A random amplified polymorphic DNA ( RAPD ) molecular marker linked to late-bolting gene in pak-choi ( Brassica campestris ssp . chinensis Makino L . )

Bulked segregant analysis (BSA) and random amplified polymorphic DNA (RAPD) methods were used to analyze F2 individuals of P-27 × P-28 to screen and characterize the molecular marker linked to latebolting gene in pak-choi (Brassica campestris ssp. chinensis Makino L.). A total of 200 random primers were used for RAPD analysis. One RAPD marker S265750 was identified to be co-segregating with the late-bolting gene, and the genetic distances between S265750 and late-bolting gene was 3.14 cM. The results of the study can be seen as a starting point for future researches on the pak-choi bolting gene mapping and molecular marker assisted breeding.


INTRODUCTION
Pak-choi (Brassica campestris ssp.chinensis Makino L.) originated from China, and is one of the most important vegetable crops in China and worldwide that has the largest planting area and total yield (Liu et al., 1998).Naturally, bolting is interrelated greatly with the yield and quality of Pak-choi cultured in spring, and has been reported as a quantitative trait, which is mostly influenced by the environment (Wang and Paterson, 1994;Wang et al., 2008;Xu and Wang, 2008).In recent years, bulked segregant analysis (BSA) method has been reported to make great progress in some important properties of marker gene location selection in various plants.Gu et al. (2006) reported a bitter Bt gene in cucumber.Lei et al. (2006) reported bacterial wilt resistance genes in potatoes.Leng et al. (2007) reported a downy mildew disease gene in Chinese cabbage (Brassica rapa ssp.

Pekinensis).
Methods for the localization of molecular markers, such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and sequence characterized amplified regions (SCAR), have been used extensively to localize genes of interest and in molecularassisted plant breeding (Zhu et al., 2004).RAPD is one of the simplest and fastest molecular marking technologies (Gabriel et al., 2005;Zhang et al., 2008), and bulked segregant analysis (BSA) is also one of the effecttive methods in finding a linked marker of a gene (Michelmore et al., 1991).In recent years, many important quality trait genes of main crops have been marked (Zhu et al., 2004).Hidetoshi et al. (2001) identified one QTL RA1255 (530 bp) controlling bolting in cabbage, and the gene position in BN007-1.Du et al. (2007) reported two SRAP markers closely linked to late-bolting in cabbage.Zhang (2007) reported one SSR marker linked to late-bolting gene in pak-choi.
Using tightly linked molecular markers to target genes to screen progenies is one of the most effective methods to carry out artificial selection in pak-choi.But until now, there have been no scientific reports focusing on the latebolting gene linked to a molecular marker in pak-choi, except one SSR marker (Zhang, 2007).In this paper, BSA and RAPD methods were used to analyze F 2 individuals of P-27 × P-28 to screen and characterize the molecular marker linked to late-bolting gene, and the preliminary results of identification of RAPD marker tightly  1), PCR reaction, the amplification profile and PCR product detection published by Anthony et al. (2008) was used.

DNA bulking and DNA extraction
To isolate RAPD markers that were tightly linked to the late-bolting locus in pak-choi, early-bolting and late-bolting bulks were used, all of which were derived from the F2 population of P-27 × P-28.Every ten plants were randomly selected from early-bolting and latebolting segregants, and leaf DNA was isolated from individual plants.
The BSA approach was used to compare two pooled DNA samples of the individuals (Michelmore et al., 1991).In order to perform BSA for identification of markers closely linked to latebolting gene, we selected ten early-bolting and ten late-bolting F2 individuals to construct early-bolting bulk and late-bolting bulk, respectively.The young leaves of parental lines and F2 plants were selected to extract genomic DNA using the CTAB method (Murray and Thompson, 1980;Saghai-Maroof et al., 1984;Rogers and Bendich, 1988).

Random amplified polymorphic DNA (RAPD) analysis
The RAPD exploration and PCR protocol were adopted from the method described by Li et al. (2001).Amplifications were carried out in 20 µl of reaction mixture, including 20 ng of template DNA, 20 ng of each random primer (Table 1), 2.5 mM MgCL2, 0.2 mM dNTPs, 1.0 U Taq polymerase and 10×PCR buffer (Shanghai Sangon Biotech Co., Ltd, China).
The cycling parameters were denatured for 5 min at 94°C, followed by 45 cycles of 94°C for 1 min, 37°C for 1 min and 72°C for 90 s.The PCR was terminated following incubation at 72°C for 10 min and then stored at 4°C until the PCR products were analyzed.
The quality and quantity of DNA extracted were estimated by electrophoresis on agarose gel (0.8 %) containing ethidium bromide using 1×TAE buffer and by UV spectrophotometry, respectively.The concentration of extracted DNA was adjusted to 10 to 20 ng/µl and samples were stored at -20°C.

Bulk segregant analysis (BSA)
BSA (Michelmore et al., 1991) was finally performed for each individual on the bulks of DNA of ten early-bolting plants or latebolting plants.Primers which amplified a DNA fragment in only one of the bulks were confirmed on the same set of bulk, followed by PCR on ten individual early-bolting and ten late-bolting plants.
RAPD markers with the best linkage to late-bolting gene were evaluated further on an additional number of individual plants.

Marker scoring and linkage analysis
The banding patterns obtained from RAPD were scored as present (1) or absent (0), each of which was treated as an independent character regardless of its intensity.Among the 200 RAPD primers, the polymorphic primers were identified between the two parents and the F1 hybrid.For each polymorphic primer, the RAPD bands of P1, P2 and 255 F2 individuals were treated as dominant markers and scored as either present (1) or absent (0).Clearly distinguishable bands ranging from 100 to 2000 bp were used in the genetic and marker analysis.Linkage between DNA markers and the late-bolting locus was determined by the recombination rate.Recombination rate (cM) was calculated as follows: recombinator / (no. of individual of F2 generation × 2) × 100 %, where 2 stand for diploid genome (Liu et al., 2002).

DNA testing
The PCR products were separated on 0.8% agarose gel according to the lengths of amplified fragments and stained with ethidium bromide (Jiang et al., 2006).The results of the inspection was electrophoresis strip clear and consistent, neat zone type and no degradation phenomenon (Figure 1), which indicates that the extraction of DNA can satisfy the RAPD amplified reactions needed.

The field performance investigation of F 2 individuals
255 F 2 plants derived from P-27 and P-28 crosses were  cultivated in the field.Investigation on the field performance of each F 2 individual plant was significant, making the phenotypic identification easier.The study showed that the separation of bolting time was obvious in the F 2 generation of P-28 × P-27 (Figure 2) and it was close to a Skewness distribution, according to quantitative traits genetic characteristic.

RAPD primer screening
According to the polymorphism between P 1 , P 2 and F 1 , 90 RAPD polymorphic primers were selected in 200 random primers.The 90 RAPD polymorphic primers provided a clear polymorphism in early-bolting bulk and late-bolting bulk (Figures 3 and 4).One RAPD primer S265 amplified about 750 bp band (Figure 4), which was then named S265 750 .
To identify RAPD marker linked to late-bolting genes, DNA bulks were composed of the latest bolting and earliest bolting plants of the segregating families of P-27 and P-28.For each set of bulks, 200 random primers were screened (Figure 3), and 90 (45%) polymorphic RAPD primers between parents were used to screen 255 individuals from F 2 population.Between S101 to S300, primers amplified RAPD markers either in the bolting or in the late-bolting bulk.These primers were examined further on F 2 individual plants.A polymorphic fragment was obtained from plant P-27 and late-bolting plants, but no corresponding band was obtained from P-28 and early-bolting plants (Figure 4).The primer S265 (5'-GGCGGATAAG-3'), which produced one RAPD marker S265 750 with about 750 bp, generated DNA fragments that were found to be linked to the late-bolting locus in F 2 population.The presence of the RAPD markers S265 750 was analyzed in the entire population of F 2 (Figures 4 and 5).

Linkage map of late-bolting gene marker
A total of 200 RAPD markers scattered on the whole pakchoi chromosomes were used to determine the linkage to late-bolting.90 RAPD polymorphic primers were   employed to amplify P 1 , P 2 and 255 F 2 plants.All clearly distinguished polymorphism bands ranging from 100 to 2000 bp were treated as dominant markers and scored as either present (1) or absent (0) (Table 2).According to Table 2, there was the ratio of 52:6:10:187 among latebolting with belt mark, late-bolting without belt mark, early-bolting with belt mark and early-bolting without belt mark.So, there were 16 recombinants in 255 F 2 individuals.Linkage between DNA markers and the late-bolting locus was determined by the recombination rate.The genetic distance between S265 750 and late-bolting gene was about 3.14 cM, calculated as described in materials and methods.

DISCUSSION
Bolting traits belong to quantitative traits.Mero and  Honma (1985) first reported that the hybrid between early-bolting or late-bolting varieties in Chinese cabbage showed early-bolting with an advantage, but late-bolting percentage can be effected by modify genes.Li and Cho, (1997) indicated that bolting traits is in accordance with dominant and addition genetic model, in which the addition effect played a more important role, while the dominant effect only had performance.Recessive genes were more than the dominant allele, and dominant allele only control part of dominant characters.Our study showed the separation of bolting time obviously in the F 2 generation of P-28 × P-27 (Figure 2), and it was close to a Skewness distribution, according to quantitative traits genetic characteristic.In this study, the BSA method provided a rapid and simple alternative technique to identify one RAPD marker linked to late-bolting.S265 (5'-GGCGGATAAG-3'), one of the 200 random primers screened, reproducibly produced polymorphism S265 750 (about 750 bp) among parents and the two bolting bulks.The results confirm primarily that late-bolting gene is linked with the RAPD marker S265 750 .The amplified products of S265 showed that there were 16 recombinants in 255 F 2 individuals.Genetic distance between S265 750 and late-bolting gene was about 3.14 cM, calculated as described in materials and methods.
The RAPD marker S265 750 in this study can be regarded as a marker tightly linked to a quantitative-character, and it can be used in the molecular-assisted breeding procedures.Molecular-assisted breeding of late-bolting in pak-choi plants can identify the late-bolting of plants in the early stage of breeding, increasing selection veracity and efficiency, and can accelerate reasonable and rapid utilization of the late-bolting gene, shortening the breeding cycle.

Figure 1 .
Figure 1.Examination of isolated DNA on agarose gel.

Figure 2 .
Figure 2. Frequency of bolting time distribution of F2 population.

Figure 5 .
Figure 5.The amplification result of the primer S265 among part of the F2 population.M, DL2000 DNA marker; 1 to 24, number of F2 individuals.

Table 1 .
Sequences of part RAPD primers used in this study.

Table 2 .
Evaluation of bolting in F2 individuals.