**3. Genetic map construction**

Genetic maps are extensively used in gene mapping, QTL mapping and assembly of whole genome sequence. High density molecular markers in genetic maps are advantageous and necessary in most applications. The detection of multiple loci in a SRAP PCR reaction can be automated through fluorescently labelled SRAP primers so it is feasible to construct a high density genetic map using SRAP technology.

In *B. napus*, we used a five-color fluorescent dye set including '6-FAM', 'VIC', 'Pet', 'NED' and 'LIZ' to perform SRAP fragment analysis with the ABI genetic analyzer (Sun et al., 2007). 'LIZ' was used as the internal standard while the other four fluorescent dyes were used to label SRAP primers and combined with unlabeled primers. After obtaining SRAP products with four fluorescently labeled and four unlabeled primers, all the products from four SRAP primer combinations were pooled to increase the detection throughput by four fold. We used 1,634 SRAP primer combinations selected from 12 labeled and 442 unlabeled SRAP primers to produce 13,472 SRAP markers. Together with 79 SSR markers, we assem‐ bled currently the most saturated genetic map in *B. napus*.

Using cDNA-SRAP technology, we first constructed a transcriptome map based on *B. olera‐ cea* cDNAs obtained from leaf tissue (Li et al., 2003). In cDNA-SRAP, one step PCR allows the amplification of single strand cDNAs after the first strand cDNA is synthesized using reverse transcriptase. Since most cDNA-SRAP markers come from differences in gene se‐ quence, these markers are considered to be functional markers. SRAP products can be easily isolated from polyacrylamide gels for sequencing so we sequenced 190 fragments that corre‐ sponded to 190 polymorphic loci from cDNA-SRAP. Through analysis of sequence similari‐ ty, 169 out of 190 cDNA marker sequences were homologous to genes reported in Arabidopsis, which allowed the identification of extensive colinearity between the two ge‐ nomes according to the gene-for-gene alignment. Later, we developed over 1,000 SRAP markers using genomic DNA from the same mapping population and assembled these SRAP markers from both genomic DNA and cDNA samples on the same genetic map (Gao et al., 2007). In addition, we integrated 10 SCAR markers using sequences of genes with known functions in the biosynthesis of glucosinolates and inflorescence architecture, and one SCAR marker flanking a resistance gene to downy mildew.

SRAP technology can be combined with other markers to construct genetic maps. For exam‐ ple, Yu et al., (2007) constructed a high-density genetic map in a cultivated allotetraploid cotton population using SSR, SRAP, AFLP, and target region amplification polymorphism (TRAP). This high density cotton genetic map consists of 697 SSR, 171 TRAP, 129 SRAP, 98 AFLP, and two morphological markers, covering a genetic distance of 4,536.7 cM with the average genetic distance of 4.1 cM per marker. Gulsen et al., (2010) reported a new citrus linkage map using SRAP, RAPD, SSR, ISSR, peroxidase gene polymorphism (POGP), resist‐ ant gene analog (RGA), and a morphological marker, Alternaria brown spot resistance gene. In total, they assembled 385 SRAP, 97 RAPD, 95 SSR, 18 ISSR, 12 POGP, and 2 RGA markers on the citrus genetic map.

In the Cucurbitaceae family, Yeboah et al., (2007) constructed genetic maps in cucumber us‐ ing SRAP and ISSR markers. They developed pseudo-testcross F1 segregating populations from a cross between two diploid parents and constructed male and female parental genetic maps separately with 164 SSR and 108 SRAP markers. More recently, Zhang et al., (2012) constructed a high density consensus genetic map in an inter-subspecific mapping popula‐ tion in cucumber. The consensus map contained over a thousand molecular markers includ‐ ing 1,152 SSR, 192 SRAP, 21 SCAR and one STS. In another cucurbit species, Levi et al., (2006) constructed an extended genetic map for watermelon using five PCR-based molecular markers SRAP, AFLP, SSR, ISSR and RAPD. As suggested by the authors, low polymor‐ phism is often observed in watermelon cultivars, combining several marker systems is nec‐ essary to construct a high density genetic map covering the whole genome.

SRAP markers have been used to construct genetic maps in a wide range of plant species. In Dendrobium plants that are used as Chinese herbs, Xue et al., (2010) constructed two genetic maps in two Dendrobium species, *D. officinale* and *D. hercoglossum* with a double pseudotestcross strategy using SRAP and RAPD methods. In root plants, Chen et al., (2010) con‐ structed a genetic map in an F1 population derived from an interspecific cross in cassava by combining AFLP, SSR, SRAP and expressed sequence tag (EST)-SSR markers. In total, they assembled 355 markers into 18 linkage groups covering a genetic distance of 1,707.9 cM, which served as a foundation for QTL mapping in this species. In grass species, Xie et al., (2011) used SSR and SRAP markers to construct two genetic maps of male and female pa‐ rental lines respectively in diploid orchardgrass (*Dactylis glomerata* L.) using a pseudo-test cross strategy. In total, they assembled 164 SSR markers and 108 SRAP markers on these two genetic maps. In a fruit tree, Luohanguo (*Siraitia grosvenorii* C. Jeffrey), Liu et al., (2011) used SRAP and ISSR markers to assemble a genetic map consisting of 170 SRAP markers and 29 ISSRs in 25 linkage groups. In a fiber crop, Chen et al., (2011) used SRAP, ISSR and RAPD markers to construct a genetic map in Kenaf (*Hibiscus cannabinus* L.) that is one of the most economically important fiber crops globally.
