficiency of the mutant,and the mutant regulates trait development through auxin and ethylene pathways.In order to explore the formation mechanism of virescent leaf,the metabolic processes of chlorophyll and carotenoid were studied,and the expression of key enzymes encoding chlorophyll breakdown was significantly increased.The expression of PDV2 genes encoding the plastid dividing protein complex subunit that determines the number of chloroplasts is significantly down-regulated.Combined with the results of leaf ultrastructure,chlorophyll fluorescence parameters and transcriptome analysis,it is shown that the PDV2 down-regulation may affect the chloroplast split,which in turn leads to the virescent leaf.3.In this paper,the map-based cloning and BSA-Seq of v-3 were carried out.Heterozygous green leaves ‘SC311’ were used to obtain F2-A population,and the ratio of green leaf : yellow leaf separation was 59:18.Due to the male sterility of the mutant,the F2-B population was constructed with ‘9930’ as the parent and ‘SC311Y’ as the mother of the mutant.The results showed that F1 was a normal green leaf,and the initial location of 93 F2-B plants was 73:20.After a chi-squared test,the ratio of offspring separation of the two populations was3:1.These results show that the virescent leaf is a mass trait controlled by a recessive gene,v-3.Two hundred and twenty primers were selected on the whole gene and the v-3 gene was localized in the 15.56 Mb-41.69 Mb of chromosome 3（based on the location of the ’Gy14’genome,the same below）using BSA-SSR.Further,15 yellow-leaved individuals from the F2-A populations and 15 green-leaved individuals were selected for BSA-Seq,and the v-3 gene was localized in the 34.66-37.53 Mb of chromosome 3.The linkage map on chromosome 3 was also constructed based on the F2-A population of 177 plants,and the v-3 gene was localized in the 32.16-35.64 Mb interval of chromosome 3.A framework genetic map was constructed by randomly selecting 93 plants in the F2-B population and localizing the v-3 gene to the 32.70-35.66 Mb interval of chromosome 3.The number of F2-B population was expanded（3903plants）,screened for polymorphic markers,and further localized the v-3 gene to the 33.54-35.66 Mb interval of chromosome 3 by stepwise transfer method with no recombinant markers in the interval.The v-3 gene was finally localized within 774.88 Kb of chromosome 3 by integrating BSA-SSR,BSA-Seq and map-based cloning.Using ’Gy14’ and ’9930’ as the reference genome,respectively,we looked for eligible SNPs in the localization interval based on resequencing data（in exons,non-synonymous mutations,"Green " pool is heterozygous,"Yellow" pool is pure,and "Yellow" pool is different from the reference genome）.Only four genes with seven SNPs were present in both genomes,but only Csa3G042730,encoding the RNA exosome supercomplex subunit RST1,was also DEG.The presence of a SNP（G→C）on exon 13 of this gene caused the conserved serine to threonine,and thus Csa3G042730 was speculated to be a candidate gene for virescent leaf（v-3）. |
