Genetic characterization and fine mapping of a chlorophyll-deficient mutant (BnaC.ygl) in Brassica napus
Lixia Zhu, Xinhua Zeng, Yanli Chen, Zonghui Yang, Liping Qi, Yuanyuan Pu, Bin Yi, Jing Wen, Chaozhi Ma, Jinxiong Shen, Jinxing Tu, Tingdong Fu (*)
National Key Laboratory of Crop Genetic Improvement, National Sub-center of Rapeseed Improvement in Wuhan, Huazhong Agricultural University, Wuhan 430070, China
* Corresponding author: Tingdong Fu
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Tel: 86-27-87281900
Fax: 86-27-87280009
Abstract
A chlorophyll-deficient mutant with yellow-green leaves of Brassica napus was obtained by treatment with the chemical mutagen ethyl methanesulfonate (EMS). Compared with the wild type at seedling stage, the mutant displayed decreased total chlorophyll content, less granal stacks and granal membranes. Genetic analysis confirmed that the mutant phenotype was controlled by a recessive gene, which was designated as BnaC.ygl. Mapping of the gene was subsequently conducted in two populations with yellow-green leaves (populationⅠBC8 and ⅡBC4, which comprised 3472 and 5288 individuals respectively) . Analysis on the public simple sequence repeat markers (SSR) showed that four SSR markers linked to BnaC.YGL gene displayed polymorphism. Based on the information of these SSR markers, the BnaC.YGL gene was mapped to the linkage group N17. From a survey of amplified fragment length polymorphism (AFLP), 15 of 47 AFLP markers were successfully converted into sequence characterized amplified region (SCAR) markers. BnY5 and CB10534, the closest flanking markers, were 0.32 cM and 0.03 cM away from the BnaC.YGL gene, respectively. And in the two populations, 18 makers cosegregated with BnaC.YGL. BLAST analysis revealed that the sequences of the makers displayed highly conserved homology with C06 of B. oleracea. The collinearity of makers to makers on N17 and on C06 showed that there might be an inversion occurring on the N17 group. These results are expected to accelerate the process of cloning the BnaC.YGL gene and facilitate the understanding of the biological processes of chloroplast development in Brassica napus.
Keywordschlorophyll-deficient mutant
Brassica napus
Fine mapping
Chloroplast development
Introduction
Chlorophyll (Chl) plays several important roles in photosynthetic light-harvesting and energy transduction both directly and indirectly. Meanwhile, its biosynthesis, accumulation and degradation are also associated with chloroplast development, photo-morphogenesis and chloroplast-nuclear signalling (Eckhardt et al. 2004; Vothknecht and Westhoff 2001). Extensive studies have been focused on the Chl metabolism in various organisms by biochemical and genetic approaches (CG and SP 1978; Gaubier et al. 1995; Oster et al. 2000). Biochemical researches on the enzymatic steps have promoted the identification of their encoding genes (Eckhardt et al. 2004). So far, almost all of Chl biosynthetic genes have been identified in higher plants (Beale 2005; Nagata et al. 2005; Tripathy and Pattanayak，GK. 2012). The whole pathway of Chl biosynthesis can be subdivided into four parts: (1) formation of 5-aminolevulinic acid (ALA), the committed step for all tetrapyrroles, (2) formation of a pyrrole ring porphobilinogen and the synthesis of the first closed tetrapyrrole having inversion of ring D, (3) formation of protoporphyrin IX (Proto IX), which is a common precursor for Chl and heme/bilin biosynthesis, and (4) formation of Chl from Proto IX (Tanaka et al. 2011; Tripathy and Pattanayak，GK. 2012). The ATP-dependent insertion of Mg2+ into Proto IX is the branch point for chlorophyll and heme biosynthesis (Tripathy and Pattanayak，GK. 2012). In this metabolic pathway, plants need to prevent excessive accumulation of the intermediate molecules because most of Chl biosynthetic intermediates are strong photosensitizers that would lead to oxidative damage or cell death under illumination (Tanaka et al. 2011).
Mutant lines are important resources for forward genetic studies of gene functions as well as for a better understanding of the regulatory mechanisms of biological metabolism because of the abnormal expression of some metabolic intermediates. Mutants can be obtained by chemical and irradiation mutagenesis such as EMS, γ-ray, or through biological methods such as tissue culture and T-DNA/ transposon insertions. The various methods used by different researchers have resulted in a large number of Arabidopsis, rice, barley, maize, soybean and rapeseed mutants which have been extensively used to study gene functions (Falbel and Staehelin 1999; Hirochika et al. 2004; Krishnan et al. 2009; Lunde et al. 2003; Zhao et al. 2000).
The mutants with chlorophyll-deficient or chlorophyll-modified leaves are very common and widely used to study the molecular mechanisms that regulate Chl biosynthesis and chloroplast development in many plants such as Arabidopsis (Eckhardt et al. 2004), rice (Kurata et al. 2005; Sakuraba et al. 2013; Wu et al. 2007), soybean (Palmer et al. 2000), barley (Falbel and Staehelin 1994; Falbel and Staehelin 1996; Falbel and Staehelin 1999; Mueller et al. 2012; Yaronskaya et al. 2003), maize (Lunde et al. 2003) and so on. Currently, at least 27 genes that encode 15 enzymes from glutamyl-tRNA to Chl b in Chl biosynthesis have been identified in Arabidopsis (Nagata et al. 2005; Nagata et al. 2007). A correlation analysis between the gene expression and the level of Mg-Proto IX in a range of mutants (gun, chlm, crd1) showed that the steady-state level of Mg-Proto IX was not a determinant of plastid-to-nucleus signalling in Arabidopsis, which revised the previous model on plastid signalling (Mochizuki et al. 2008). In rice, more than 70 chlorophyll mutants that exhibit albino, chlorina, stripe, virescent, yellow-green and zebra leaves have been reported (Kurata et al. 2005). However, only a few genes involved in the Chl biosynthesis have been cloned and characterized, such as OsChlD, OsChlH, OsChlI, OsDVR, OsCAO, OsGluRS, and OsPOR (Jung et al. 2003; Liu et al. 2007; Sakuraba et al. 2013; Wang et al. 2010; Wu et al. 2007; Zhang et al. 2006). In Brassica napus, only one chlorophyll- deficient mutant (Cr) has been reported. The yellow-green leaf phenotype in Cr is controlled by a recessive gene and has been successfully turned into CMS lines (Zhao et al. 2000). Moreover, it has a decreased amount of light-harvesting complexes but an increased amount of some core polypeptides of PSII (Guo et al. 2007). However, the mapping of the recessive gene which is responsible for the mutational phenotype in Cr was not reported so far.
Currently, a combination of map-based cloning strategy and comparative genome analysis is usually used for gene mapping, which is an effective approach for gene mapping in cultivated Brassicas described in “triangle of U” (Kowalski et al. 1994; Parkin et al. 2005; Wang et al. 2011; Xia et al. 2012). However, only several genes have been cloned in cultivated Brassicas by this way, such as the dwarf gene dwf2，seed coat color genes BrTT8 and TTG1 homologue in B. rapa (Li et al. 2012; Muangprom and Osborn 2004; Zhang et al. 2009), the high-β-carotene gene Or in B. oleracea (Lu et al. 2006), the RGMS genes (BnMs1, BnMs2, BnMs3), the dwarf gene BnRGA and the cleistogamy gene Bn-CLG1A in B. napus (Dun et al. 2011; Lei et al. 2007; Liu et al. 2010; Lu et al. 2012; Yi et al. 2010). Since a large amount of B. rapa, B. oleracea and B. napus sequence information has been released to the public databases, and especially with the completion of B. rapa genome sequencing, cloning genes with this approach would be more effective in cultivated Brassicas.
In this study, we isolated a rapeseed Chl-deficient mutant, which exhibited a yellow-green leaf phenotype, reduced Chl level, and affected chloroplast development. The objectives of our study were to: (1) analyse the inheritance model of the mutant, (2) develop the molecular markers linked to the BnaC.YGL gene, and (3) fine map the BnaC.YGL gene through a classic map-based cloning strategy in combination with comparative mapping among B. napus and other Brassica species. The results will promote the map-based cloning of the BnaC.YGL gene as well as the understanding of the biological processes of chloroplast development in Brassica napus
Materials and Methods
Plant materials
The chlorophyll-deficient mutant (BnaC.ygl) was obtained from the B. napus inbred line T6 (+/+) whose seeds were treated with 1% EMS for 8h. The mutant with a yellow- green leaf phenotype at seedling stage (Fig.1a)was identified at the M2 generation and was selfed togeneratean inbredline. Two breeding lines (B409 and 1161) with normal green leaves were used to construct the populations.
Chlorophyll content determination and transmission electron microscopy analysis
Total Chl was determined with UVmini-1240 (Shimadzu) according to the method of Arnon (Arnon 1949). The leaf samples of the wild-type (T6) and chlorophyll-deficient mutant (BnaC.ygl) were harvested from the plants at different stages in normal conditions. Leaves of the wild-type and the mutant (approximately of 30 mg fresh weight respectively) were cut and homogenized in 5 mL 80% (V/V) aqueous acetone for 48h, and then centrifuged at 3,000 g for 10 min. The combined supernatants were diluted with acetone for spectrophotometric analysis.
For transmission electron microscopy analysis, leaves were cut into 1×1 cm sections and fixed in 2.5% (w/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and further fixed in 1% OsO4 in the same buffer. The subsequent steps were carried out as described by Yi et al (2010).
Genetic analysis
The cross between the mutant (BnaC.ygl) line and B409 was carried out (hereafter referred to as populationⅠ). The F2 population was derived from the self-pollination of F1 plants. The BC1 was derived by the backcrosses of F1 to the mutant line. The phenotype of the reciprocal hybrid F1 and the segregation ratio of ⅠF2 andⅠBC1 population were used to detect the genetic pattern of the chlorophyll-deficient mutant. The other similar hybrid population (F1) was obtained by crossing 1161 with the mutant line (hereafter referred to as population Ⅱ). The ⅡF2 and ⅡBC1 were also obtained. Data from the above experiments were analysed using the SAS system (SAS 8.1). All the measurements in the experiments were analysed as completely random design.
DNA extraction and marker analysis
Total DNA was extracted individually from the leaves at seedling stage using the CTAB method with some modifications (Doyle and Doyle 1990). Based on the measurement of DNA concentration by a Beckman spectrophotometer (Beckman, Fullerton, USA), final DNA concentration was set as 50 ng/μl in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).
The SSR markers available in public were used to identify the polymorphisms between the parents (the mutant and B409). SSR analysis was performed as described previously by Piquemal et al (2005). Equivalent amounts of DNA (12 chlorophyll-deficient leaf plants and 12 normal green leaf plants) from populationⅠwere randomly selected to construct three chlorophyll-deficient bulks (CB) and three normal green leaf bulks (GB). All the SSR markers were used to firstly analyse the two parents of populationⅠand then the two bulks. The amplification products were separated on a 6% polyacrylamide denaturing sequencing gel and visualised using a silver staining system (Lu et al. 2004) with some modifications.
For AFLP technique, the CB and GB DNA samples were digested with the enzyme combinations (EcoRI, MseI and SacI) to produce 12.5 μl of digested solution, respectively. Subsequently, the digested restriction fragment ends were ligated to specific double-stranded adapters by T4 DNA ligased. The adapter-ligated DNA was diluted five-fold and then pre-amplified with AFLP primers (EA/MC, EA/MG, EC/MC, EC/MG, SA/MG) into 25 μl solution. The pre-amplified products were analysed in a 1.0% agarose gel electrophoresis, and diluted 10 to 30 folds for selective amplification (Negi et al. 2000). The products of the selective amplification were separated and silver stained as described for AFLP markers.
The polymorphic AFLP fragments were cloned and sequenced as described previously by Ke (Ke et al. 2004) and Yi (Yi et al. 2006). These sequences of markers linked to the BnaC.YGL gene were extended using BLAST searches in http://brassica.bbsrc.ac.uk/IMSORB/. Based on these sequences, the specific primers were designed by the software Primer3 (Rozen and Skaletsky 2000). These primers were used to detect the polymorphisms in the CB and GB bulks.
Genetic map of the BnaC.YGL gene
To construct a rough flanking map, we used the BC3 population with 500 individuals which showed the BnaC.ygl mutant phenotype (populationⅠ). For a fine map of the region around the BnaC.YGL gene, we enlarged the backcross population I BC8 ((ygl×B409)×ygl) to 3472 plants showing the BnaC.ygl mutant phenotype and constructed the backcross Population ⅡBC4 ((1161×ygl)×ygl) with 5288 individuals showing the BnaC.ygl mutant phenotype. The data of these markers and individual phenotypes were analysed with the MAPMAKER/EXP 3.0 program (Lander et al. 1987; Lincoln et al. 1992). The map order was estimated by maximum-likelihood.
Development of markers and comparative mapping with Arabidopsis thaliana and B. oleracea
The genome sequence from B. oleracea became available for blast since April in 2011 (http://brassicadb.org/brad/). All the polymorphic fragments linked to the BnaC.ygl gene were sequenced to determine their putative homology using BLAST search against the B. oleracea genome database (Brassica database, BRAD; http://brassicadb.org/brad/). To identify the putative syntenic region around the BnaC.YGL gene in Arabidopsis genome, the scaffolds in B. oleracea genome with which the markers had homology were used to identify their homologous regions in the Arabidopsis genome database [http://www.arabidopsis.org/]. IP markers were developed according to the information on the corresponding Arabidopsis loci. The detailed procedures of developing IP makers were performed as previously described by Xia et al (2012).
Results
Reduced Chl accumulation and affected chloroplast development in BnaC.ygl mutant
The BnaC.ygl mutant exhibited a yellow-green leaf phenotype at seeding stage and became green at the later stage (Fig. 1a, d). Compared with the wild type, it showed deficiency in chlorophyll and exhibited higher level of Chl a/b (Table 1) at seedling stage. The leaves of the BnaC.ygl mutant had 7.02% to 35.19% reduction of total Chl compared with those in the wild type at different stages (Table 1). The Chl a and Chl b levels in BnaC.ygl mutant and the wild type were significant differences at 6-week-old and 14-week-old plants, but not at 20-week-old plants. In BnaC.ygl mutant, the Chl a/b ratio appeared highest at 6-week-old plants, which was possibly due to that the synthesis of Chl b was more severely declined than that of Chl a. However, at the later stages, the Chl a /b ratio declined to almost the level of the wild-type (Table 1). The above results suggested that the BnaC.ygl mutant showed delayed greening during photomorphogenesis due to the slow rate of Chl accumulation. Thus, it can be speculated that when the mutant plants accumulated a sufficient amount of Chl, which almost reached the level of the wild type, the leaves looked almost the same as the wild-type in the later development.