Fine Mapping and Candidate Gene Identification for Wax Biosynthesis Locus, BoWax1 in Brassica oleracea L. var. capitata

Cuticular waxes play important roles in plant protection against various biotic and abiotic environmental stresses. The cuticular wax covering gives normal cabbage a glaucous appearance, but the appearance of waxless mutant is glossy green. Based on the present study, inheritance of the glossy green character of mutant HUAYOU2 follows a simple recessive pattern. Genetic analysis of an F2 population comprising 808 recessive individuals derived from HUAYOU2 (P1, maternal parent) and M36 (P2, paternal parent) revealed that a single recessive locus, BoWax1 (Brassica oleracea Wax 1), controls glossy green trait in B. oleracea. This locus was mapped to a region of 158.5 kb on chromosome C01. Based on nucleotide sequence analysis, Bol013612 was identified as the candidate gene for BoWax1. Sequencing results demonstrated that there is a deletion mutation of two nucleotides in the cDNA of Bol013612 of HUAYOU2, which may account for its glossy green trait. These results lay the foundation for functional analysis of BoWax1 and may accelerate research on wax metabolism in cabbage.


INTRODUCTION
The aerial epidermis of terrestrial plants is coated with a cuticle that consists of cutin and cuticular wax . Cutin is composed of modified fatty acids and provides mechanical strength to the surface layer (Kerstiens, 1996;Beisson et al., 2012) The cuticular wax, which embeds in and covers the cutin polymer, is composed of very long chain fatty acids (VLCFAs) and their derivatives, including primary and secondary alcohols, alkanes, aldehydes, ketones, and wax esters (Rashotte et al., 1997;Lee and Suh, 2015). Together with cutin, cuticular wax plays an important role in the protection of plants from damage caused by environmental stresses, such as water deficiency (Kerstiens, 1996;Mawlong et al., 2015), insect attacks (Stork, 1980), pathogen infection (Wang et al., 2006), and UV irradiance (Shepherd and Wynne Griffiths, 2006).
Wax composition and content can vary according to species, ontogeny, and environment (Jenks and Ashworth, 1999;Laila et al., 2016). As noted previously, the major components of wax are VLCFAs and their derivatives, including alkanes, aldehydes, primary and secondary alcohols, ketones, and esters (Riederer and Müller, 2006;Bernard and Joubès, 2013). C16 and C18 acyl-CoAs are precursors in the biosynthesis of C20-C34 VLCFA-CoAs, which are formed by the addition of C2 units derived from malonyl-CoA by the enzymes of the fatty acid elongase (FAE) complex in the endoplasmic reticulum (ER) (Kunst and Samuels, 2009;Li-Beisson et al., 2013). The FAE complex consists of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and trans-2,3-enoyl-CoA reductase (ECR), The FAE complex catalyzes four consecutive enzymatic reactions, resulting in a two-carbon extension of the acyl chain during each elongation cycle (Kunst and Samuels, 2009;Li-Beisson et al., 2013). The produced VLCFA-CoAs produced by the FAE complex are modified by two distinct pathways: an alkane-forming pathway and an alcohol-forming pathway. Most of the wax components, including aldehydes, alkanes, secondary alcohols, and ketones, are biosynthesized in the alkane-forming pathway, while the products of the alcohol-forming pathway, including primary alcohols and wax esters, account for only 10-15% of the total wax in Arabidopsis stems (Rowland et al., 2006).
Many genes associated with cuticular wax metabolism and its regulation in Arabidopsis and other plants have been identified with the help of waxless mutants. These genes include the FAE-related enzymes KCS1, KCS2, KCS6, KCS9, KCS20, and KCR1 (Millar and Kunst, 1997;Millar et al., 1999;Todd et al., 1999;Fiebig et al., 2000;Pruitt et al., 2000;Hooker et al., 2002;Beaudoin and Kunst, 2009;Franke et al., 2009;Lee et al., 2009;Kim et al., 2013) and enzymes involved in the modification of VLCFA-CoAs, such as CER1, CER3, MAH1, CER4, and WSD1 (Rowland et al., 2006(Rowland et al., , 2007Greer et al., 2007;Li et al., 2008;Bourdenx et al., 2011), which play various roles in wax biosynthesis. CER4 encodes an alcohol-forming fatty acyl-CoA reductase (FAR) that was reported to be responsible for the synthesis of primary alcohols in the epidermis of aerial tissues and in roots (Rowland et al., 2006). Several genes implicated in wax biosynthesis of Brassica species have been reported in the recent studies. For example, the BrWax1 gene in Brassica rapa was mapped to linkage group A01, and Bra013809 was predicted to be the candidate gene for BrWax1 (Zhang et al., 2013). The BnaA.GL gene in Brassica napus was finely mapped to a linkage group close to the end of chromosome A9 (Pu et al., 2013). In cabbage, the BoGL1 gene was delimited to the end of chromosome C08 by a flanking marker, SSRC08-76, at a genetic distance of 0.2 cM (Liu D.et al., 2017). Although several genes in the alkane-forming pathway in Brassica species were isolated (Zhang et al., 2013;, no study concerning primary alcohol-forming pathway genes has been reported. In the present study, the BoWax1 gene was mapped to chromosome C01, and Bol013612 was identified as the candidate BoWax1 gene. Based on the lower expression level of Bol013612 and the differences in its sequence in the glossy green mutant HUAYOU2, the Bol013612 gene was predicted to participate in wax biosynthesis in the plant through the alcohol-forming pathway. These results provide insight into the elucidation of the alcohol-forming pathway and the molecular mechanism of wax biosynthesis in Brassica species.

Plant Materials
The plant materials in this study were provided by our team named the Cabbage and Broccoli Research Group, Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS) and were grown in the greenhouse in Changping (39 • 54 N,116 • 13 E,Beijing,China).
Five glossy green cabbage mutants have been reported previously (Tang et al., 2015). HUAYOU2 is also a cabbage mutant with a glossy green appearance. The mutant plant HUAYOU2 was initially discovered from the highly inbred line HUA2, which was used as the wild type (WT) line in this research. HUAYOU2 (P 1 , maternal parent) was crossed with M36 (P 2 , paternal parent), an inbred line of Chinese kale (Brassica oleracea var. alboglabra) with a glaucous appearance, to generate the F 1 , F 2 , BC 1 P 1 , and BC 1 P 2 populations for inheritance and mapping studies. The BC 1 P 1 and BC 1 P 2 populations were created by backcrossing the F 1 plant (maternal parent) with HUAYOU2 (paternal parent) and M36 (paternal parent), respectively. The same F 1 plant was used as the female parent in the backcrosses and was also used to obtain the F 2 population.

SEM Analysis
Scanning electron microscopy (SEM) was employed to study the surfaces of the leaves of HUAYOU2 and WT plants. Fresh leaves from five-leaf-stage plants were fixed overnight in 2% glutaraldehyde, mounted on specimen stubs using double-sided tape and coated with gold particles in a SEMPrep2 sputter coater (Nanotech, England, United Kingdom). The phenotype was analyzed by SEM (S-4800, Hitachi, Japan) with a secondary electron detector at high voltage (10 kV).

DNA Isolation and PCR Amplification
Genomic DNA was extracted from fresh leaves using the cetyltrimethylammonium bromide (CTAB) method (Doyle, 1990). The concentration of each sample of genomic DNA was measured spectrophotometrically and adjusted to approximately 30 ng/µl.

Inheritance Analysis and Mapping Strategy
The glossy and glaucous phenotypes in plants were identified visually and recorded at the five-leaf stage when the difference in appearance could be easily distinguished. Segregation ratios of the F 2 and BC 1 P 1 populations were analyzed with the Chi-square (χ 2 ) test. Two DNA pools were constructed by mixing equal amounts of DNA from 12 glossy green and 12 glaucous F 2 individuals. Then, 2000 pairs of simple sequence repeat (SSR) primers in the laboratory were used to successively screen the parental lines and the DNA pools. Primers that demonstrated polymorphism between the parents were subsequently used to screen the two pools. The linkage relationship between the target gene and the polymorphic primers was confirmed in 128 F 2 individuals using JoinMap 4.0 (Van Ooijen, 2006). Linkage between markers and the BoWax1 gene was determined with the Kosambi mapping function, and a genetic map was constructed using MapDraw 2.1 software (Liu and Meng, 2003;Kosambi, 2016). To finely map the mutant gene, 50 pairs of new primers based on primary mapping results were developed. The new primers were used to analyze the parental lines and recombinants of 808 F 2 recessive individuals. The sequences of all polymorphic markers used in this study are listed in Table 1 Sequence and Expression Analysis of the Bo13612 Gene The InterPro annotations for B. oleracea and GO annotations for Arabidopsis thaliana for all the genes located within the mapped region were obtained from BRAD 1 (Cheng et al., 2011). The expression level of the Bo13612 gene in WT and HUAYOU2 plants was determined by qPCR. Total RNA was extracted from fresh leaves at the five-leaf stage using the EasyPure R Plant RNA Kit (TRANS) as described by the manufacturer. First-strand cDNAs were synthesized in a 20-µl reaction volume containing approximately 7 µg of RNA and oligo(dT) primers using the TransScript One-Step gDNA Removal and cDNA Synthesis Kit (TRANS). Real-time PCR was performed with ABI SYBR Green on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. The β-actin gene was used as an internal control (Okazaki et al., 2007).
The 2 − C t method was used to analyze the data (Livak and 1 http://brassicadb.org/brad/index.php Schmittgen, 2001). Three technical replicates were performed in this study. The sequence of the primers for qPCR, qPCR-013612 and qPCR-actin, are listed in Table 1. Each sample was tested in triplicate. The sequences of the Bo13612 gene were amplified using the gDNA and cDNA as templates with the KAPPA HiFi HotStart ReadyMix PCR Kit (Kappa Biosystems). The PCR products were sequenced by BGI TechSolutions, Co., Ltd., and the sequences were aligned using Multalin software 2 (Corpet, 1988). Primers used to amplify the gDNA sequence were designed using Primer Premier 5 software and date from the B. oleracea genome database (BRAD 1 ). The primers were named Bol013612g1, Bol013612g2, Bol013612g3, Bol013612g4, and Bol013612g5 (Table 1) (Lalitha, 2000). The primer used to amplify the cDNA of the Bol013612 sequence was designed based on data from the B. oleracea transcriptome database 3 and was named Bol013612c1 (Table 1) (Liu et al., 2014).

The Morphological Appearance Is Altered in HUAYOU2
Regarding the coverage of cuticular wax, the appearance of the leaves in the WT cabbage was glaucous (Figure 1a). In contrast to WT, the leaves of HUAYOU2 tended to be glossy green, with little wax on the plant surface (Figure 1b). SEM was used to investigate the micro-characteristics and density of the wax crystals on the leaf surface. The results showed that the surface of the WT leaf is covered with compact wax crystals with a scale-and rod-like shape (Figure 1c). Unlike the WT, there are very few wax crystals covering the leaf surface of HUAYOU2 leaves, and these wax crystals tended to have a particle-type shaped (Figure 1d). This indicates that the glossy phenotype of HUAYOU2 is caused by a decrease in cuticular wax crystals, which is consistent with observations of many wax-deficient mutants in other plants (Pu et al., 2013).

Inheritance Analysis of the Glossy Green Trait in HUAYOU2
The appearance of all 100 F 1 plants from the crosses of HUAYOU2 and M36 was glaucous, indicating that the glaucous trait was dominant over the glossy green trait. Among 3328 F 2 individuals, 2520 had glaucous leaves, and 808 had glossy green leaves, corresponding to a segregation ratio of 3:1 by the Chi-square test. The segregation ratio in the BC 1 P 1 population was 1:1 (210 glaucous:222 glossy), and all the BC 1 P 2 plants had a glaucous appearance ( Table 2). These results demonstrated that inheritance of the glossy green trait in HUAYOU2 follows a monogenic recessive pattern.

Fine Genetic and Physical Mapping of the BoWax1 Gene
A total of 2000 SSR markers available in the laboratory were used to identify polymorphisms in the parental lines (HUAYOU2 3 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE42891 and M36) and in the glossy and glaucous DNA bulks. As a result, 114 pairs of primers were identified that showed polymorphism between the parental lines, but only BolD000046 was identified as being polymorphic between two DNA pools. Marker BolD000046 was mapped close to BoWax1 using 128 segregating F 2 population plants (Figure 2A).
To saturate the genetic map containing BoWax1, 50 pairs of primers near primer BolD000046 were designed based on the physical position of marker BolD000046. Eight of the 50 newly developed primers showed polymorphism between parental lines HUAYOU2 and M36. The eight SSRs were subsequently screened to investigate recombination using 808 F 2 recessive individuals. Based on information from the polymorphic markers and the recombinants, the genetic distance between the two closest flanking makers, C01gSSR148 and C01gSSR150 (Figure 2A),   was found to be 0.2 cM. The physical distance between primers C01gSSR148 and C01gSSR150 is 158.5 kb ( Figure 2B). All of the primers are on chromosome C01 and the order of the markers on the genetic map is consistent with that on the physical map.

Candidate Gene Analysis
Analysis of the genomic region between C01gSSR148 and C01gSSR150 was carried out. Gene annotations in BRAD and alignments with A. thaliana showed that gene Bol013612 contains the conserved FAR domain, which plays important roles in wax biosynthesis and is present in proteins that are responsible for primary alcohols synthesis (Rowland et al., 2006). At4g3370 is an example of an Arabidopsis gene that contains the conserved FAR domain and exhibits the same glossy stem wax phenotype in two mutant lines (cer4-1 and cer4-2). A deletion and/or rearrangement of the promoter region occurs in the cer4-1 mutant and a T-DNA insertion is present in the fourth intron of At4g33790 (at nucleotide 1,960 relative to the start codon) in the cer4-2 mutant (Rowland et al., 2006). According to the homologous analysis results, the Bol013612 gene was predicted to participate in the alcohol-forming pathway. Aside from Bol013612, no other genes in the mapped region were found to be related to plant wax metabolism (Gene analysis and annotations are supplied in Additional file 1: Supplementary 2). The Bol013612 gene was selected as the candidate for expression analysis using qPCR. The results showed that the expression level of Bol013612 is significantly decreased compared with that of the WT, implying that transcription of the Bol013612 gene was affected (Figure 3).
The Bol013612 gene was sequenced to further determine whether the glossy green trait of HUAYOU2 is caused by a mutation in Bol013612. The Bol013612 gene, numbered MG808275 in NCBI database, is 5,448 bp in length, including 10 exons and 9 introns ( Figure 4A). The The coding sequence (CDS) of Bol013612 is 1,482 bp in length. The sequencing results showed that there was a deletion of two bases in the eighth exon of HUAYOU2 (sequences of WT and HUAYOU2 are supplied in Additional file 1: Supplementary 1). The deletion mutation was suspected of causing a frameshift mutation in the Bol013612 gene and a premature stop codon (Figures 4B,C). This might disrupt the function of this gene, which may influence the wax biosynthesis and subsequently cause the glossy appearance of HUAYOU2.

DISCUSSION
Waxes are secreted onto the surface or into the interior of cuticles to form the first barrier against various environmental stresses (Millar et al., 1999). Since Dellaert discovered the first waxy cuticle eceriferum mutant (cer) in A. thaliana (Dellaert et al., 1979), many genes related to wax synthesis have been isolated, including wax synthesis-related enzymes (CER4, CER6, CER10, FATB, and GL8) (Fiebig et al., 2000;Bonaventure and Ohlrogge, 2003;Dietrich et al., 2005;Zheng et al., 2005;Rowland et al., 2006), wax transporters (CER5 and WBC11) (Pighin and Samuels, 2004;Bird et al., 2007;Panikashvili et al., 2007), and transcription factors (SHN1, MYB30, MYB96, and WXP1) (Broun et al., 2004;Zhang et al., 2005;Raffaele et al., 2008;Seo et al., 2011). Several genes implicated in Brassica plant wax biosynthesis pathway were reported in recent studies. Though these studies identify the same glossy waxless appearance, the inheritance patterns are different. For example, the waxless trait of the Chinese cabbage mutant 08A235-2 is controlled by a single recessive gene (Zhang et al., 2013), same as that of HUAYOY2 in this study. By contrast, a dominant mutant allele controls the waxless trait in Brassica napus mutant 6-1025 (Pu et al., 2013). The glossy appearance of HUAYOU2 in this study is also determined by a single recessive gene, but sequence and functional analysis of the candidate gene revealed that the novel locus for glossiness on chromosome C01 is involved in the primary alcohol-forming pathway, in contrast with the gene BnaA.GL in Brassica napus (Pu et al., 2013) and the gene BrWax1 in Brassica rapa (Zhang et al., 2013).
Cer4 mutant and cabbage mutant LD10GL are fertile in contrast with the cabbage glossy green mutant 10Q-961, in which only a few seeds were produced after self-pollination . Enzymes containing the conserved FAR domain usually act on medium-and long-chain fatty acids and have been reported to be involved in the biosynthesis of plant cuticular wax (Rowland et al., 2006). In Arabidopsis, FAR has specificity for very-long-chain fatty acids and is present in enzymes responsible for the synthesis of primary alcohols in the epidermal cells of aerial tissues and in the roots (Rowland et al., 2006). CER4 is one of the genes encoding an FAR domain in Arabidopsis. The glossy wax-deficient trait is displayed in Arabidopsis mutant cer4 due to decreases in the production primary alcohols and wax esters.
By studying the cabbage mutant HUAYOU2, BoWax1 was finely mapped and the target gene was identified. In the F 2 population, gene BoWax1 was localized to a region of 158.5 kb on cabbage chromosome C01. The sequence variation and abnormal expression of the Bol013612 gene imply that Bol013612 may be the target gene that accounts for the glossy green trait of HAUYOU2. These results will help us further elucidate the formation of the glossy trait in HUAYOU2 and accelerate wax biosynthesis studies in cabbage. More research needs to be carried out in subsequent work, such as functional studies of BoWax1, investigation of the metabolic mechanism of cabbage wax biosynthesis and identification of other genes involved.

AUTHOR CONTRIBUTIONS
DL, LY, and ZF designed and supervised the study. XD, ZZL, JT, MZ, YZ, HL, YL, and ZSL participated in its design. DL participated in the mapping, RT-PCR, and sequence analysis. JT, ZZL, YL, and ZSL participated in the statistical analysis. DL and LY wrote the manuscript. All the authors discussed the results and contributed to the manuscript. All authors read and approved the final manuscript.