Brassica rapa L. ssp. chinensis (L.) Hanelt, known as nonheading chinese cabbage in China, is an important vegetable in eastern Asia and its genetic improvement requires a genetic linkage map. The first genetic linkage map of nonheading chinese cabbage using 112 doubled haploid lines derived from a released F1 hybrid cultivar Shulü between two lines SW-3 and Su-124 was constructed in this paper. One hundred thirty-eight molecular markers were mapped into 14 linkage groups. Among these markers, there were 77 sequence-related amplified polymorphism markers, 27 simple sequence repeat markers, 21 random amplification polymorphic DNA markers, and 13 intersimple sequence repeat markers. Chi-square tests showed that 54 markers are distorted from Mendelian segregation ratios, and the direction of the distortion is mainly toward the maternal parent SW-3. The distortion affects not only the estimation of genetic distance, but also the order of distorted markers on a same linkage group. Given a specific marker order, the authors proposed a multipoint approach to correct the linkage map in an unbiased manner in an F2 population while considering distorted, dominant, and missing markers. A new method was used to correct the linkage map in the doubled haploid population mentioned earlier considering new, distorted, and missing markers. The total length of the corrected linkage map was 1923.75 cM, with an average marker spacing of 15.52 cM. The map will facilitate selective breeding and mapping of quantitative trait loci.
The genus Brassica includes many important vegetables: chinese cabbage [B. rapa ssp. pekinensis (Lour.) Hanelt], nonheading chinese cabbage (B. rapa ssp. chinensis), cabbage (B. oleracea L. var. capitata L.), broccoli (B. oleracea var. italica Plenck), cauliflower (B. oleracea var. botrytis L.), kale (B. oleracea var. acephala DC.), and rape [B. napus L. ssp. oleifera (Delile) Sinskaya] (Nozaki et al., 1997). Nonheading chinese cabbage, which originated from China, is one of the most important vegetables in eastern Asia. However, there are fewer detailed selective breeding programs worldwide. Recently, we have established a selective breeding program focusing on the improvement of important agronomic traits (e.g., disease resistance and quality). Such a breeding program requires the development of a large number of DNA markers for the detection of quantitative trait loci (QTL), and marker-assisted selection to speed up the breeding process and to increase the selection efficiency.
Genetic linkage maps that are essential to the detection of QTL and other applications have been constructed for nearly all economically important plants. Although there are a number of genetic maps reported in B. rapa (Ajisaka et al., 1995; Nozaki et al., 1997; Song et al., 1991; Zhang et al., 2006), a linkage map for nonheading chinese cabbage is unavailable.
Sequence-related amplified polymorphism (SRAP) recently developed by Li and Quiros (2001) is aimed at amplifying the open reading frames with particular primer pairs. The primers consist of core sequences and three selective nucleotides, and the core sequences contain “filler” sequences (nonspecific constitution) and specific sequences. The forward primer is 17 bp (10-bp filler + CCGG + three selective nucleotides) whereas the reverse primer is 18 bp (11-bp filler + AATT + three selective nucleotides). They can pair with exons and promoters (or introns) respectively. The polymorphisms revealed by these primer pairs originate from the variations of the lengths of the involved introns, promoters, and spacers among individuals and species. Sequence-related amplified polymorphisms are easily amplified in crops (Li and Quiros, 2001), and may combine simplicity, reliability, moderate throughput ratio, and facile sequencing of selected bands, so it has been widely used in the comparative analysis of biotype (Budak et al., 2004a), in the evaluation of genetic diversity (Budak et al., 2004b; Ferriol et al., 2003), and in the construction of genetic maps (Li and Quiros, 2001; Sun et al., 2007). In theory, moreover, SRAP can detect any kind of sequence differences, including base changes and insertions and deletions, so there are as many of these markers as single nucleotide polymorphisms (SNPs) in a genome. In contrast to SNPs, however, a primer combination in the SRAP protocol can detect multiple loci in a genome without known sequence information. Intersimple sequence repeat (ISSR) is derived from simple sequence repeat (SSR), which amplifies specifically the region between two microsatellite motifs (Zietkiewicz et al., 1994). Compared with random amplified polymorphic DNA (RAPD), ISSR produces more reliable and reproducible result. Intersimple sequence repeat has been broadly applied to investigate genetic diversity, phylogenetic relationships and germplasm identification, and construction of genetic linkage maps (Blair et al., 1999; Kantety et al., 1995; Kojima et al., 1998; Moreno et al., 1998). Therefore, it is feasible to make use of SRAP, ISSR, and other markers in the construction of a moderately saturated genetic map.
Microspore culture and anther culture are commonly used to produce doubled haploid (DH) lines. The DH lines have been widely used to explore the genetic architecture of complex traits (Kuginuki et al., 1997; Voorrips et al., 1997), and to develop further elite parental lines for hybrid seed production (Chen and Beversdorf, 1990), because genetically homozygous DH lines are usually obtained in a single generation (Burr et al., 1988). However, high distortion segregation frequencies are frequently reported in the DH populations of B. rapa (Ajisaka et al., 1999; Zhang et al., 2006), B. oleracea (Voorrips et al., 1997), and B. napus (Cloutier et al., 1995).
Segregation distortion is usually observed for some or all markers in both experimental and natural populations (Lyttle, 1991). The distortion affects linkage tests (Garcia-Dorado and Gallego, 1992) and the estimation of recombination fractions between distorted markers (Lorieux et al., 1995a, b). Currently, most statistical methods used for map construction ignore the fact that some molecular markers are distorted (Jiang and Zeng, 1997; Lander and Green, 1987). Recently, Zhu et al. (2007) proposed a multipoint approach to correct unbiasedly a linkage map given a specified marker order in an F2 population, which is especially useful to deal with distorted, dominant, and missing markers.
In this paper, therefore, we establish the first genetic linkage map of nonheading chinese cabbage containing new, distorted, and missing markers using the method of Zhu et al. (2007). The map will facilitate selective breeding and mapping of QTL.
Materials and Methods
One hundred twelve DH lines were derived from a newly released F1 hybrid cultivar Shulü of nonheading chinese cabbage between other two lines SW-3 and Su-124. The maternal parent SW-3, a self-incompatible line, was developed from cultivar Aijiaohuang by molecular-assisted selection; the paternal parent Su-124, an inbred line, was derived from cultivar Suzhouqing. There are a number of different traits between these two parents, including leaf color, peduncle color, disease resistance, hot tolerance, cold hardiness, and quality.
About 0.3 g fresh bud of flower was used to extract genomic DNA using the cetyl-trimethyl-ammonium bromide (CTAB) method. DNA was quantified by comparing with λDNA through running agarose gels.
Sequence-related amplified polymorphism analysis.
The sequences of SRAP primers were provided by G. Li at the University of Manitoba, Canada, and are shown in Table 1. Reactions were performed in a 10-μL volume containing 30 ng DNA, 0.5 umol·L−1 forward and reverse primers, 0.2 mmol·L−1 deoxyribonucleotide triphosphate (dNTPs), 1.5 mmol·L−1 MgCl2, 1× polymerase chain reaction (PCR) buffer (Tri-HC; pH, 8.3; 100 mm; KCl, 500 mm), and 0.75 U Taq DNA polymerase. The protocol of PCR reaction was as follows: 94 °C for 3 min, then 94 °C for 30 s, 37 °C for 30 s, 72 °C for 90 s for five cycles; 94 °C for 30 s, 48 °C for 30 s, 72 °C for 90 s for 33 cycles; and finally 72 °C for 7 min. The products were separated on 5% vertical polyacrylamide gel. The gel was run at 70 W constant power for 1.5 to 2 h until xylene cyanol reached toward the bottom of the gel. After electrophoresis, the gel was stained by AgNO3 solution.
Sequences of sequence-related amplified polymorphism primers used in the development of genetic linkage maps for nonheading chinese cabbage.
Simple sequence repeat analysis.
The sequences of SSR primers were shown in Table 2. The SSR reactions were performed in a 10-μL volume containing 20 ng DNA, 0.5 μmol·L−1 forward and reverse primer, 0.2 mmol·L−1 dNTPs, 1.0 mmol·L−1 MgCl2, 1 × PCR buffer, and 0.5 U Taq DNA polymerase. The PCR procedure was the following: 94 °C for 5 min, then 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s for 35 cycles, then 72 °C for 7 min. The products were separated on 5% vertical polyacrylamide gel. The gel was run at 60 W constant power for 1 to 1.5 h until xylene cyanol reached toward the bottom of the gel. After electrophoresis, the gel was stained by AgNO3 solution.
Sequences of simple sequence repeat primers used in the construction of genetic linkage maps for nonheading chinese cabbage.
Random amplified polymorphic DNA analysis.
The sequences of RAPD primers were obtained from Nanjing Sunshine Biotechnology Co., Ltd. (Nanjing, China). The RAPD reaction was performed in a total volume of 20 μL, with 1 × PCR buffer, 2.0 mmol·L−1 MgCl2, 0.25 mmol·L−1 dNTPs, 0.2 μmol·L−1 primer, and 0.5 U Taq DNA polymerase, and 20 ng template DNA. The procedure of PCR amplification was as follows: 94 °C for 3 min, then 94 °C for 30 s, 37 °C for 45 s, 72 °C for 90 s for 42 cycles, then 72 °C for 7 min. The PCR products were electrophoresed on 1% horizontal agarose gel in 1× Tris-Acetate-EDTA (TAE) buffer with a voltage of 5 V·cm−1 and visualized under ultraviolet light after staining in 1 μg·mL−1 ethidium bromide.
Intersimple sequence repeat analysis.
The sequences of RAPD primers were obtained from Nanjing Sunshine Biotechnology Co., Ltd. The ISSR reaction was performed in a total volume of 20 μL, with 1 × PCR buffer, 2.0 mmol·L−1 MgCl2, 0.2 μmol·L−1 primer, 0.25 mmol·L−1 dNTPs, and 0.5 U Taq DNA polymerase, and 20 ng template DNA. The PCR amplification procedure was as follows: 94 °C for 4 min, then 95 °C for 45 s, 52 °C for 45 s, 72 °C for 45 s for 40 cycles, then 72 °C for 7 min. The PCR products were electrophoresed on 1.5% horizontal agarose gel in 1× TAE buffer with a voltage of 5 V·cm−1 and visualized under ultraviolet light after staining in 1 μg·mL−1 ethidium bromide.
Reconstruction of genetic linkage map.
Let the order of m markers on the same chromosome be M 1, M 2, …, Mm in a DH population, rk be the recombination fraction between the kth and (k + 1)th markers, sk (0 ≤ sk < ∞; k = 1, 2, …, m) be the viability coefficients of MkMk relative to mkmk at the kth marker, x be a dummy variable defined as xk = 1 and −1 for a homozygote of P1 and P2 respectively, and z be an indicator of the phenotype of the kth marker Mk. If the distortion is incited by viability selection, the markers linked to the viability locus show distorted Mendelian segregation ratios. Sometimes the locus is known as segregation distortion locus (or SDL). In the construction of a linkage map, therefore, it is necessary to estimate 2m − 1 coefficients, including sk (k = 1, 2, …, m) and rk (k = 1, 2, …, m − 1). The case where sk = 1 (k = 1, 2, …, m) represents typical Mendelian segregation.
where n represents population size;
Maximum likelihood estimates for all parameters in Eq. (1) may be directly solved for if there are no missing genotypes; if this is not the case, an expectation and maximization algorithm (Dempster et al., 1977) should be adopted, including the following steps:
Initialization: The viability coefficient
is initialized with 1, and the initial value of the recombinant fraction along with the order of markers is obtained from Mapmaker 3.0 software (Lander et al., 1987).
E-step: Let A k = (ak,icd), denoted by P(x k x k+1|z 1, …, z m) in a 2 × 2 matrix form, where ak,icd is the cth row and dth column element of Ak for the ith individual. Using the multipoint method, the posterior probabilities P(x k x k+1|z 1, …, z m) for each individual can be calculated by
M-step: The recombination fraction between the kth and (k + 1)th markers and the selection coefficients (sk) can be updated as
where X(k) and W(k) are indicator variables, X(k) = 1 if 1 ≤ k < m or takes a value of zero if this condition is not met; W (k) = 1 if 1 < k ≤ m or takes a value of zero if this condition is not met. If
, Eq. (3) and Eq. (4) are unavailable. In this case, the iterated algorithm [i.e., Newton–Raphson (Lorieux et al., 1995b)] works well. In addition, sk = 1 if there are no significant differences for all chi-square tests. The E-step and M-step are iterated until convergence occurs.
Polymorphism screening of primers between parents.
Before the construction of a linkage map, the two parents were screened for polymorphism with 160 SRAP primer combinations, 200 SSR primer combinations, 120 RAPD primers, and 58 ISSR primers (Table 3). In total, 160 of 538 primers (combinations) (29.7%) could produce polymorphic loci, including 90 SRAP primer combinations, 40 SSR primer combinations, 22 RAPD primers, and eight ISSR primers. With 90 polymorphic SRAP primer combinations, a total of 154 polymorphic loci were selected at an average of 1.7 loci per primer combination. Meanwhile, 43 SSR polymorphic loci, 32 RAPD polymorphic loci, and 16 ISSR polymorphic loci were obtained. All these polymorphic markers were performed in the DH line population.
Molecular markers analysis for distorted, linked, and unlinked markers in nonheading chinese cabbage.
Construction of genetic linkage map.
One hundred twelve DH lines derived from a released F1 hybrid cultivar Shulü were genotyped for 138 linked molecular markers, including 77 SRAP markers, 27 SSR markers, 21 RAPD markers, and 13 ISSR markers. The results of a chi-square test showed that 54 of 138 markers were distorted from Mendelian segregation ratios, and the direction of distortion was mainly toward the maternal parent SW-3. As an illustrative example, the results for both chi-square tests and the genetic distances between adjacent markers with and without considering segregation distortion for the eighth linkage group are listed in Table 4. Results from Table 4 show that the distortion affects the estimation of genetic distance between distorted markers. Therefore, it is necessary to correct the bias of genetic distances between distorted markers.
Test for goodness-of-fit for Mendelian segregation ratios and genetic distances (GD) with and without considering marker segregation distortion for the eighth linkage group in nonheading chinese cabbage.
In the construction of a genetic linkage map using molecular markers, there are three steps. The first step is to cluster markers into linkage groups. One hundred thirty-eight molecular markers were assembled into 14 linkage groups with Mapmaker 3.0 software. The second step is to estimate pairwise genetic distances in each of the linkage groups. In this paper, the genetic distances between adjacent markers were calculated twice. The first time was using Mapmaker 3.0 without considering segregation distortion, and the second time was adopting the new multipoint method mentioned earlier while considering segregation distortion. The results in Table 5 show that the length of each linkage group ranges from 21.57 to 542.98 cM and all these molecular markers covered 1923.75 cM for the whole genome, with an average marker spacing of 15.52 cM. The genetic distances between distorted markers with the consideration of segregation distortion were usually longer than those without the consideration of segregation distortion, although there were one or two exceptions that were observed on linkage groups LG 1 to LG 3. Therefore, it is important and necessary to correct the bias of genetic distances between distorted markers. The third and last step is to optimize the orders of all mapped markers in all linkage groups. The optimized linkage groups were showed in Fig. 1.
Main characteristics of markers and genetic distances (GD) per linkage group in nonheading chinese cabbage.
Genetic maps for B. rapa have been published (Ajisaka et al., 1999; Chyi et al., 1992; Foisset et al., 1996; Song et al., 1991; Voorrips et al., 1997). The total length of the current map in this study was comparable with those reported, although we mapped fewer markers. However, the number of linkage groups on the current map does not match the 10 expected chromosomes in the genome. To integrate the linkage groups with the 10 chromosomes, further research needs to be done through adding more individuals in the DH population, developing more molecular markers, and using different types of mapping populations such as F2, recombinant inbred lines, and backcrosses.
Marker segregation distortion is a common phenomenon in vegetable crops (Foisset et al., 1996; Kim et al., 1999; Voorrips et al., 1997). Fifty-four distorted markers, accounting for 39.1% of the total markers, were observed in this study. In DH populations, a skewed segregation ratio has been often observed in many other plants (Foisset et al., 1996), and their existence probably results from gametic or zygotic selection, or from a specific selection derived from the production of plants by in vitro microspore culture. The distortion is known to bias the estimation of recombination fraction between consecutive markers and the order of distorted markers on the same linkage group (Lorieux et al., 1995a, b). Most quantitative geneticists who are interested in QTL mapping do not prefer to use distorted markers for QTL mapping, because the basic assumption of Mendelian segregation is violated. Too many distorted markers will cause tremendous information loss in QTL mapping if these markers are removed from the marker map (Luo et al., 2005). Hence, it is necessary to develop a new statistical method to reconstruct a molecular linkage map to adjust the bias of the estimates of the genetic distances between adjacent markers.
Murigneux et al. (1993) suggest that a more strict linkage test standard be used when segregation distortion existed (i.e., relatively higher logarithm of odds (LOD) value). Lorieux et al. (1995b) developed software called MapDisco to reconstruct a molecular linkage map considering distorted markers. The software by Manly and Olson (1999) also offers options to compute the recombination fractions in case of deviation from the Mendelian hypothesis. However, nearly all cited programs cannot determine whether the corrected values are close to the true ones. The reason is that the previously mentioned investigations are seldom addressed in theoretical simulation studies. More recently, we developed an alternative algorithm for reconstructing a molecular linkage map given a specified marker order while considering distorted, dominant, and missing markers using an F2 population as an example (Zhu et al., 2007). Our analysis is similar to the method of Lander and Green (1987) in spirit, but we extend construction of a genetic linkage map to more general situations, considering distorted, dominant, and missing markers. In this paper we simplify our method from an F2 population to a DH population. These results are fundamentals for QTL mapping in nonheading chinese cabbage.
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