Cgl2 plays an essential role in cuticular wax biosynthesis in cabbage (Brassica oleracea L. var. capitata)

The aerial parts of most land plants are covered with cuticular wax which is important for plants to avoid harmful factors. There is still no cloning study about wax synthesis gene of the alcohol-forming pathway in Brassica species. Scanning electron microscopy (SEM) showed that, compared with wild type (WT), wax crystal are severely reduced in both the adaxial and abaxial sides of cabbage (Brassica oleracea L. var. capitata L.) leaves from the LD10GL mutant. Genetic analysis results revealed that the glossy trait of LD10GL is controlled by a single recessive gene, and fine mapping results revealed that the target gene Cgl2 (Cabbage glossy 2) is located within a physical region of 170 kb on chromosome 1. Based on sequence analysis of the genes in the mapped region, the gene designated Bol013612 was speculated to be the candidate gene. Gene Bol013612 is homologous to Arabidopsis CER4, which encodes fatty acyl-coenzyme A reductase. Sequencing identified a single nucleotide substitution at an intron/exon boundary that results in an insertion of six nucleotides in the cDNA of Bol013612 in LD10GL. The phenotypic defect of LD10GL was confirmed by a functional complementation test with Arabidopsis mutant cer4. Our results indicated that wax crystals of cabbage mutant LD10GL are severely reduced and mutation of gene Bol013612 causes a glossy phenotype in the LD10GL mutant.

In Arabidopsis, wax biosynthesis begins with the elongation cycle, which converts fatty acids (C16-C18) into VLCFAs by transferring a C2 unit from malonyl-CoA to long-chain acyl-CoA [13][14][15][16][17]. The generated VLCFAs are subsequently modified by the alcoholforming pathway (also known as the acyl reduction pathway) or the alkane-forming pathway (also known as the decarbonylation pathway) to produce the main components of cuticular wax, including primary alcohols, esters, secondary alcohols, aldehydes, alkanes, and ketones [18,19]. In Arabidopsis, primary alcohols and wax esters, the main products of the alcohol-forming pathway, account for 10-15% of the total wax on the stems and 15-25% on the leaves, while the alkane-forming pathway produces the majority of wax compounds on the stems (84%) and on the leaves (60%) [20].
Some genes involved in wax biosynthesis of Brassica species have been reported in recent studies. For example, gene BrWax1 in Brassica rapa was mapped to an 86.4-kb physical interval on linkage group A1, and gene Bra013809, which is orthologous to the CER2 gene in Arabidopsis, was predicted to be the candidate gene [32]. In Brassica napus, the BnaA.GL gene was finemapped using cDNA microarray chip analysis [33]. In cabbage, Cgl1 was fine-mapped, and a candidate gene orthologous to CER1 was identified [34]. Although several genes involved in wax biosynthesis have been identified in Brassica species, few genes of the alcoholforming pathway have been cloned. In the present study, with help of wax-deficient cabbage mutant LD10GL, a high-resolution genetic map was constructed. The Bol013612 gene was predicted to be the candidate gene associated with the glossy phenotype, and the prediction was proved by gene sequencing and functional analysis results. Based on the homology analysis with Arabidopsis CER4 which encodes fatty acyl-coenzyme A reductase, Bol013612 was speculated to be involved in the alcoholforming pathway in cabbage. These results will help to illustrate the alcohol-forming pathway and accelerate research concerning wax metabolism in Brassica species.

Results
Wax is significantly reduced in LD10GL WT (LD10) material is a high-generation cabbage (B. oleracea L. var. capitata) inbred line with glaucous appearance, and it is the near-isogenic line (NIL) of LD10GL. Surfaces of all the aerial organs of WT, such as the leaves (Fig. 1a), seedpods (Fig. 1c), stems, and flower buds (Fig. 1e), were glaucous with cuticular wax. In contrast, appearance of the corresponding organs tended to be glossy green in LD10GL (Fig. 1b, d and f).
To study microscopic character of wax crystals, SEM was used to observe the adaxial and abaxial sides of the leaves. Result showed that both leaf sides of WT are covered with compact wax crystals and the crystals are rod shaped ( Fig. 2a and c). In contrast, the amount of wax crystals in both the adaxial and abaxial sides of LD10GL a b c d e f was markedly reduced, and the wax crystals tended to be rodlet shaped ( Fig. 2b and d).
To dissect chemical components changes in LD10GL, wax composition of WT and LD10GL was analyzed by GC-MS. As shown in Fig. 3 and Additional file 1, similar content of alkanes, ketones, fatty acids, aldehydes, and secondary alcohols was observed by WT and LD10GL. But significant reduction of primary alcohols and wax esters was detected in LD10GL compared with WT. For example, the primary alcohol and wax ester content is about sixtyfold and fourfold in WT of LD10GL.
The glossy green character of the LD10GL mutant is controlled by a single recessive gene To investigate the inheritance of the glossy green trait in LD10GL, seeds of the parental materials, LD10GL(B. oleracea L. var. capitata)and M36 (B. oleracea var. alboglabra), and the derived F1, F2, BC1 and BC2 populations were sown. The glossy green individuals were identified visually. Results showed that surface of leaves and stems of all F 1 plants is glaucous, suggesting that the glossy green trait is recessive to the glaucous trait. In the F 2 population, 2130 of 2834 individuals showed a glaucous appearance, and 704 individuals showed a glossy green phenotype. The segregation ratio was confirmed to be 3:1 (χ2 = 0.24 < χ2 0.05 = 3.84, P > 0.05) by the Chi-square test. In BC 1 population, which was obtained through backcross of F 1 with LD10GL plants, the numbers of glaucous plants and glossy plants were 202 and 192, respectively, displaying a ratio of 1:1 (χ2 = 0.03 < χ2 0.05 = 3.84, P > 0.05). Furthermore, surface of all BC 2 plants, which were achieved through backcrossing the F 1 plant with M36, was glaucous ( Table 1). All results above indicate that the glossy green trait of LD10GL is controlled by a single recessive gene. Two DNA pools were constructed by mixing equal amounts of DNA from 12 glossy green and 12 glaucous F 2 individuals. To obtain the markers linked to gene Cgl2, 1064 pairs of primers were screened with the parents and DNA pools successively. As a result, only primer BoID000046 with polymorphism both between the parents and the two DNA pools was obtained. To further verify the linkage between BoID000046 and Cgl2, gDNA of 2834 F 2 individuals was amplified as a template with primer BoID000046. As expected, genetic distance between primer BoID000046 and Cgl2 was confirmed to be 4.7 cM (Fig. 4a). To acquire more markers linked to the Cgl2 gene, another 200 pairs of primers on the same chromosome (chromosome C01) were analyzed with F 2 individuals. Consequently, four more pairs of markers linked to Cgl2 were obtained, and Cgl2 was mapped to a genetic interval of 6.2 cM with markers BoID000046 and BoID000065 (Fig. 4a).

Marker development and fine mapping of Cgl2
To narrow the mapped region, 50 pairs of new primers within the mapped region were developed according to B. oleracea genome database. Screened with the parental lines, 11 pairs of polymorphic primers were identified. These markers were used to screen 704 F 2 individuals with the glossy phenotype for the recombinant plants.
Based on the information of the polymorphic markers and the recombinants, a fine linkage map comprising 13 DNA markers was constructed (Fig. 4b). Gene Cgl2 was delimited to a region between markers C01SSR147 and C01SSR150 with genetic distances of 0.2 cM and 0.1 cM, respectively, covering a physical distance of 170 kb (2503307-2,752,299) (Figs. 4b and 5a and b).

Identification of the candidate gene for Cgl2
To identify the candidate gene, BLAST searches against the NCBI conserved domain database with genes in the mapped region were conducted. Result revealed that the gene Bol013612 (C01:2,609,971:2,615,418), which contains the fatty acyl-coenzyme A reductase (FAR) domain, may be a candidate gene. The FAR enzyme usually acts on medium-and long-chain fatty acids and has been reported to be involved in the biosynthesis of plant primary alcohols and cuticular wax [20].
To examine transcriptional level of the candidate gene, expression of Bol013612 was tested using qPCR. Results showed that the expression level in LD10GL is lower than that in WT, which may be induced by mutation and abnormal function of gene Bol013612 in LD10GL (Fig. 6). The cDNA, gDNA, and nucleotide sequence approximately 2000 bp upstream of the gDNA of Bol013612 were sequenced. Results showed that, compared with the WT, there exists a nucleotide substitution from adenine to guanine at nucleotide position 622 in the gDNA and an insert of six nucleotides in the cDNA of gene Bol013612 ( Fig. 7a and b; Additional file 2). According to the GT-AG rule, which implies that an intron should start with a 'GT' at the 5′ side and end with an ' AG' at the 3′ side [35], the mutational pattern in LD10GL was illustrated as follows: a single nucleotide substitution at position 622 in the gDNA brings a new terminal ' AG' to the first intron and thus generated an alternative 3′ splice site in intron 1. Because of the earlier ending of the first intron, the six additional nucleotides were included within the 5′ portion of exon 2 ( Fig. 7a and b). As a result, six nucleotides are inserted into the cDNA sequence at nucleotide position 92 (Fig. 7b). Because of the insertion of the six nucleotides, one amino acid is modified and another two additional amino acids are introduced (Fig. 7c), which is just within the FAR domain of this protein (Additional file 3). The resulting change of amino acid sequence is considered Several independent transgenic lines were obtained from each process, and the Bol013612 gene was ectopically expressed in Arabidopsis in the cgl2-LD10GL and Cgl2-LD10 plants (Fig. 6). As a result, the phenotypic defect of Arabidopsis mutant cer4 was restored in Cgl2-LD10 (Fig. 8d), but the glossy appearance was still exhibited in cgl2-LD10GL (Fig. 8b) and cer4-3301 (Fig. 8c) plants; the wax load and morphology were restored to wildtypelevels in transgenic plants expressing Cgl2, but not cgl2-LD10GL (Fig. 9). The functional complementation test result and high sequence homology between gene Bol013612 and known FAR-coding gene implicates protein Bol013612 is a wax biosynthesis enzyme. Collectively, these results illustrate that it is the mutation of Bol013612 that influences wax biosynthesis and causes the glossy green trait in LD10GL.

Discussion
Appearance of many plants is glaucous for the covering of cuticular wax, which constitutes the outermost barrier of plants. Compared with glaucous wild-type plants, most land plants with metabolic defects in wax biosynthesis are readily detectable with naked eye. With T-DNA insertion, many Arabidopsis cer (eceriferum) mutants were isolated and the corresponding genes were identified. Same as other Arabidopsis cer mutants, cer4 has a glossy green appearance. But different from some sterile cer mutants, such as cer1, cer3, cer6, and cer10, cer4 mutant is fertile [36].In contrast to the glaucous appearance in WT, surface of the leaves, seedpods, flower buds and stems is glossy green in cabbage LD10GL mutant. Changes of wax crystal density and shape were observed through SEM, indicating that it is the reduction of wax leads to glossiness in LD10GL. Similar as cer4 mutant, cabbage mutant LD10GL is fertile, different from another cabbage glossy green mutant 10Q-961, in which few seeds were produced after self-pollination [34].

kb, and
Bra013809 was predicted to be the candidate gene for BrWax1 [32]. The BnaA.GL gene in Brassica napus was proved to be located at the end of the A9 chromosome [33]. In cabbage, the BoGL1 gene was delimited to the end of chromosome C08 by flanking marker SSRC08-76 with genetic distance of 0.2 cM and Bol018504 was selected as the candidate gene [34]. Although several wax biosynthesis-related genes have been identified in Brassica species, little research about the alcoholforming pathway and the related genes were reported.
In Arabidopsis, FAR catalyzes the two-step reduction of VLCFAs to produce primary alcohols. The primary alcohols together with C16:0 acyl-CoAs combine to produce alkyl esters. Primary alcohols and alkyl esters are two of the main cuticular wax components [20,37]. CER4 is one of the key genes encoding FAR in Arabidopsis. For reduction of primary alcohols and wax esters, glossy wax-deficient trait is displayed in cer4 [20]. High level of protein sequence homology is exhibited between cabbage gene Bol013612 and CER4. Similar as cer4, primary alcohol and wax esters are the main reduced wax compositions in mutant LG10GL.
As helpful material for metabolic mechanism research, the cabbage mutant LD10GL was applied to the wax biosynthesis study. Using a standard molecular genetic mapping strategy, the Cgl2 gene was delimited to the interval between markers C01SSR147 and C01SSR150. Based on sequence analysis results, gene Bol013612 was speculated to be the candidate gene. Concerning sequence homology and similar reduced wax composition in LD10GL and cer4, gene Bol013612 was speculated to encode FAR. Linkage relationship of the glossy character in LD10GL and Bol013612 was further confirmed by a functional complementation test. All results provided evidence that Bol013612 may be the target gene for Cgl2.
All of these results will help us to further illustrate the formation of the glossy trait in LD10GL and wax biosynthesis in cabbage. More research needs to be carried out in future work, such as the metabolic mechanism of cabbage wax biosynthesis and identification of other genes involved.

Conclusions
We fine-mapped a wax synthesis gene using the F 2 populations, analyzed the genes present in the candidate region, and identified Bol013612, which is homologous to CER4, as a candidate gene for Cgl2. A 6-bp insertion resulted in the abnormal function of the FAR domain in the LD10GL glossy mutant line. The identification of Cgl2 gene will enhance our understanding about its role in cabbage wax biosynthesis and lays a foundation for further study of cabbage wax metabolism.

Plant materials and genetic study
Plant materials in this study are all maintained in our lab. F 1 , F 2 , BC 1 , and BC 2 populations were constructed using the female LD10GL parent (P 1 ) and male M36 parent (P 2 ) for inheritance and fine mapping studies. The glossy cabbage mutant LD10GL is a natural glossy mutant of LD10 cabbage. The gene responsible for the glossy green trait in LD10GL is named as Cgl2 for the reason that Cgl2 is the 2nd gene which was fine-mapped and cloned in cabbage (the 1st gene fine-mapped and cloned was named Cgl1). LD10 is a high-generation cabbage (B. oleracea L. var. capitata) inbred line with glaucous appearance and is referred as WT in this study. Same agronomic traits, such as shape of the head and plant type are exhibited by LD10 and LD10GL, except the glossy green appearance. M36 is a Chinese kale (Brassica oleracea var. alboglabra) material with glaucous appearance. F 1 plants were obtained by crossing LD10GL and M36. One F 1 plant was used as the female parent material in the backcrosses with LD10GL and M36, and F 2 plants were obtained by selfing the same F 1 plant. All the plant materials were provided by the Cabbage and Broccoli Research Group, Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS) and grown in the greenhouse in Changping (39°54′N, 116°13′E, Beijing, China) according to local crop management practices. The glossy phenotype was assessed and recorded at the fiveleaf stage. The segregation ratios of the F 2 and BC 1 populations were analyzed with the Chi-square test (χ 2 ).

SEM and GC-MS analysis
Fresh leaves from the five-leaf-stage plants were fixed overnight in 2% glutaraldehyde, mounted on specimen stubs using double-sided tape and then coated with gold particles in a SEMPrep2 sputter coater (Nanotech). The phenotype was analyzed by SEM (S-4800, Hitachi, Japan) with a secondary electron detector at a high voltage (10 kV). GC-MS analysis of cuticular wax was carried out as described as Tang [38].

The mapping strategy
Total genomic DNA from fresh leaves was extracted with the cetyltrimethylammonium bromide (CTAB) protocol, and the concentration of genomic DNA was adjusted to 30 ng/μl [39]. (PCR) and polyacrylamide gel electrophoresis (PAGE) was performed with 20 μl of reaction mixture [40]. Using the bulked segregant analysis (BSA) method, 12 glaucous and 12 glossy individuals from the F 2 population were selected to construct two DNA bulks. One thousand two hundred sixty-four pairs of SSR primers that were designed according to data of the B. oleracea genome database (Brassica Database, BRAD; http://bras sicadb.org/brad/index.php) [41] were screened by the parental materials and two DNA pools successively for mapping analysis. The linkage relationships between the Cgl2 gene and the polymorphic primers were confirmed with F 2 individuals. Linkages between markers and the Cgl2 gene were determined using the software Joinmap 4.0 (Kyasma, NL). To finely map Cgl2, 50 pairs of SSR primers near the preliminary mapping region were developed and used to screen for the recombinants among 704 recessive F 2 individuals. The individuals whose gel bands were the same as those of M36 or the F 1 were used as the recombinants and selected for further analysis. The linkage between the markers and the Cgl2 gene was determined with the Kosambi mapping function, and a genetic map was constructed using the software Mapdraw 2.1 [42,43]. The sequences of all polymorphic markers used in this study are listed in Table 2.
Identification and sequence analysis of the candidate gene Conserved domains of genes between the two closest markers were analyzed using the BLAST program (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [44]. Genes with wax-related conserved domains were selected as the candidate genes. The level of protein sequence homology between Bol013612 and Arabidopsis CER4 was analyzed with software MultAlin (http://mul talin.toulouse.inra.fr/multalin/multalin.html) [45] and DNAMAN software (http://www.lynnon.com/dnaman. html). The expression level of the candidate gene in WT and LD10GL was tested by qPCR. Sequences of the candidate genes were amplified using the gDNA and cDNA as templates with a KAPPA HiFi HotStart ReadyMix PCR Kit (Kappa Biosystems). The PCR products were sequenced by BGI TechSolutions Co., Ltd. Sequences were aligned with the software MultAlin (http://multalin.toulouse.inra.fr/multalin/multalin.html)