Genome-wide Association Analysis Reveals a Novel QTL CsFS1 for Fruit Skin Color in Cucumber

doi:10.21203/rs.3.rs-578494/v1

Download PDF

Research Article

Genome-wide Association Analysis Reveals a Novel QTL CsFS1 for Fruit Skin Color in Cucumber

https://doi.org/10.21203/rs.3.rs-578494/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

Fruit skin color is a crucial external trait that affects consumer preference in cucumber. In this study, an F₂ population was constructed from a cross between the inbred lines G35 (with light-green fruit skin) and Q51 (with dark-green fruit skin) and used to investigate the inheritance patterns of fruit skin color in cucumber. Genetic analysis showed that dark-green fruit skin was dominant to light-green skin. A major QTL, Fruit skin 1 (CsFS1), was identified between 36.62 Mb and 39.77 Mb on chromosome 3 by BSA-seq and GWAS. We further narrowed down the CsFS1 locus to a 94-kb interval containing 15 candidate genes in three F₂ recombinant individuals with light-green skin color and one BC₄F₂ recombinant individual with dark-green skin color. Among these genes, Csa3G912920, which encodes a GATA transcription factor, was expressed at a higher level in the pericarp of the NIL-1334 line (with light-green fruit skin) than in that of the NIL-1325 line (with dark-green fruit skin). This study provides a novel allele for the improvement of fruit skin color in cucumber breeding.

Plant Molecular Biology and Genetics

Cucumber

fruit skin color

BSA-seq

GWAS

GATA transcription factor

A major QTL that controls fruit skin color in cucumber, CsFS1, was identified in a 94-kb region that harbors the strong candidate gene CsGATA1.

Fruit skin color is a valuable trait in the horticulture industry because it strongly influences consumer preference and exhibits extensive phenotypic variation that can be used in breeding. Many quantitative trait loci (QTLs) and genes related to fruit skin color have been detected and/or cloned in crops. In melon, skin color is determined by the composition and content of pigments such as carotenes, flavonoids, and chlorophylls (Freilich et al. 2015). In yellow casaba muskmelon, CmKFB, which encodes a kelch domain-containing F-box protein, was identified on chromosome 10 and shown to downregulate the accumulation of naringenin chalcone (Feder et al. 2015). MEL03C003375 on chromosome 4 and MELO3C003097 on chromosome 8 were also shown to be closely associated with skin color (Zhao et al. 2019). In watermelon, qrc-c8-1 on chromosome 8 controls the green shade of fruit skin; it was identified by high-density genetic mapping of recombinant inbred lines and explained 49.942% of the phenotypic variation in skin color (Li et al. 2018). Cla002755 and Cla002769 on chromosome 4 are markers for yellow skin and were identified by bulked segregant analysis sequencing (BSA-seq) and genome-wide association studies (GWAS) (Dou et al. 2018). In tomato, SlMYB12 was mapped to chromosome 1; it corresponded to the pink gene y and controlled the accumulation of yellow-colored flavonoids in the tomato fruit epidermis (Adato et al. 2009; Ballester et al. 2010). In pepper, three independent pairs of genes (y, c1, and c2) and two QTLs (pc8.1 and pc10) were identified as controlling ripe fruit color and chlorophyll content (Hurtado-Hernandez and Smith 1985).

Cucumber (Cucumis sativus L., 2n = 2x = 14) is an economically important cucurbitaceous crop worldwide, with a total global production of 75.2 million tons, of which 56.2 million tons (74.7 %) were produced throughout the Chinese mainland in 2018 (data available at http://www.fao.org/). The skin color of cucumber fruit is an important agronomic character that affects consumer choice. The locus w that controls the white fruit skin of cucumber was mapped to an 8.2-kb region on chromosome 3 between the LH39253 and ASPCR39250 markers and contains only one gene, Csa3G904140 (APRR2) (Liu et al. 2015). APRR2 encodes a nuclear localization transcription factor and controls fruit skin color by reducing the content of chlorophyll and chloroplasts (Anne et al. 2015; Jiao et al. 2017). Cucumber Csa7G051430 was identified by BSA-seq of extreme-phenotype F₂ individuals from a cross between the light-green skin mutant lgp and the wild type 406. It is homologous to Arabidopsis ARC5, which plays an important role in chloroplast division (Gao et al. 2003; Zhou et al. 2015). Similarly, Csa6G133820, mapped through the light-green leaf and fruit skin mutant M218, encodes a Ycf54-like protein required for chlorophyll synthesis named CsYcf54 (Bollivar et al. 2014; Lun et al. 2015). Csa2G352940 (CsMYB36), encoding the transcription factor MYB36, regulates yellow-green peel color in cucumber (Hao et al. 2018). To date, the mechanism that controls green fruit skin color in cucumber remains unclear. Further study of skin color inheritance and identification of candidate genes associated with green skin color will therefore provide valuable information.

BSA-seq and GWAS are simple and effective methods for the identification of molecular markers associated with target genes and QTLs that control traits of interest (Atwell et al. 2010; Michelmore et al. 1991). This study was designed to determine the inheritance pattern of green fruit skin color and to map major skin color QTLs. BSA-seq analysis detected a genomic region harboring a major fruit skin color QTL, CsFS1, on chromosome 3, and it was further validated by GWAS analysis. This study also provides preliminary evidence that Csa3G912920 is the probable candidate gene in the CsFS1 locus.

Plant materials and phenotype evaluation

Two cucumber inbred lines, G35 (light-green skin color) and Q51 (dark-green skin color), were crossed to create F₁ progeny and then self-pollinated to generate an F₂ population. The F₁ progeny was backcrossed four times to the recurrent inbred parent G35 and then self-crossed to yield the BC₄F₂ generation. Chlorophyll a and chlorophyll b were extracted from fruit skins of G35, Q51, and F₁ progeny with ethyl alcohol and quantified by a spectrophotometric method. Two parental lines, together with the F₁ and F₂ generations, were used to describe and validate the inheritance pattern of skin color traits in immature fruit. Twenty F₂ individuals with extremely light-green skin color and 20 with extremely dark-green skin color were selected for BSA-sEq. Two hundred seventy-eight individuals from the F₂ population were used for trait evaluation and QTL analysis. Fruit skin color in the F₂ population was independently evaluated by three persons. NIL-1334 (light-green skin) and NIL-1325 (dark-green skin) from the BC₄F₂ generation were used for gene expression analysis. Based on fruit skin color, 289 cucumber accessions were classified into eight categories (white, yellow-white, white-green, yellow-green, light-green, green, dark-green, and black-green) (Fig. S1) and used for GWAS analysis. All the plants were grown in plastic greenhouses under natural sunlight from spring 2016 to summer 2020 at the Tianjin Kernel Cucumber Research Institute.

Genomic DNA and total RNA extraction

Genomic DNA was extracted by the cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson 1980) from fresh young leaves of P₁, P₂, and F₂ individuals and used for BSA-seq and QTL analyses.

Pericarp tissues were harvested from NIL-1334 and NIL-1325 at 0 days post-anthesis (DPA), 5 DPA, and 10 DPA. Each sample consisted of at least three fruits from different plants, and three replicate samples were used for gene expression analysis. Total RNA was extracted using the Quick RNA Isolation Kit (Huayueyang Biotechnology (Beijing) Co., Beijing, China) following the manufacturer’s instructions. The concentration of total RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Delaware, USA).

BSA-seq

Two DNA pools, the light-green pool (QL-pool) and dark-green pool (SL-pool), were created by mixing equal amounts of DNA from 20 individuals with light-green fruit skins and 20 individuals with dark-green fruit skins, respectively. Paired-end sequencing libraries (150-bp read length) with insert sizes of approximately 400 bp were prepared for sequencing on the Illumina NovaSeq 6000 platform. The short reads from the two pools were aligned to the reference genome of the 9930 line (Huang et al. 2009) using BWA software with default parameters (Li and Durbin 2009). SNP-calling was performed using SAMtools and BCFtools (Li and Durbin 2009). Low-quality SNPs with base quality value < 30, read depth < 2×, and mapping quality value < 30 were excluded to minimize false positives caused by repetitive genomic sequence or sequencing and alignment errors.

Two parameters, SNP-index and ∆(SNP-index) (Takagi et al. 2013), were calculated to identify candidate regions for fruit skin color QTLs. SNP-index is the proportion of reads covering a given SNP that differ from the reference sequence. Thus, SNP-index = 0 if all short reads covering a given nucleotide position contain the reference SNP (9930 line), whereas SNP-index = 1 if all the short reads at that position contain the mutant SNP. ∆(SNP-index) is obtained by subtracting the SNP-index of the QL-pool from that of the SL-pool. The average SNP-index at a given genomic interval was calculated using a sliding window with a 1-Mb window size and a 10-kb increment. SNP-index graphs for the QL-pool and SL-pool, as well as the corresponding ∆(SNP-index), were plotted. The ∆(SNP-index) should not differ significantly from 0 in a genomic region with no major QTL (Takagi et al. 2013). We used a Rscript simulation to generate confidence intervals around the SNP-index under the null hypothesis of no QTL. First, we created two pools of progeny with a given number of individuals by random sampling. From each pool, a given number of alleles were sampled, corresponding to the read depth. Second, the SNP-index for each pool and the Δ(SNP-index) were calculated, and the process was iterated 10,000 times for each read depth to generate confidence intervals. Finally, these intervals were plotted for all genomic regions with variable read depths.

GWAS

Re-sequencing data from 289 cucumber accessions were obtained, with an average genome coverage of 98.27% and an average sequencing depth of 19.728×. We obtained 2,352,638 SNPs, and 399,352 high-quality SNPs were retained, with a deletion rate of less than 0.2. The association between fruit skin color and each SNP was tested using a unified mixed model (Yu et al. 2006; Zhang et al. 2010) that includes principal components (Price et al. 2006) as a fixed effect to account for the population structure and kinship matrix (Loiselle et al. 1995) and to explain familial relatedness. Using the Bayesian information criterion, a backward elimination procedure was implemented to determine the optimal number of principal components to include in the mixed model (Schwarz 1978). The false discovery rate was controlled at 5% using the Benjamini and Hochberg procedure (Benjamini and Hochberg 1995). A likelihood ratio-based r² statistic was used to assess the goodness-of-fit of each SNP (Sun et al. 2010). All analyses were performed using the Genome Association and Prediction Integrated Tool (GAPIT) package (Lipka et al. 2012).

Marker development and QTL analysis

The SNPs were filtered from the re-sequencing data of the two parents, G35 and Q51. The sequence context of the candidate SNPs was examined in the 9930 reference genome using BLAST alignment to obtain longer sequences for marker development. In total, 35 kompetitive allele specific PCR (KASP) SNP markers on chromosome 3 were developed using the BSA-seq and GWAS data and created using Primer 5.0 (PREMIER Biosoft International, USA) (Table S3). The genotypes of the F₂ population were analyzed using an Infinite M1000 microplate reader (Tecan, Switzerland) and the online tool “snpdecoder” (http://www.snpway.com/snpdecoder/). Linkage analysis was performed using JoinMap 4.0 (Ooigen 2011), and QTL analysis was performed in MapQTL6.0 using the multiple QTL model (MQM mapping) procedure (Van Ooijen, 2009).

Quantitative real-time PCR (qRT-PCR)

Single-stranded cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Bio Inc., Dalian, China) following the manufacturer’s instructions. qRT-PCR was performed in a 10-µl reaction volume consisting of 5 µl TB Green Premix Ex Taq (Tli RNaseH Plus) (TaKaRa), 0.25 µl ROX Reference Dye (50×), 0.25 µl each of forward and reverse primers (10 µM), 1 µl cDNA templates, and 3.25 µl purified water. Thermal cycling began with an initial step at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s, and it was performed on the QuantStudio Flex 6 Real-Time PCR System (Applied Biosystems, California, USA). All samples were performed in triplicate, and CsACTIN (Csa2G018090) was used as the internal reference gene. Relative expression values were determined using the comparative Ct method (2^−ΔΔCt). Primers used for qRT-PCR are listed in Table S4.

Phylogenetic analysis

CsGATA1 and its homologous amino acid sequences were retrieved from public databases: SolGenomics (https://solgenomics.net/) and the Cucurbit Genomics Database (http://cucurbitgenomics.org). Known GATA transcription factors from rice, maize, and Arabidopsis were added to the analysis. Sequence alignments and a neighbor-joining tree with 1000 bootstrap replicates were constructed in MEGA X (Kumar et al. 2016).

Phenotypic analysis of fruit skin color in cucumber

The inbred lines G35 (light-green cucumber) and Q51 (dark-green cucumber) were used as parents for fine mapping of fruit skin color. The fruit skin color of all F₁ individuals was darker green than G35 and lighter green than Q51, but it inclined more towards dark green (Fig. 1a). Pigment content analysis showed that chlorophyll a and chlorophyll b contents were significantly lower in G35 than in Q51 (Fig. 1b). These results indicated that fruit skin color was determined by chlorophyll content.

Identification of a major QTL locus, CsFS1, on chromosome 3 by BSA-seq and GWAS

To rapidly identify loci for skin color in the F₂ population, two bulks consisting of 20 dark-green (SL-pool) and 20 light-green (QL-pool) progenies were sequenced on the Illumina platform. A total of 12.9 Gb of raw reads were generated, with an average depth of approximately 20.4×. The short reads were aligned to the cucumber reference genome (Huang et al. 2009), and 145,804 SNPs were identified between the dark-green and light-green parents. Based on the SNP-indices of the QL- and SL-pools, the ∆(SNP-index) of a genomic region from 36.62 Mb to 39.77 Mb on chromosome 3 was greater than the threshold value and close to 1.00 (Fig. 2a). This region may therefore harbor a major QTL for the fruit skin color trait in cucumber.

To independently confirm that this region was indeed related to fruit skin color, GWAS was performed on 289 cucumber accessions (average depth of 19.73× and 98.27% coverage of the cucumber reference genome) (Huang et al. 2009). A total of 2,352,638 SNPs were identified using GATK software with default parameters (McKenna et al. 2010). To reduce the incidence of false-positive signals, a high-resolution variation map of 399,352 SNPs with minor allele frequency > 5% and missing rate < 0.2% was generated and used for genome-wide association analysis of fruit skin color with a unified mixed linear model that controlled for population structure and familial relatedness. A Manhattan plot for cucumber fruit skin color showed the strongest association signal (SNP_fs) on the distal arm of chromosome 3, overlapping with the genomic region identified by QTL-seq (Fig. 2b). This indicated that a major QTL controlling fruit skin color resided on the distal arm of chromosome 3, and it was named CsFS1 (Fruit skin 1).

Fine mapping narrowed down CsFS1 to a 94-kb interval

To identify the candidate gene(s) in the CsFS1 locus, classical QTL analysis was performed using 278 F₂ progenies. A total of 35 SNP markers were developed between 15.66 and 39.77 Mb on chromosome 3 and used for genotypic analysis of the F₂ segregating population (Table S3). QTL analysis using an MQM showed that the LOD peak from 64.85 to 69.05 cM was consistent with the physical distance from 39.0 to 39.77 Mb on chromosome 3 (Fig. 3a). In this interval, the highest LOD marker explained 35.6% of the phenotypic variation in the F₂ segregating population (Table S1). The genomic interval of CsFS1 was further narrowed down to between two SNP markers (39,531,980 and 39,626,163 bp) using four recombinant individuals from the F₂ and BC₄F₂ populations (Fig. 3b). We therefore confirmed that the CsFS1 locus lay within a 94-kb interval on chromosome 3.

Identification of a candidate gene related to fruit skin color

According to the cucumber genome database (http://www.icugi.org/), 12 of the 15 predicted protein-coding genes in the 94-kb interval have functional annotations (Table S2). qPCR experiments were performed to investigate the expression patterns of three possible candidate genes associated with fruit skin traits in NIL-1334 (light-green) and NIL-1325 (dark-green). In the pericarp, only the expression of Csa3G912920 differed significantly between NIL-1334 and NIL-1325 (P < 0.05) (Fig. 3c, Fig. S2). The Csa3G912920 gene encodes a plant GATA transcription factor and has a conserved zinc finger domain. A phylogenetic tree and sequence alignment showed that Csa3G912920 homologs from melon (MELO3C003335), watermelon (Cla97C09G175500), and wax gourd (Bhi05M000420), highlighted in the gray-shadowed box, all encode GATA transcription factors (Fig. 4a and b). Secondary structural element analysis showed that the zinc finger domains include four 𝛽-folds and one 𝛼-spiral by looking up the literature (Fig. 4b). Csa3G912920 was designated as a candidate gene for CsFS1.

Previous studies have shown that Arabidopsis GNC (GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED) and CGA1 (CYTOKININ-RESPONSIVE GATA1), members of the GATA transcription factor family, play a major role in the regulation of chlorophyll synthesis (Chiang et al. 2012). Under light, overexpression of GNC promotes chloroplast development and the production of chlorophyll in roots (Richter et al. 2013). We therefore inferred that Csa3G912920 is the probable candidate gene for CsFS1 and named it CsGATA1.

In this study, we combined QTL-seq (Takagi et al. 2013) of an F₂ segregating population with GWAS to identify a major QTL CsFS1 for fruit skin color in cucumber. The major advantage of QTL-seq is that DNA marker development and marker genotyping are not necessary. The SNPs available between the parental lines serve as such markers, reducing cost and time. In addition, the use of the SNP-index allows for accurate evaluation of the frequencies of the parental alleles. These advantages make QTL-seq an attractive method for rapid identification of genomic regions that harbor major QTLs.

Fruit skin color is an essential agronomic trait in cucumber that affects exterior quality and consumer preferences. In this study, we detected the major QTL CsFS1 on chromosome 3 between 39,531,980 and 39,626,163 bp. Previously, the w locus controlling the white fruit skin trait was also mapped to chromosome 3 (Liu et al. 2016), residing 281 kb upstream of the CsFS1 locus. In the w locus, Csa3G904140 (APRR2) harbors a single-nucleotide insertion that causes a frameshift mutation and a truncated protein in the white cucumber. Here, we found no sequence differences in APRR2 between the two parents, G35 and Q51. Therefore, CsFS1 is a novel QTL that controls green fruit skin in cucumber.

Through classical genetic mapping, CsFS1 was narrowed to a 94-kb physical interval that contains 15 predicted protein-coding genes. The Csa3G912920 gene encodes a GATA-type transcription factor, and its expression differed significantly between near isogenic lines with light- and dark-green fruit skins. Previous studies have shown that the GATA transcription factor families in Arabidopsis, rice, and other plants are highly conserved (Reyes et al. 2004). The GATA transcription factors are evolutionarily conserved transcriptional regulators that recognize promoter elements with a G-A-T-A core sequence (Behringer and Schwechheimer 2015). The paralogous LLM-domain B-GATA transcription factors GNC and GNL contribute to chlorophyll biosynthesis and chloroplast formation in light-grown Arabidopsis seedlings (Bastakis et al. 2018; Bi et al. 2005; Chiang et al. 2012). Together, GNC and GNL control germination, greening, flowering time, and senescence downstream of auxin, cytokinin, gibberellin, and light signaling (Ranftl et al. 2016). Studies have confirmed that some GATA genes are preferentially expressed in the leaf (Ao et al. 2015). Leaves are the main organs for photosynthesis and light stress response in plants. High expression of a GATA transcription factor in leaves is consistent with its influence on chlorophyll synthesis. Therefore, it is reasonable to suggest that Csa3G912920 is the candidate gene for fruit skin color in cucumber. Nonetheless, additional experiments are required to provide evidence for Csa3G912920 gene function and robustly evaluate this hypothesis.

In conclusion, we identified a novel QTL, CsFS1, that controls green fruit skin color in cucumber and proposed a candidate gene, Csa3G912920, that may be responsible for the green color phenotype. Our results provide insight into the biological and molecular mechanisms of fruit skin color formation and can promote the development of attractive cucumber varieties with enhanced nutrients in the future.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2016YFD0101900) and the Tianjin Natural Science Foundation Project (20JCYBJC00720). The authors would like to thank TopEdit (www.topeditsci.com) for linguistic assistance during preparation of this manuscript.

Author contributions statement

YL and HH designed the research; XG, LZ, and AW performed the experiments and analyzed the data; QY and HW analyzed the data; WK, JL, SD, RY, and HH conducted the field trials; QY, XG, DS, YR and TL wrote the manuscript, and all authors read and approved the manuscript.

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Adato A, Mandel T, Mintz-Oron S, Venger I, Levy D, Yativ M, Domínguez E, Wang Z, De Vos RCH, Jetter R (2009) Fruit-Surface Flavonoid Accumulation in Tomato Is Controlled by a SlMYB12-Regulated Transcriptional Network. PLOS Genetics 5:e1000777
Anne, Cortleven, Thomas, Schmülling (2015) Regulation of chloroplast development and function by cytokinin. Journal of experimental botany
Ao T, Liao X, Xu W, Liu A (2015) Identification and characterization of GATA gene family in castor bean (Ricinus communis). Plant Diversity and Resources 37:453-462
Atwell S, Huang YS, Vilhjálmsson BJ, Willems G, Horton M, Li Y, Meng D, Platt A, Tarone AM, Hu TT (2010) Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines. Nature 465:627-631
Ballester A-R, Molthoff J, de Vos R, Hekkert BtL, Orzaez D, Fernández-Moreno J-P, Tripodi P, Grandillo S, Martin C, Heldens J (2010) Biochemical and Molecular Analysis of Pink Tomatoes: Deregulated Expression of the Gene Encoding Transcription Factor SlMYB12 Leads to Pink Tomato Fruit Color. Plant Physiology 152:71-84
Bastakis E, Hedtke B, Klermund C, Grimm B, Schwechheimer C (2018) LLM-Domain B-GATA Transcription Factors Play Multifaceted Roles in Controlling Greening in Arabidopsis. Plant Cell 30:582-599
Behringer C, Schwechheimer C (2015) B-GATA transcription factors - insights into their structure, regulation, and role in plant development. Front Plant Sci 6:90
Benjamini Y, Hochberg Y (1995) Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society: Series B (Methodological) 57:289-300
Bi YM, Zhang Y, Signorelli T, Zhao R, Zhu T, Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor gene family reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity. Plant J 44:680-692
Bollivar D, Braumann I, Berendt K, Gough SP, Hansson M (2014) The Ycf54 protein is part of the membrane component of Mg-protoporphyrin IX monomethyl ester cyclase from barley (Hordeum vulgare L.). Febs j 281:2377-2386
Chiang YH, Zubo YO, Tapken W, Kim HJ, Lavanway AM, Howard L, Pilon M, Kieber JJ, Schaller GE (2012) Functional characterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development, growth, and division in Arabidopsis. Plant Physiol 160:332-348
Dou J, Lu X, Ali A, Zhao S, Zhang L, He N, Liu W (2018) Genetic mapping reveals a marker for yellow skin in watermelon (Citrullus lanatus L.). PLoS One 13:e0200617
Feder A, Burger J, Gao S, Lewinsohn E, Katzir N, Schaffer AA, Meir A, Davidovich-Rikanati R, Portnoy V, Gal-On A (2015) A Kelch Domain-Containing F-Box Coding Gene Negatively Regulates Flavonoid Accumulation in Muskmelon. Plant Physiol 169:1714-1726
Freilich S, Lev S, Gonda I, Reuveni E, Portnoy V, Oren E, Lohse M, Galpaz N, Bar E, Tzuri G (2015) Systems approach for exploring the intricate associations between sweetness, color and aroma in melon fruits. BMC Plant Biol 15:71
Gao H, Kadirjan-Kalbach D, Froehlich JE, Osteryoung KW (2003) ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery. Proc Natl Acad Sci U S A 100:4328-4333
Hao N, Du Y, Li H, Wang C, Wang C, Gong S, Zhou S, Wu T (2018) CsMYB36 is involved in the formation of yellow green peel in cucumber (Cucumis sativus L.). Theoretical and Applied Genetics 131:1659-1669
Huang S, Li R, Zhang Z, Li L, Gu X, Fan W, Lucas WJ, Wang X, Xie B, Ni P (2009) The genome of the cucumber, Cucumis sativus L. Nat Genet 41:1275-1281
Hurtado-Hernandez H, Smith PG (1985) Inheritance of mature fruit color in Capsicum annuum L. Journal of Heredity 76:211-213
Jiao J, Liu H, Liu J, Cui M, Xu J, Meng H, Li Y, Chen S, Cheng Z (2017) Identification and functional characterization of APRR2 controlling green immature fruit color in cucumber (Cucumis sativus L.). Plant Growth Regulation 83:233-243
Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol 33:1870-1874
Li B, Lu X, Dou J, Aslam A, Gao L, Zhao S, He N, Liu W (2018) Construction of A High-Density Genetic Map and Mapping of Fruit Traits in Watermelon (Citrullus Lanatus L.) Based on Whole-Genome Resequencing. Int J Mol Sci 19
Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754-1760
Lipka AE, Tian F, Wang Q, Peiffer J, Li M, Bradbury PJ, Gore MA, Buckler ES, Zhang Z (2012) GAPIT: genome association and prediction integrated tool. Bioinformatics 28:2397-2399
Liu H, Meng H, Pan Y, Liang X, Jiao J, Li Y, Chen S, Cheng Z (2015) Fine genetic mapping of the white immature fruit color gene w to a 33.0-kb region in cucumber (Cucumis sativus L.). Theor Appl Genet 128:2375-2385
Loiselle BA, Sork VL, Nason J, Graham C (1995) Spatial genetic structure of a tropical understory shrub, PSYCHOTRIA OFFICINALIS (RuBIACEAE). American Journal of Botany 82:1420-1425
Lun Y, Wang X, Zhang C, Yang L, Gao D, Chen H, Huang S (2015) A CsYcf54 variant conferring light green coloration in cucumber. Euphytica 208:509-517
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297-1303
Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci U S A 88:9828-9832
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321-4325
Ooigen V (2011) Multipoint maximum likelihood mapping in a full-sib family of an outbreeding species. Genet Res (Camb) 93:343-349
Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, Reich D (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38:904-909
Ranftl QL, Bastakis E, Klermund C, Schwechheimer C (2016) LLM-Domain Containing B-GATA Factors Control Different Aspects of Cytokinin-Regulated Development in Arabidopsis thaliana. Plant Physiol 170:2295-2311
Reyes JC, Muro-Pastor MI, Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice. Plant Physiol 134:1718-1732
Richter R, Bastakis E, Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNL/CGA1 in the control of greening, cold tolerance, and flowering time in Arabidopsis. Plant Physiol 162:1992-2004
Schwarz G (1978) Estimating the Dimension of a Model. Ann Statist 6:461-464
Sun G, Zhu C, Kramer MH, Yang SS, Song W, Piepho HP, Yu J (2010) Variation explained in mixed-model association mapping. Heredity (Edinb) 105:333-340
Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, Uemura A, Utsushi H, Tamiru M, Takuno S (2013) QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J 74:174-183
Yu J, Pressoir G, Briggs WH, Vroh Bi I, Yamasaki M, Doebley JF, McMullen MD, Gaut BS, Nielsen DM, Holland JB (2006) A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nat Genet 38:203-208
Zhang Z, Ersoz E, Lai CQ, Todhunter RJ, Tiwari HK, Gore MA, Bradbury PJ, Yu J, Arnett DK, Ordovas JM (2010) Mixed linear model approach adapted for genome-wide association studies. Nat Genet 42:355-360
Zhao G, Lian Q, Zhang Z, Fu Q, He Y, Ma S, Ruggieri V, Monforte AJ, Wang P, Julca I (2019) A comprehensive genome variation map of melon identifies multiple domestication events and loci influencing agronomic traits. Nat Genet 51:1607-1615
Zhou Q, Wang S, Hu B, Chen H, Zhang Z, Huang S (2015) An ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5 gene mutation confers light green peel in cucumber. J Integr Plant Biol 57:936-942

FigS1.pdf
Fig. S1 Eight categories of fruit skin colors were defined to evaluate phenotypes of 289 cucumber accessions. a White, b Yellow-white, c White-green, d Yellow-green, e Light-green, f Green, g Dark-green, h Black-green.
FigS2.pdf
Fig. S2 Relative expression of three candidate genes in fruit pericarp of the light-green near isogenic line NIL-1334 and the dark-green near isogenic line NIL-1325 at 0 days post-anthesis (DPA), 5 DPA, and 10 DPA. The relative expression is shown as the mean ± standard deviation, and statistical significance was determined using Student’s t-tests (*P < 0.05).
TableS1.xlsx
Table S1 QTL analysis of fruit skin color in the cucumber F2 population.
TableS2.xlsx
Table S2 Information on 15 candidate genes between 39,531,980 and 39,626,163 bp on chromosome 3.
TableS3.xlsx
Table S3 Information on 35 KASP SNP markers for QTL analysis.
TableS4.xlsx
Table S4 Primers used in qRT-PCR.

Download PDF

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

Genome-wide Association Analysis Reveals a Novel QTL CsFS1 for Fruit Skin Color in Cucumber

Archived Versions:

Version 1

Abstract

Figures

Key message

Introduction

Materials And Methods

Plant materials and phenotype evaluation

Genomic DNA and total RNA extraction

BSA-seq

GWAS

Marker development and QTL analysis

Quantitative real-time PCR (qRT-PCR)

Phylogenetic analysis

Results

Discussion

Declarations

References

Supplementary Files

Archived Versions:

Version 1