Fine Mapping of Cla015407 Controlling Plant Height in Watermelon

in Journal of the American Society for Horticultural Science
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  • College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin, Heilongjiang 150030, China; and Key Laboratory of Biology and Genetic Improvement of Horticulture Crops (Northeast Region), Ministry of Agriculture and Rural Affairs, Harbin, Heilongjiang 150030, China

The plant compact and dwarf growth habit is an important agronomic trait when breeding watermelon (Citrullus lanatus) cultivars because of their reduced vine length, high-density planting, and better land utilization; however, the genetic basis of the dwarf growth habit is not well-known. In this study, the plant population of six generations, P1, P2, F1, F2, BC1P1, and BC1P2, were studied. A genetic segregation analysis demonstrated that dwarfism is mainly controlled by a single recessive Cldw gene. Furthermore, whole-genome sequencing of two distinct watermelon cultivars, W1-1 (P1) and 812 (P2), was performed and preliminarily mapped through a bulked segregant analysis of F2 individuals that revealed the Cldw gene locus on chromosome 9. Two candidate genes, Cla015407 and Cla015408, were discovered at the delimited region of 43.2 kb by fine mapping, and gene annotation exposed that the Cla015407 gene encodes gibberellic acid 3β-hydroxylase protein. In addition, a comparative analysis of gene sequence and cultivars sequences across the reference genome of watermelon revealed the splice site mutation in the intron region of the Cldw gene in dwarf-type cultivar 812. The quantitative real-time polymerase chain reaction exhibited a significantly higher expression of the Cla015407 gene in cultivar W1-1 compared with 812. There was no significant difference in the vine length of both cultivars after gibberellic acid treatment. In brief, our fine mapping demonstrated that Cla015407 is a candidate gene controlling dwarfism of watermelon plants.

Abstract

The plant compact and dwarf growth habit is an important agronomic trait when breeding watermelon (Citrullus lanatus) cultivars because of their reduced vine length, high-density planting, and better land utilization; however, the genetic basis of the dwarf growth habit is not well-known. In this study, the plant population of six generations, P1, P2, F1, F2, BC1P1, and BC1P2, were studied. A genetic segregation analysis demonstrated that dwarfism is mainly controlled by a single recessive Cldw gene. Furthermore, whole-genome sequencing of two distinct watermelon cultivars, W1-1 (P1) and 812 (P2), was performed and preliminarily mapped through a bulked segregant analysis of F2 individuals that revealed the Cldw gene locus on chromosome 9. Two candidate genes, Cla015407 and Cla015408, were discovered at the delimited region of 43.2 kb by fine mapping, and gene annotation exposed that the Cla015407 gene encodes gibberellic acid 3β-hydroxylase protein. In addition, a comparative analysis of gene sequence and cultivars sequences across the reference genome of watermelon revealed the splice site mutation in the intron region of the Cldw gene in dwarf-type cultivar 812. The quantitative real-time polymerase chain reaction exhibited a significantly higher expression of the Cla015407 gene in cultivar W1-1 compared with 812. There was no significant difference in the vine length of both cultivars after gibberellic acid treatment. In brief, our fine mapping demonstrated that Cla015407 is a candidate gene controlling dwarfism of watermelon plants.

Watermelon (Citrullus lanatus) is an important cucurbit crop that covers 7% of the total vegetable crop production area (Guo et al., 2012). The dwarf-type plants have significant advantages in agricultural crops because of their high-density planting, resistance to wind storms, lodging condition tolerance, and reduced major losses during mechanical harvesting operations (Amasino et al., 2003).

Numerous dwarfism-associated traits such as short internodes, reduced main stem length, and bushy-type growth habits of different crops have been studied to identify the candidate genes controlling the compact and dwarf growth habit. It was reported that the compact and dwarf growth habit of watermelon is controlled by allelism of two genes, dw-1 and dw-1s, and three independent loci, dw-2, dw-3, and dw-4 (Huang et al., 1998; Loy and Liu, 1972; Yang et al., 2009). The first recessive gene, dw-1, was discovered in dwarf-type watermelon; it causes mutation in the standard-type watermelon cultivar WB-2 by synthesis of abnormal internodal cells (short internodes) (Mohr, 1956). The second genetically inherited recessive gene, dw-1s, was detected in short-vine watermelon cultivar Somali Local (Dyutin and Afanas’eva, 1987). The third recessive gene, dw-2, was identified in the short-internode vine cultivar Bush Desert King (BDK), which has shortened internode cells, and the fourth gene, dw-3, was discovered in the dwarf male-sterile watermelon cultivar DMSW (Huang et al., 1995). The gene dw-4 was identified in a self-pollinated population of short-vine watermelon cultivar 5-6y (Yang et al., 2009). Recently, potential candidate genes encoding gibberellic acid (GA) 20-oxidase-like protein and controlling dwarfism have been mapped on watermelon chromosome 7 (Dong et al., 2018). Moreover, the dwarfism gene Cldw-1 encoding an ATP-binding cassette transporter (ABC transporter)-related protein was also identified in watermelon line WM102 on chromosome 9 (Zhu et al., 2019).

Many genes controlling the compact and dwarf growth habit have been mapped in few cucurbit crops, and those findings strongly indicated the differential causes of the plant compact and dwarf growth habit. Regarding cucumber (Cucumis sativus), a dwarf-type mutant was obtained from cucumber ‘Lemon’ because of the mutagenesis resulting from the dw gene (Robinson and Mishanec, 1965). Then, the recessive gene cp was identified in cucumber line PI308915, which causes a shorter internodal length and controls the angle between leaves and side branches of compact plants (Kauffman and Lower, 1976). A compact mutant, cp-2, was also identified in ethyl methanesulfonate–treated cucumber plants, but the allelism of both cp and cp-2 was not completely understood (Kubicki et al., 1986). Then, the mutant gene cp was also identified in a short-vine cucumber cultivar and fine-mapped at the 220-kb genetic region located at chromosome 4, which is highly homologous with cytokinin oxidase (CKX) (Li et al., 2011). Regarding melon (Cucumis melo), three recessive genes, si-1, si-2, and si-3, have been reported to control the compactness or dwarfism of three comparative cultivars, Maindwarf, Persia 202, and UC Top Mark bush, respectively (Knavel, 1988, 1990; Paris et al., 1984). Among these genes, the si-1 gene is mainly connected with yellow virescence (Pitrat, 1991) and displays bushy-type plant phenotypes with compact growth as well as a short internodal length. Furthermore, the mdw1 gene was identified on a 1.8-cM region in the dwarf-type mutant line PNU-WT1, which exhibited high homology with erecta and ubiquitin (Hwang et al., 2014). During a recent study of melon, a putative genetic region associated with short internodal length was mapped on chromosome 7, and gene annotation predicted erecta as a recessive gene for controlling dwarfism (Zhang et al., 2019).

The crop morphological traits are regulated by multiple genes, and the compact and dwarf growth habit genes are mostly involved in biosynthesis or signal transduction pathways of plant hormones. Among these types of hormone, GAs are the most widely studied hormones (Nagai et al., 2018; Peng, 1999) because dwarf-type plants are mostly divided into the GA-deficient type or insensitive type based on the location of their mutant genes during hormonal biosynthesis or metabolic pathways. To date, numerous genes encoding GA synthesis–related enzymes have been cloned (Chiang et al., 1995; Sun, 1992; Winkler, 1995) and expressed as mutant genes in different plant types (Hedden and Phillips, 2001). Mainly, GA 3β-hydroxylase catalyzes the absolute activity in the GA biosynthesis pathway (Yamaguchi, 2008), and its pivotal role in maize (Zea mays) and rice (Oryza sativa) has been reported (Chen et al., 2014; Itoh et al., 2001; Teng et al., 2013).

The bulked segregant analysis (BSA) is a rapid technique for mapping genes related to the measured phenotype of different crops and identifying the tightly closed genetic markers linked to the major genes and that phenotype. In 1991, the BSA was primarily used to map disease resistance genes (Michelmore et al., 1991). It has been widely used for rapid gene mapping and the identification of genetic markers linked with quantitative or qualitative traits of interest in horticultural crops such as watermelon (Dong et al., 2018), melon (Li et al., 2017; Liu et al., 2019), cucumber (Zhang et al., 2015), and tomato [Solanum lycopersicum (Zhao et al., 2016)].

During this study, whole-genome sequencing and BSA were performed for primary and fine mapping of genes controlling dwarfism. In addition, cleaved amplified polymorphism sequence (CAPS) and kompetitive allele-specific polymerase chain reaction (KASP) markers were developed and used. The genetic inheritance analysis was also performed by using plant populations of six generations. The present study results will provide additional insight for marker-assisted breeding of dwarf watermelon cultivars in China.

Materials and Methods

Plant materials and population development.

Two distinct watermelon cultivars, W1-1 and 812, were selected as plant materials based on their distinguished plant characteristics. The cultivar W1-1 is a female with standard-type vine length and moderate internodes, whereas cultivar 812 is a male with a dwarf-type bushy vine length and short internodes (Fig. 1). These distinct cultivars were cross-pollinated to develop the further generations (F1, F2, BC1P1, and BC1P2); they were also used for whole-genome resequencing, BSA, and genetic inheritance analyses. All experiments were continued over 2 years (2018 and 2019) in a plastic green house at XiangYang Experimental Farm, Northeast Agricultural University, Harbin, China (lat. 44°04′N, long. 125°42′E). The standard horticultural practices were also performed according to the needs and symptoms of plant cultivation, plant growth, and typical climatic conditions.

Fig. 1.
Fig. 1.

Differences in the plant morphological characteristics of two distinct watermelon cultivars: (A) 35 d after sowing W1-1 (P1, standard type) grown in the green house and (B) 35 d after sowing 812 (P2, dwarf type) grown in a greenhouse.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 2021; 10.21273/JASHS04934-20

In 2018, the watermelon cultivars were grown and crossed to acquire their F1 hybrid. Then, a single F1 hybrid was further self-crossed and back-crossed to produce the F2, BC1P1, and BC1P2 generations separately. For plant cultivation, seedlings of all generations were germinated in small polyethylene bags filled with an organic mixture of soil and peat moss; then, well-developed seedlings at the fully expanded leaf stage were transferred to ridges in the plastic greenhouse with a row space of 50 cm and plant space of 12 cm.

The plant phenotypes were visually observed at the seedling stage and plant reproductive stage and classified as the standard type or dwarf type based on their main stem length (MSL). When the MSL of all plants was ≈50 cm long, the side stems were trimmed and the data of the main stem (still attached to the plant) of the plant population of six generations were recorded on same day by using a simple measuring tape. The genetic inheritance analysis was performed by investigating the segregation ratio of standard-type and dwarf-type plant phenotypes of six generations, P1 (n = 15), P2 (n = 15), F1 (n = 15), F2 (n = 196), BC1P1 (n = 150), and BC1P2 (n = 150). Furthermore, a total of 184 of 196 F2 individuals were used for primary mapping. In 2019, a total of 1439 F2 individuals were grown according to the same planting geometry and trait-measuring methods used during 2018. The fine-mapping region and marker–trait association were obtained from total of 1623 (184 and 1439) F2 individuals.

Whole-genome resequencing of bulked DNA.

Total DNA was isolated by using the improved cetyltrimethylammonium ammonium bromide (CTAB) technique (Allen et al., 2006). The DNA concentration was measured by using a spectrophotometer (SMA3000; Plextech, Shenzhen, China). DNA purity was checked using 1% agarose gel electrophoresis. A total of two bulked samples were arranged by mixing an equal proportion of extracted DNA from 30 standard-type and 30 dwarf-type F2 plants. Whole-genome sequencing (HiSeqTM 2500 platform; Illumina, San Diego, CA) of two bulked DNA samples was performed to generate the sequencing library of 200-bp paired-end reads.

BSA analysis and primary mapping.

The total resequenced reads were analyzed by removing the low-quality reads, adaptors, and reads with >10% unknown bases. The clean end reads were used across the watermelon reference genome (97103) available in the Cucurbit Genomics Database (Guo et al., 2012, 2019; Zheng et al., 2018), using the Burrows-Wheeler Aligner (BWA) software package (Li and Durbin, 2009). The raw data reads of single-nucleotide polymorphisms (SNPs) and insertion and deletions (InDels) were sorted and low-quality reads (<20 read depth) were removed using Samtool’s rmdup command (Li et al., 2009). The Unified Genotyper module of GATK was used to detect the SNPs in multiple samples (McKenna et al., 2010), and the InDels were filtered with variant filtration and annotated with ANNOVAR (Wang et al., 2010). The primary genetic region of the Cldw gene controlling dwarfism was determined by the delta (Δ) SNP index derived from locally estimated scatterplot smoothing (LOESS) regression (P ≤ 0.01) curves at each SNP position of both bulks (dwarf-type and standard-type) according to the following previously reported equations (Li et al., 2017):

SNPindex(aa)=(Xaa+Maa)SNPindex(bb)=Xaa/(Xbb+Mbb)Δ(SNPindex)=SNPindex(aa)SNPindex
where X represents the cultivar 812, M represents the cultivar W1-1, and aa and bb are the dwarf-type bulk and standard type bulk genotypes, respectively. Xaa and Maa indicate the depth of the aa population resulted from X and M, respectively. Xbb and Mbb represent the depth of the bb population resulting from X and M, respectively. The Δ(SNP index) of 1 showed striking correlations between SNPs and dwarf traits, whereas Δ(SNP index) = 0 represented no association of the SNPs within the dwarf trait. Additionally, the Δ(SNP index) analysis of each chromosome was conducted for both types of bulk based on the read depth to test the significance of the SNPs at P ≤ 0.01 and LOESS regression (0.62). The detected region above the threshold value was designated as the main region responsible for controlling dwarfism.

CAPS markers development.

Initially, the genetic region controlling dwarfism was mapped on targeted chromosome by BSA. All the filtered resequenced data were loaded into SNP2CAPS software (Thiel et al., 2004), and the major SNP loci and differential cutting sites of restriction endonucleases were chosen and transformed into CAPS markers by designing the polymerase chain reaction (PCR) assays across the whole-genome chromosomes (Amanullah et al., 2020). The best CAPS markers for PCR amplification were exported using software (Primer Premier V6.0; Premier Biosoft, San Francisco, CA), and the final sequences were synthesized by Sangon Biotech Co. (Shanghai, China).

CAPS marker verification using PCR.

The PCR amplification was performed in a 20-μL mixture using the touchdown PCR system (Amanullah et al., 2018). The mixture contained 2 μL of template of gDNA, 0.4 μL Taq endonuclease, 1 μL each of forward and reverse primer, 2 μL Taq buffer, 0.6 μL dNTPs, and 13 μL nuclease-free water. Amplification was started by preheating at 94 °C for 7 min, followed by 30 heating cycles at 94 °C for 1 min, denaturation at 60 °C for 30 s, cooling at 0.5 °C gradients, extension at 72 °C for 90 s, and final elongation at 72 °C for 10 min. All the fragmented PCR products were subsequently separated using 1% agarose gel electrophoresis and digested with four different restriction enzymes (TaqI, XhoI, EcoRI, and RsaI). The enzyme digestion reaction contained 5 μL of PCR-yielded product, 0.3 μL of restriction enzymes, 1 μL of enzyme buffer, and 8.7 μL of nuclease-free water. A total of 15 μL of digestion reaction was incubated at a specific temperature of 37 or 65 °C, depending on the required temperature for endonuclease activity, in an incubator for 3 to 4 h. The final digested products were verified by the detection of distinct bands in both watermelon cultivars and offspring populations.

Fine mapping.

The KASP system and PCR reaction procedures were conducted as described previously (Zhao et al., 2017). First, primary linkage mapping was performed on chromosome 9 by using KASP markers genotyping within 184 F2 individuals (137 standard type and 47 dwarf type). Linkage mapping software (JoinMap; Kyazma, Wageningen, the Netherlands) was used by incorporating the data of standard-type (recorded as D) and dwarf-type (recorded as B) F2 individuals. The second-year data of 230 F2 individuals (all dwarf phenotype) were verified for primary mapping by using seven polymorphic CAPS markers (Table 1). Next, two identified major CAPS markers flanking the Cldw gene region were used to identify the recombinant individuals from a total of 390 F2 individuals (all dwarf phenotypes). Furthermore, new KASP markers (Supplemental Table 1) were designed between the detected primary mapping interval and further validated by using the 43 identified recombinant individuals.

Table 1.

Primer sequences of the cleaved amplified polymorphism sequences (CAPS) markers used to map the Cldw gene controlling compact and dwarf growth habit in watermelon.

Table 1.

Exogenous GA3 treatment, endogenous GA3 measurements, and relative expression analysis.

Initially, the two cultivars W1-1 and 812 were exogenously treated with GA3 in a large plastic greenhouse. The GA3 used for exogenous treatment was obtained from Solarbio Science and Technology (Beijing, China). The GA3 powder was dissolved in a small quantity of ethanol and diluted in double-distilled water (ddH2O) to make the final solution. All plants were exogenously treated with various GA3 concentrations (0.3, 0.9, and 1.5 mmol⋅L−1); control plants were simply sprayed with a mixture of ethanol and ddH2O. The vine length was measured once per week after the seed germination.

When the plants reached the same visual growth rate as that during their reproductive growth stage, the eighth internodes from three plants were sampled. The endogenous hormone contents of sampled internodes were extracted by using liquid chromatography-mass spectrometry according to a previously described method (Chen et al., 2011). The actual measurement of endogenous GA3 was performed for the same harvested node samples.

The gene expression level was analyzed using a quantitative real-time (qRT) PCR system (QTOWER; Analytik Jena, Jena, Germany) with the SYBR Green Master Mix reagent (Novogene, Beijing, China) according to the manufacturer’s instructions. The qRT-PCR reaction mixture was composed of 1 μL of cDNA (50 ng⋅μL−1 of total RNA), 1 μL of each primer (10 μM), 10 μL of 2× SYBR Green Master Mix, and nuclease-free water, with a final volume of 20 μL. The amplification reactions included 1 cycle of 95 °C for 1 min, followed by 35 amplification cycles at 95 °C for 15 s, 58 °C for 20 s, and 72 °C for 15 s. The specific transcript amplification was verified by a single peak of the melting curve analysis obtained after the completion of the amplification reaction. The negative controls without cDNA templates were performed for overall runs to verify the potential impurity.

RNA extraction and cloning of CLA015407.

Plants adjacent to the eighth internode during the reproductive growth period (ovary expansion stage) were independently harvested from both watermelon cultivars for the tissue-specific analysis. Total RNA from the harvested samples was isolated using Trizol (Zhang et al., 2006). Complementary DNA (cDNA) was synthesized using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan).

Cloning of the candidate gene Cla015407 sequence was performed for both cultivars. Amplification was performed as follows: 1 cycle of 95 °C for 5 min, followed by 30 amplification cycles at 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 90 s. Then, the amplified targeted fragments were aligned to the vector pMD18-T and sent to Sangon Biotech Co. for sequencing.

Data analysis.

All the numerical data of plant phenotypes were entered and preliminarily sorted using spreadsheet software (Excel; Microsoft, Redmond, WA). The standard descriptive analysis of means and genetic segregation analysis (χ2 test) were performed using statistical analysis software (SPSS 23.0; IBM, Armonk, NY).

Results

Analysis of phenotypes and genetic inheritance of watermelon dwarfism.

The vine length of the cultivars W1-1 and 812 clearly exhibited significant differences in dwarfism at multiple growth points, standard stem lengths, and fewer growth points during the entire growth period, respectively (Fig. 1). The average stem lengths of cultivars 812 and W1-1 were 53.3 cm and 171.10 cm in 2018, and the stem lengths were 56.6 cm and 173.90 cm in 2019, respectively. Overall, the stem length did not show any type of changes in the F2-derived individuals; the maximum stem length was 186.3 cm and the minimum stem length was 40.7 cm.

The results of the analysis of the genetic segregation ratio of the population of six generations are shown in Table 2. In 2018, the expressed the ratio of standard (n = 149) to dwarf (n = 47) types of 196 F2-derived progenies corresponded to a 3:1 Mendelian segregation ratio (χ2 = 0.1429; P > 0.05). In 2019, a total of 1096 standard-type and 343 dwarf-type F2 plants were perfectly segregated among 1439 F2-derived individuals, which also supported the previously observed phenotypic segregation ratio of 3:1 (χ2 = 1.0718; P > 0.05). The plants of both F1 and BC1P1 populations behaved as the standard type; however, the plants of the BC1P2 population exhibited a 1:1 segregation ratio of the standard type (n = 73) and dwarf type (n = 77). Overall, the observed segregation ratios of the standard-type and dwarf-type phenotypes in all derived generations indicated that the internode length is primarily controlled by a single recessive gene, Cldw; however, the standard-type internode length was dominant over that of the dwarf-type, and the genetic inheritance of the observed individuals was unaffected in the environment during 2018 and 2019.

Table 2.

Verification of P1, P2, F1, F2, BC1P1, and BC1P2 segregation ratios of the standard-type and dwarf-type watermelon individuals.

Table 2.

Identification of the CLDW gene using BSA and primary mapping.

The results of whole-genome sequencing and BSA of two constructed DNA bulks (30 dwarf types and 30 standard types) revealed the primary genetic region of the Cldw gene on chromosomes 9 with a fluctuating peak (Fig. 2, Supplemental Table S2). However, the main genetic region of Cldw on chromosome 9 ranged from 1.20 to 3.57 Mb (Fig. 3). A total of 10 KASP markers were developed in the candidate gene region, which was detected by the BSA analysis; primary linkage mapping was performed according to the observed phenotypic data of 184 F2-derived individuals (137 standard types and 47 dwarf types). The main genetic locus of the dwarfism controlling gene was spotted between the adjacent KASP markers K1692577 and K1906994 within a 4.8-cM interval (Fig. 4), and the physical distance was 214.4 kb (range, 1,692,577–1,906,994 bp).

Fig. 2.
Fig. 2.

Delta single-nucleotide polymorphism (SNP) index of the whole-genome chromosomes to identify the compact growth habit gene locus in watermelon. The scattered points indicate the delta SNP index calculated for each specific SNP position. The x-axis represents the position of the watermelon chromosomes; the y-axis represents the SNP index. The curved red lines on the x-axis represent an average of each chromosome and indicate the SNP index. The green line is the threshold level determined using locally estimated scatterplot smoothing (LOESS) regression. The green circle on chromosome 9 represents the significant fluctuation peak detected in the main genetic region.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 2021; 10.21273/JASHS04934-20

Fig. 3.
Fig. 3.

Delta single-nucleotide polymorphism (SNP) index plots across chromosome 9 to identify the compact growth habit gene locus in watermelon. The x-axis represents the position of watermelon chromosome 9; the y-axis represents the SNP index. The curved red lines on the x-axis represent the average of each chromosome and indicate the SNP index. The candidate gene region is indicated by a green circle and ranges from 1.20 to 3.57 Mb. The blue line is the threshold level (0.50) determined by locally estimated scatterplot smoothing (LOESS) regression (0.62) on chromosome 9.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 2021; 10.21273/JASHS04934-20

Fig. 4.
Fig. 4.

Primary mapping of compact growth habit gene Cldw in watermelon using (A) kompetitive allele-specific polymerase chain reaction (KASP) and (B) cleaved amplified polymorphism sequence (CAPS) marker genotyping. (A) Linkage map of 10 KASP markers in the candidate region. The entire map distance was 39.10 cM, and the average distance was 3.91 cM. The Cldw gene was preliminarily mapped between markers K1692577 and K1906994 on chromosome 9 using a total of 184 F2 individuals (137 standard type and 47 dwarf type). The KASP markers are on the right side and the genetic interval for each marker is on the left side. (B) The Cldw gene was further mapped at marker C1763379 between two markers C1652670 and C1906994 on chromosome 9 using a total of 230 F2 dwarf-type individuals. Marker genotypes of the recombinants near the dwarf gene Cldw were between markers C1652670 and C1906994. The alleles are abbreviated according to their origin: B represents the dwarf-type, H represents the dwarf-type recombinants, and X represents the missing date. The blue column shows F2 individuals. The blue row shows CAPS markers.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 2021; 10.21273/JASHS04934-20

In 2019, additional primary mapping was performed by further developing seven CAPS markers at the chromosome 9 region to screen the 230 F2 individuals with the dwarf phenotype. Furthermore, the Cldw gene was delimited between two CAPS markers, C1652670 and C1906994, from three and five recombinants (H), respectively (Fig. 4); however, a single marker, C1763379, seemed perfectly co-segregated by exhibiting the dwarf-type (B) trait. The CAPS markers showed more recombinants, but there were no recombinants in marker C1763379. The results of primary mapping were similar for watermelon grown during the experiments performed in 2018 and 2019, which indicated the perfect mapping interval.

Fine mapping.

Initially, a large group of 390 F2 individuals (all dwarf-types) was used for screening and verification of two CAPS markers, C1652670 and C1906994, and a total of 43 recombinants were mainly filtered from primary mapping analysis. Then, 5 KASP markers, K1692577, K1764972, K1822206, K1865395, and K1906994, were further developed and used for fine mapping using a genotypic analysis of 43 recessive recombinants (Wang et al., 2019). Finally, the Cldw gene was mapped between two KASP markers, K1822206 and K1865395 (Fig. 5), at the delimited region of 43.2 kb.

Fig. 5.
Fig. 5.

Fine mapping of the Cldw gene controlling the compact and dwarf growth habit in watermelon through recessive recombinants. The gene Cldw was delimited in the 43.2 kb region of chromosome 9 between kompetitive allele-specific polymerase chain reaction (KASP) markers K1822206 and K1665395. The white shows bar W1-1 (P1), the black bar shows 812 (P2), the gray bar shows F1, and 2D006, 2D039, 2D216, Z9D102, 2D097, 2D161, 2D210, 3D090, Z9D003, Z9D018, Z9D112, 3D093, and Z9D035 are dwarf-type recessive recombinants.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 2021; 10.21273/JASHS04934-20

According to the annotated online version of the reference genome of watermelon, only two homologous genes, Cla015407 and Cla015408, were found in fine-mapped regions between 1,822,206 and 1,865,395 bp, and these two genes were predicted to encode GA 3β-hydroxylase protein, which mainly triggers the catalytic activity of conversion of nonbioactive GA into bioactive GA (Teng et al., 2013). Bioactive GA promotes elongation in the vine length, but the dwarfism is caused by the absence of bioactive GA. Many studies reported that a gene mutation of GA3 hydroxylase causes the plant compact and dwarf growth habit (Itoh et al., 2001). In this study, various exogenous GA3 treatments were applied to identify the relationship of dwarfism and GA in watermelon cultivar 812. The endogenous GA3 quantification (nanograms per gram) indicated that GA3 deficiency is the only factor that causes dwarfism in cultivar 812.

Furthermore, a KASP marker was successfully developed to identify the candidate gene for controlling dwarfism by using the detected SNP mutations in gene Cla015408. Then, this KASP marker, KCla015408, was genotyped in 43 recombinants, but genetically recombinant individuals were found. Therefore, the gene Cla015408 was ignored and the gene Cla015407 was specified as a candidate gene for controlling dwarfism in cultivar 812.

Expression and cloning of dwarfism gene CLA015407 and GA3 measurement.

To further explore whether the Cla015407 gene sequence and structure are mutated, we amplified the full-length gene sequence (including the intron sequence) in two cultivars, W1-1 and 812. According to the comparative analysis of the Cla015407 gene sequence, watermelon cultivar sequences, and the published watermelon reference genome (97103), the structure of the Cla015407 gene contains two exons and one intron. The coding sequence (including intron sequence) of the Cla015407 gene showed two single base mutations and a single base insertion in cultivar 812; however, this coding sequence had a single base mutation that occurred at the splice site of the intron in cultivar 812. The three SNP mutation sites were located in the intron region of the Cla015407 gene (Fig. 6C).

Fig. 6.
Fig. 6.

Validation of the Cldw gene that controls the compact and dwarf growth habit of watermelon. (A) Measurement of the endogenous gibberellic acid (GA3) content (nanograms per gram) in the eighth internodes during the reproductive growth period of two cultivars, W1-1 (P1; standard type) and 812 (P2; dwarf-type). Error bar indicates the sd of three repeats (n = 3). Values are means ± sd (n = 3). (B) The expression levels of the Cla015407 gene in the eighth internodes during the reproductive growth period of two cultivars, P1 and P2. The expression level of the Cla015407 gene in P2 was set to a value of 1 and used as a reference. Error bar indicates the sd of three repeats (n = 3). Values are means ± sd (n = 3). (C) Comparative analysis of genomic variations of the Cla010407 gene between cultivars and the reference watermelon genome (97103). The large gray boxes indicate the exon and the small gray boxes indicate the intron. The red letters indicate mutations and insertion of bases. Two single-base mutations and one single-base insertion in the intron were detected for P2. One of the single-base mutations occurred at the splice site of the intron.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 2021; 10.21273/JASHS04934-20

In general, single-base mutations and insertions or deletions in introns did not affect gene expression. However, regarding the three mutations detected, one site in the Cla015407 gene was changed from G to A (G→A) in cultivar 812, and the main mutation site was located at the splice site region of the intron of the Cla015407 gene (Fig. 6C). Interestingly, the original splicing site of the Cla015407 gene in cultivar 812 mutated from AG to AA, and the new splicing site AG appeared to alter the original exon length during the transcription process. Therefore, we speculated that this new altered splicing site allowed normal expression of the affected gene. Therefore, the splice site region mutation in the Cla015407 gene in cultivar 812 was mainly caused by gene structure mutation that affected the normal gene expression and exhibited dwarfism in cultivar 812.

If the watermelon plants exhibit the same dwarfism mechanism, then they must be deficient in bioactive GA. For further confirmation of whether this dwarfism is related to bioactive GA, we measured endogenous GA3 synthesis at the eighth internode of cultivars W1-1 and 812; the results indicated that more bioactive GA3 was synthesized in cultivar W1-1 than in cultivar 812 (Fig. 6A). The expression level of the Cla015407 candidate gene in both cultivars was also analyzed by qRT-PCR; the results revealed a significantly higher expression level in W1-1 than in 812 (Fig. 6B). However, the expression level of the Cla015407 gene and GA3 contents indicated improved synthesis of bioactive GA3 in cultivar W1-1 and that the gene expression level was also positively correlated with the endogenous GA3 contents.

Exogenous GA3 treatment.

If the Cla015407 gene controls dwarfism, then dwarfism should be induced by gene mutation and endogenous bioactivity of GA3. Therefore, we hypothesized that exogenous treatment of GA3 can exhibit phenotypic differences in both cultivars. Three distinct GA3 treatments (0.3, 0.9, and 1.5 mmol⋅L−1) were exogenously applied on plants of both cultivars. Cultivar 812 showed a significant response to increased GA3 exogenous treatment. No significant difference in the vine length of both cultivars was induced by the maximum GA3 concentration (1.5 mmol⋅L−1). The treatment was continued until the plants reached the reproductive growth stage. Cultivars W1-1 and 812 exhibited no significant differences in vine length in response to the GA3 treatment (1.5 mmol⋅L−1); however, the number of side branches was significantly decreased in response to the maximum GA3 concentration (Fig. 7). These results strongly indicated that the vine length differences of cultivars W1-1 and 812 occurred because of the differences in the GA3 concentration.

Fig. 7.
Fig. 7.

The differences in plant morphological characteristics (height and internodal length) of watermelon cultivars W1-1 (P1; standard type) and 812 (P2; dwarf type) after various concentrations of gibberellic acid (GA3) treatment. (A–D) Control check (CK) treatment and various exogenous GA3 treatments for P2. (E–H) CK treatment and various exogenous GA3 treatments for P1. Control plants were treated with a simple spray of an equal volume of a mixture of ethanol and double-distilled water. Photographs were obtained when the plants reached the reproductive growth period.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 2021; 10.21273/JASHS04934-20

Discussion

The compact and dwarf growth habit is one of the most important phenotypes of watermelon plants, and dwarf-type plants are suitable for high-density cultivation because of their compact nature (Dong et al., 2018). From the farmer’s point of view, dwarf plants use more natural light, cover less land area, save labor costs, need no pruning, have reduced production costs, and result in increased profits, among other benefits. Therefore, the selection of excellent cultivars of dwarf plants should be considered for the important selection standard of production of watermelon or any other crop. Cultivation of long-vine watermelon cultivars is performed worldwide, and dwarf watermelon cultivars are cultivated less frequently because of improper availability or poor knowledge of farmers. Hence, further molecular studies should be performed to develop and use the dwarf watermelon cultivars for further commercial cultivation.

During previous studies, allelic genes dw-1 and dw-1s and independent loci dw-2, dw-3, and dw-4 controlling the compact and dwarf growth habit have been identified (Huang et al., 1998; Loy and Liu 1972; Yang et al., 2009). GA 3β-hydroxylase has been reported to catalyze the absolute activity in the GA biosynthesis pathway (Yamaguchi, 2008), and it is reason why the compact and dwarf growth nature experiences rarely synthesis or no synthesis of bioactive GA (Itoh et al., 2001; Teng et al., 2013). The effectiveness of GA 3β-hydroxylase has been reported for maize and rice (Chen et al., 2014; Itoh et al., 2001; Teng et al., 2013).

During this study, a fine mapping analysis indicated that the Cldw region is located on chromosome 9, and that two main genes Cla015407 and Cla015408 are directly involved in the synthesis of bioactive GA. Two experiments were conducted to verify whether the dwarfism of cultivars W1-1 and 812 is related to bioactive GA. Initially, exogenous GA3 treatment at a maximum concentration of 1.5 mmol⋅L−1 at the plant reproductive growth stage demonstrated that the vine length of cultivar 812 retained the wild-type phenotype. This result also indicated that the dwarf-type cultivar 812 has hormonal mutations because of gene dysfunction in the GA synthesis pathway. Subsequently, the endogenous GA3 content of cultivar 812 was significantly decreased at the eighth internode compared with that of W1-1, which strongly indicated that GA3 was not synthesized properly in 812. These two conclusions also indicated that plant dwarfism is caused by a loss of function or abnormal function of homologous genes Cla015407 and Cla015408.

We developed a KASP marker based on the SNP positions of the Cla015408 gene to precisely identify these two homologous genes as targeted genes of dwarfism. The Cla015408 gene was not effective because of the co-segregation of genetic recombination in 43 recombinants. Therefore, the Cla015408 gene was excluded and the Cla015407 gene was considered the final candidate gene. Then, gene expression analyses were conducted using qRT-PCR for cultivars W1-1 and 812, and the results demonstrated that the expression of the Cla015407 gene was significantly greater in cultivar W1-1 than in 812, and strongly indicated that the quantity of bioactive GA synthesis was greater in cultivar W1-1.

During this study, the GA 3β-hydroxylase gene was found in the fine-mapped region of 43.2 kb. The comparative analysis of the Cla015407 gene coding sequence, sequencing data of both cultivars W1-1 and 812, and the reference genome of watermelon (97103) exhibited a total of three SNP mutation sites in the intron region and a splice site mutation was found in the Cla015407 gene sequence. According to the GT→AG rule (Xu et al., 1995), the two nucleotides starting at the 5′ end of any intron were GT, but the two nucleotides present at the 3′ end were AG. Interestingly, the original splice site region of the Cla015407 gene in cultivar 812 mutated from AG to AA and appeared as a new AG splicing site. In summary, all evidence presented herein strongly suggest that the GA 3β-hydroxylase homolog of Cla015407 might be a causal gene that controls dwarfism in watermelon cultivar 812.

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Supplemental Table 1.

Developed kompetitive allele-specific PCR (KASP) markers used for primary mapping and fine mapping of Cldw gene, respectively.

Supplemental Table 1.
Supplemental Table 2.

Bulked segregant analysis (BSA) based delta (Δ) single nucleotide polymorphism (SNP)_index (1.2-3.6 Mb) of chromosome 9; Standard type bulk is abbreviated by S-bulk and Dwarf type bulk is abbreviated by D-bulk.

Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.Supplemental Table 2.

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

We sincerely acknowledge the editors and reviewers for their valuable comments that have helped to improve this manuscript.

This work was supported by the National Nature Science Foundation of China (no. 31672177, and “Academic Backbone” Project of Northeast Agricultural University (no. 16XG06).

P.G. is the corresponding author. E-mail: gaopeng_neau@163.com.

  • View in gallery

    Differences in the plant morphological characteristics of two distinct watermelon cultivars: (A) 35 d after sowing W1-1 (P1, standard type) grown in the green house and (B) 35 d after sowing 812 (P2, dwarf type) grown in a greenhouse.

  • View in gallery

    Delta single-nucleotide polymorphism (SNP) index of the whole-genome chromosomes to identify the compact growth habit gene locus in watermelon. The scattered points indicate the delta SNP index calculated for each specific SNP position. The x-axis represents the position of the watermelon chromosomes; the y-axis represents the SNP index. The curved red lines on the x-axis represent an average of each chromosome and indicate the SNP index. The green line is the threshold level determined using locally estimated scatterplot smoothing (LOESS) regression. The green circle on chromosome 9 represents the significant fluctuation peak detected in the main genetic region.

  • View in gallery

    Delta single-nucleotide polymorphism (SNP) index plots across chromosome 9 to identify the compact growth habit gene locus in watermelon. The x-axis represents the position of watermelon chromosome 9; the y-axis represents the SNP index. The curved red lines on the x-axis represent the average of each chromosome and indicate the SNP index. The candidate gene region is indicated by a green circle and ranges from 1.20 to 3.57 Mb. The blue line is the threshold level (0.50) determined by locally estimated scatterplot smoothing (LOESS) regression (0.62) on chromosome 9.

  • View in gallery

    Primary mapping of compact growth habit gene Cldw in watermelon using (A) kompetitive allele-specific polymerase chain reaction (KASP) and (B) cleaved amplified polymorphism sequence (CAPS) marker genotyping. (A) Linkage map of 10 KASP markers in the candidate region. The entire map distance was 39.10 cM, and the average distance was 3.91 cM. The Cldw gene was preliminarily mapped between markers K1692577 and K1906994 on chromosome 9 using a total of 184 F2 individuals (137 standard type and 47 dwarf type). The KASP markers are on the right side and the genetic interval for each marker is on the left side. (B) The Cldw gene was further mapped at marker C1763379 between two markers C1652670 and C1906994 on chromosome 9 using a total of 230 F2 dwarf-type individuals. Marker genotypes of the recombinants near the dwarf gene Cldw were between markers C1652670 and C1906994. The alleles are abbreviated according to their origin: B represents the dwarf-type, H represents the dwarf-type recombinants, and X represents the missing date. The blue column shows F2 individuals. The blue row shows CAPS markers.

  • View in gallery

    Fine mapping of the Cldw gene controlling the compact and dwarf growth habit in watermelon through recessive recombinants. The gene Cldw was delimited in the 43.2 kb region of chromosome 9 between kompetitive allele-specific polymerase chain reaction (KASP) markers K1822206 and K1665395. The white shows bar W1-1 (P1), the black bar shows 812 (P2), the gray bar shows F1, and 2D006, 2D039, 2D216, Z9D102, 2D097, 2D161, 2D210, 3D090, Z9D003, Z9D018, Z9D112, 3D093, and Z9D035 are dwarf-type recessive recombinants.

  • View in gallery

    Validation of the Cldw gene that controls the compact and dwarf growth habit of watermelon. (A) Measurement of the endogenous gibberellic acid (GA3) content (nanograms per gram) in the eighth internodes during the reproductive growth period of two cultivars, W1-1 (P1; standard type) and 812 (P2; dwarf-type). Error bar indicates the sd of three repeats (n = 3). Values are means ± sd (n = 3). (B) The expression levels of the Cla015407 gene in the eighth internodes during the reproductive growth period of two cultivars, P1 and P2. The expression level of the Cla015407 gene in P2 was set to a value of 1 and used as a reference. Error bar indicates the sd of three repeats (n = 3). Values are means ± sd (n = 3). (C) Comparative analysis of genomic variations of the Cla010407 gene between cultivars and the reference watermelon genome (97103). The large gray boxes indicate the exon and the small gray boxes indicate the intron. The red letters indicate mutations and insertion of bases. Two single-base mutations and one single-base insertion in the intron were detected for P2. One of the single-base mutations occurred at the splice site of the intron.

  • View in gallery

    The differences in plant morphological characteristics (height and internodal length) of watermelon cultivars W1-1 (P1; standard type) and 812 (P2; dwarf type) after various concentrations of gibberellic acid (GA3) treatment. (A–D) Control check (CK) treatment and various exogenous GA3 treatments for P2. (E–H) CK treatment and various exogenous GA3 treatments for P1. Control plants were treated with a simple spray of an equal volume of a mixture of ethanol and double-distilled water. Photographs were obtained when the plants reached the reproductive growth period.

  • Allen, G.C., Flores-Vergara, M.A., Krasynanski, S., Kumar, S. & Thompson, W.F. 2006 A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide Nat. Protoc. 1 2320 2325 doi: 10.1038/nprot.2006.384

    • Search Google Scholar
    • Export Citation
  • Amanullah, S., Saroj, A., Osae, B.A., Liu, S., Liu, H.Y., Gao, P. & Luan, F.S. 2020 Detection of putative QTL regions associated with ovary traits in melon using SNP-CAPS markers Scientia Hort. 270 109445 doi: 10.1016/j.scienta.2020.109445

    • Search Google Scholar
    • Export Citation
  • Amanullah, S., Liu, S., Gao, P., Zhu, Z.C., Zhu, Q.L., Fan, C. & Luan, F.S. 2018 QTL mapping for melon (Cucumis melo L.) fruit traits by assembling and utilization of novel SNPs based CAPs markers Scientia Hort. 236 18 29 doi: 10.1016/j.scienta.2018.02.041

    • Search Google Scholar
    • Export Citation
  • Amasino, R.M., Schomburg, F.M., Michaels, S.D. & Bizzell, C.M. 2003 Dwarfism genes and dwarf plants Wisconsin Alumni Res. Foundation 1 2

  • Chen, Y., Hou, M.M., Liu, L.J., Wu, S., Shen, Y., Ishiyama, K., Kobayashi, M., McCarty, D.R. & Tian, B.C. 2014 The maize dwarf1 encodes a gibberellin 3-oxidase and is dual localized to the nucleus and cytosol Plant Physiol. 166 2028 2039 doi: 10.1104/pp.114.247486

    • Search Google Scholar
    • Export Citation
  • Chen, M.L., Huang, Y.Q., Liu, J.Q., Yuan, B.F. & Feng, Y.Q. 2011 Highly sensitive profiling assay of acidic plant hormones using a novel massprobe by capillary electrophoresis-time of flight-mass spectrometry J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 879 938 944 doi: 10.1016/j.jchromb.2011.03.003

    • Search Google Scholar
    • Export Citation
  • Chiang, H.H., Hwang, I. & Goodman, H.M. 1995 Isolation of the Arabidopsis GA4 locus Plant Cell 7 195 201 doi: 10.2307/3869995

  • Dong, W., Wu, D., Li, G., Wu, D. & Wang, Z. 2018 Next-generation sequencing from bulked segregant analysis identifies a compact growth habit gene in watermelon Sci. Rep. 8 2908 doi: 10.1038/s41598-018-21293-1

    • Search Google Scholar
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