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.
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