Fine Mapping of CsVYL, Conferring Virescent Leaf Through the Regulation of Chloroplast Development in Cucumber

Abstract

Leaf color mutants in higher plants are ideal materials for investigating the structure and function of photosynthetic system. In this study, we identified a cucumber vyl (virescent-yellow leaf) mutant in the mutant library, which exhibited reduced pigment contents and delayed chloroplast development process. F₂ and BC₁ populations were constructed from the cross between vyl mutant and cucumber inbred line 'Hazerd' to identify that the vyl trait is controlled by a simply recessive gene designated as CsVYL. The CsVYL gene was mapped to a 3.8 cM interval on chromosome 4 using these 80 F₂ individuals and BSA (bulked segregation analysis) approach. Fine genetic map was conducted with 1542 F₂ plants and narrowed down the vyl locus to an 86.3 kb genomic region, which contains a total of 11 genes. Sequence alignment between the wild type (WT) and vyl only identified one single nucleotide mutation (C→T) in the first exon of gene Csa4G637110, which encodes a DnaJ-like zinc finger protein. Gene Expression analysis confirmed the differences in transcription level of Csa4G637110 between wild type and mutant plants. Map-based cloning of the CsVYL gene could accelerate the study of chloroplast development and chlorophyll synthesis of cucumber.

Keywords: Cucumis sativus L.; DnaJ proteins; chloroplast function; leaf color mutant; virescent.

FIGURE 1

Phenotypic analysis of the vyl mutant. (A) Cotyledons color of WT and vyl; (B) Detailed observations of leaf color of WT and vyl grown in field; (C) Leaf color of vyl at plant adult stage.

FIGURE 2

The leaf color phenotype and pigment contents of vyl and WT under different light intensity treatments. L1 of vyl and WT planted under 100% (A), 68% (B) and 3% (C) of full light conditions was used for leaf color observation and Chl measurement. Values are the mean ± SD, ^∗∗P < 0.01.

FIGURE 3

Chloroplast structure and photosynthesis parameters of vyl and WT at different leaf position. Electron microscopy of chloroplasts ultrastructures in L1-L4 of vyl (A–D) and WT (E–H). TM, thylakoid membrane; Sg, starch grain; P, plastoglobulus. Scale bar = 500 nm. (I) Net photosynthetic rate (P_n); (J) Stomatal conductance (Gs); (K) Intercellular CO₂ concentration (Ci); (L) Photosystem II (F_v/F_m); Values are the mean ± SD, and ^∗(P < 0.05) and ^∗∗(P < 0.01) indicate significant difference between the treatments.

FIGURE 4

Positional cloning and sequence alignment of the CsVYL gene. (A) Preliminary mapping with BSA based on 80 plants from the F₂ population placed CsVYL locus on chromosome 4 at a 3.8 cM region between UW804200 and SSR05515. Numbers on chromosome left are genetic distance (cM); (B) Further genetic position analysis with 480 F₂ individuals delimited the CsVYL locus at a 0.8 cM region; (C) Fine mapping of the CsVYL locus with 1542 individuals. The CsVYL gene was narrowed down to an 86.3 kb genetic region. Numbers on chromosome left are recombinants of each interval; (D) 11 annotated genes were predicted in the 86.3 kb region. Csa4G637110 is shown as the red arrow. (E) Gene and protein sequence alignment of CsVYL gene in 9930, Hazerd, ccmc and vyl Gray boxes, black boxes and lines between them indicate 5′ and 3′UTR, exons and introns respectively.

FIGURE 5

Relative expression of Cs4G637110 gene at different leaf color stages (yellow and green stage) of WT and vyl mutant by qRT-PCR. Data were displayed using the CsActin gene as an internal control with three biological and three technical replicates. Values are the mean ± SD. ^∗∗P < 0.01.

FIGURE 6

Expression evaluation of genes associated with Chl biosynthesis and photosynthesis through qRT-PCR. The L1 Leaves of vyl and WT were sampled. CsActin was used as the internal control. The transcript levels of all tested genes in wild type (WT) were set to 1.0. Values are the mean ± SD with three biological and three technical replicates. ^∗∗P < 0.01.