**5. Gene tagging and cloning**

In canola, we used SRAP and SSR to construct a genetic map in a doubled haploid (DH) line population that was developed from a synthetic yellow-seeded line and a conventional can‐ ola cultivar through microspore culture (Chen et al., 2009). Data for three complex traits in‐ cluding days to flowering, oil content and seed yield at three locations for three years were collected and used in QTL mapping. For oil content, 27 QTL on 14 linkage groups and for seed yield, 18 QTL on 11 linkage groups were identified while days to flowering was sug‐ gested to be controlled by a single genetic locus in this mapping population. In rapeseed, Chen et al., (2007) used 208 SSR and 189 SRAP markers to construct a genetic map for a DH line population and performed QTL mapping of yield-related traits in *B. napus*. They also developed a fixed immortalized population from randomly permutated intermating of these DH lines. They collected data for six yield-related traits, plant height, height of lowest pri‐ mary effective branch, length of main inflorescence, silique length, number of primary branches and silique density. After QTL mapping in the DH line and immortalized popula‐ tions, they identified 29 common QTL between the two populations, suggesting that there are some chromosomal regions containing QTL for multiple traits. In another QTL mapping report, Fu et al., (2007) constructed a genetic map in *B. napus* to map gene loci controlling the yellow seeded trait. They developed 420 SSR, RAPD and SRAP markers and assembled two genetic maps, of which one contained 26 linkage groups and another which had 20 linkage groups. After QTL analysis, they identified 19 QTL with one common in the two mapping populations. Further analysis allowed them to identify the collinear genomic region of chro‐

In cotton, Lin et al., (2005) developed a mapping population by crossing *G. hirsutum* and *G. barbadense* and performed QTL mapping with a genetic map constructed with SRAP, SSR and RAPD markers. Using 437 SRAP, 107 RAPD and 205 SSR markers, they constructed a genetic map with 566 markers assembled into 41 linkage groups, of which 28 were assigned to the corresponding known chromosomes. This genetic map was used to perform QTL mapping for fiber traits. In total, 13 QTL for fibre traits including two QTL for fibre strength, four QTL for fibre length and seven QTL for micronaire value were identified, of which six QTL were assigned into the A-subgenome, another six QTL into the D-subgenome while one QTL was not assigned. Three QTL for micronaire value were identified to cluster on linkage group 1, suggesting that the flanking molecular markers of these three QTL might

Similarly, Zhang et al., (2009b) reported on QTL mapping in cotton using SRAP and other markers. They assembled a genetic map containing 509 SSR, 58 intron targeted intron/exon splice junction (IT-ISJ), 29 SRAP and 8 morphological loci in 60 linkage groups. Among these 60 linkage groups, 54 were assigned into 26 chromosomes. This genetic map was used to identify QTL for fiber quality traits in five environments. In total, thirteen QTL including four QTL for fiber length, two QTL for fiber strength, two QTL for fiber fineness, three QTL for fiber length uniformity, and two QTL for fiber elongation were identified. Eleven out of 13 QTL were assigned into the A-subgenome and other two QTL, into the D-subgenome.

In chrysanthemum (*Dendranthema morifolium*), Zhang et al., (2011b) performed QTL map‐ ping of inflorescence-related traits using SRAP markers. They constructed two genetic maps

mosome 5 in Arabidopsis.

28 Plant Breeding from Laboratories to Fields

be useful in marker-assisted selection for this trait.

SRAP technology has several merits for gene tagging. Since SRAP detection uses unlimited primer combinations and there are multiple loci detected in a single SRAP PCR reaction, SRAP technology is advantageous over other molecular marker systems for gene tagging. After many genetic loci in a genome are screened quickly, closely linked SRAP markers to a trait of interest can be identified easily. We intensively used SRAP to perform gene tagging and cloning in Brassica species and worked on several economically important traits such as yellow-seeded canola and rapeseed, disease resistance and glucosinolates.

Yellow-seeded oilseeds in Brassica species are suggested to be related to high oil content so it is worthwhile to characterize the genes controlling seed coat color. Using SRAP technolo‐ gy, we cloned and characterized a gene controlling seed coat color and plant hairiness traits in *B. rapa* (Zhang et al., 2009a). We used 1,100 SRAP primer combinations to screen pooled DNA from yellow-seeded and black-seeded individuals based on the bulk segregant analy‐ sis (BSA) strategy and found 48 SRAP primer combinations that produced polymorphic loci in the pooled DNA samples from yellow-seeded and black-seeded individuals. Then we tested more pooled DNA and identified 13 SRAP markers that were linked to the gene of interest. Sequencing these SRAP markers allowed the identification of a chromosomal re‐ gion that was further used to develop new SCAR markers. With new SCAR markers and chromosome walking, we eventually identified the candidate gene and characterized the gene by complementary transformation of the corresponding mutant in Arabidopsis. The functional copy of the candidate gene recovered the phenotype of the Arabidopsis mutant and the non-functional copy in *B. rapa* mutant did not so the candidate was confirmed to underpin the yellow-seeded mutation in *B. rapa*.

Similarly, we used SRAP to tag other genes controlling the seed-coat color trait in yellow sarson, another yellow-seeded *B. rapa* and also in yellow-seeded *B. napus* canola (Rahman et

al., 2007, 2010). Yellow sarson is bright yellow-seeded and there is no color variation under different environments while all yellow-seeded canola accessions developed with yellow sarson are not pure yellow and the seed coat color varies due to differences in maturity and environments. Using SRAP screening, we identified several SRAP markers linked to one seed coat color gene. After extended flanking regions were sequenced, one closely-linked SRAP marker was successfully converted into SNP and SCAR markers. Meanwhile, we ana‐ lyzed a yellow-seeded canola line that was developed with yellow sarson and found that three genes controlled the yellow-seeded color in this *B. napus* line (Rahman et al., 2010). We identified one SRAP marker that was linked closely to one seed color locus and confirmed that this locus was located on linkage group N9 of our ultradense genetic map of *B. napus*. The second locus was mapped on linkage group N13 of our ultradense genetic map. To identify SRAP markers for the third locus, we screened 768 SRAP primer combinations and eventually found one SRAP that was linked closely to the yellow seeded color locus. These SRAP, SNP and SCAR markers can be used in marker assisted selection of yellow-seeded trait in oilseed crops of *B. rapa* and *B. napus*.

SRAP technology is an effective molecular marker system to analyze qualitative and quantitative resistance to plant diseases. In general, qualitative and quantitative resistan‐ ces are conferred by oligogenic or multigenic loci, respectively. In canola, blackleg is a major disease and qualitative resistance is available. We used the previously described ultradense genetic map to tag resistance genes to blackleg in *B. napus*. After screening 384 SRAP primer combinations, we identified two SRAP markers that were linked to a blackleg resistance gene. By compared the linked SRAP markers with the molecular markers on the ultradense *B. napus* genetic map that was constructed with another map‐ ping population, we found that one SRAP marker corresponded to a SRAP marker on N10. Therefore we took the flanking SRAP markers of the mapped resistance locus on N10 and identified other SRAP markers on the genetic map that were also polymorphic in the mapping population of the blackleg resistance gene. Eventually, further analysis allowed us to identify two blackleg resistance genes in the region where one resistance gene was suggested by other researchers (Long et al., 2010).

In several reports, SRAP markers were used to map genes controlling resistance to plant dis‐ eases in several crop species. For instance, Yi et al., (2008) used SRAP, STS and SSR markers to tag a resistance gene (*Pm4b*) to powdery mildew in wheat. They tested 240 SRAP primer combinations and identified two SRAP markers linked to the *Pm4b* gene. Eventually, they mapped the *Pm4b* gene on chromosome 2AL that was flanked by SRAP, STS and SSR mark‐ ers. In another study on gene mapping in wheat, Chen et al., (2012) used SSR, SRAP and TRAP markers to tag a wheat strip rust resistance gene. Using 400 SSR, 315 pairs of SRAP primers, and 40 pairs of TRAP primers to screen F1, F2 and BC1 mapping populations, they constructed a fine map flanking the resistance gene locus on chromosome arm 2AS and sug‐ gested that the mapped resistance gene should be a novel one.

In rice, Zhao et al., (2010) searched for SSR markers linked to a dominant resistance gene (RSV1) to rice stripe virus and then used the SRAP method to find closely linked markers. They located RSV1 into a region flanked by SSR and SRAP markers. In maize, a new domi‐ nant resistance gene to maize head smut was tagged by SSR-BSA and SRAP-BSA methods (Li et al., 2012). Closely linked molecular markers were identified and used to transfer the resistance gene from the resistant source to elite lines via marker assisted selection to breed head smut resistant hybrid cultivars in maize.

al., 2007, 2010). Yellow sarson is bright yellow-seeded and there is no color variation under different environments while all yellow-seeded canola accessions developed with yellow sarson are not pure yellow and the seed coat color varies due to differences in maturity and environments. Using SRAP screening, we identified several SRAP markers linked to one seed coat color gene. After extended flanking regions were sequenced, one closely-linked SRAP marker was successfully converted into SNP and SCAR markers. Meanwhile, we ana‐ lyzed a yellow-seeded canola line that was developed with yellow sarson and found that three genes controlled the yellow-seeded color in this *B. napus* line (Rahman et al., 2010). We identified one SRAP marker that was linked closely to one seed color locus and confirmed that this locus was located on linkage group N9 of our ultradense genetic map of *B. napus*. The second locus was mapped on linkage group N13 of our ultradense genetic map. To identify SRAP markers for the third locus, we screened 768 SRAP primer combinations and eventually found one SRAP that was linked closely to the yellow seeded color locus. These SRAP, SNP and SCAR markers can be used in marker assisted selection of yellow-seeded

SRAP technology is an effective molecular marker system to analyze qualitative and quantitative resistance to plant diseases. In general, qualitative and quantitative resistan‐ ces are conferred by oligogenic or multigenic loci, respectively. In canola, blackleg is a major disease and qualitative resistance is available. We used the previously described ultradense genetic map to tag resistance genes to blackleg in *B. napus*. After screening 384 SRAP primer combinations, we identified two SRAP markers that were linked to a blackleg resistance gene. By compared the linked SRAP markers with the molecular markers on the ultradense *B. napus* genetic map that was constructed with another map‐ ping population, we found that one SRAP marker corresponded to a SRAP marker on N10. Therefore we took the flanking SRAP markers of the mapped resistance locus on N10 and identified other SRAP markers on the genetic map that were also polymorphic in the mapping population of the blackleg resistance gene. Eventually, further analysis allowed us to identify two blackleg resistance genes in the region where one resistance

In several reports, SRAP markers were used to map genes controlling resistance to plant dis‐ eases in several crop species. For instance, Yi et al., (2008) used SRAP, STS and SSR markers to tag a resistance gene (*Pm4b*) to powdery mildew in wheat. They tested 240 SRAP primer combinations and identified two SRAP markers linked to the *Pm4b* gene. Eventually, they mapped the *Pm4b* gene on chromosome 2AL that was flanked by SRAP, STS and SSR mark‐ ers. In another study on gene mapping in wheat, Chen et al., (2012) used SSR, SRAP and TRAP markers to tag a wheat strip rust resistance gene. Using 400 SSR, 315 pairs of SRAP primers, and 40 pairs of TRAP primers to screen F1, F2 and BC1 mapping populations, they constructed a fine map flanking the resistance gene locus on chromosome arm 2AS and sug‐

In rice, Zhao et al., (2010) searched for SSR markers linked to a dominant resistance gene (RSV1) to rice stripe virus and then used the SRAP method to find closely linked markers. They located RSV1 into a region flanked by SSR and SRAP markers. In maize, a new domi‐

trait in oilseed crops of *B. rapa* and *B. napus*.

30 Plant Breeding from Laboratories to Fields

gene was suggested by other researchers (Long et al., 2010).

gested that the mapped resistance gene should be a novel one.

In eggplant, Mutlu et al., (2008) tagged a Fusarium wilt resistance gene using SRAP, SRAP-RGA, RAPD, and SCAR markers. They used 2316 primer combinations to identify molecular markers linked to the resistance gene, of which two SRAP markers were closely linked to the resistance gene. The SRAP markers were converted into SCAR markers and used in marker assisted selection of the Fusarium wilt resistance in eggplant.

Besides plant disease resistance, genes underpinning other traits have been tagged using SRAP technology, For instance, genes controlling two important traits, sex determination and tuberculate fruit in cucumber were tagged using SRAP technology (Li et al., 2008; Zhang et al., 2010a). In cucumber, there are three major gene loci, F/f, M/m, and A/a that determine various sex types. Li et al., (2008) analyzed M/m gene locus and identified 8 SRAP markers linked to this gene locus. Additionally, they used SRAP markers to per‐ form chromosome walking and converted some SRAP markers to co-dominant SCAR markers through sequencing SRAP fragments. Eventually they identified very closely linked SRAP markers at a genetic distance of less than one cM. Similarly, Zhang et al., (2010a) performed gene tagging of cucumber tuberculate fruit. They found that the tuber‐ culate fruit (Tu) was controlled by a dominant gene and used a BSA strategy to identify molecular markers linked to this dominant gene. After testing 736 SRAP primer combi‐ nations, they found 9 SRAP markers that were linked to the Tu gene and used SSR markers to anchor this gene on chromosome 5, further indicating that they would use the mapping results to clone the Tu gene later.

Male sterility is a commonly used method to produce hybrid seeds for exploiting hetero‐ sis in crops. Since genic male sterility is usually controlled by a few genes, SRAP tech‐ nology is useful to tag the genes underpinning male sterility. For example, Zhang et al., (2011c) used SRAP and SSR markers to tag a dominant genic male sterile gene in *B. oler‐ acea*. They performed BSA analysis with SRAP and SSR markers. By screening polymor‐ phisms between fertile bulks and sterile bulks with 26,417 SRAP primer pairs, they identified 14 SRAP markers that were linked to the male sterility gene MS-cd1. After se‐ quencing the SRAP fragments, three SRAP markers were converted into SCAR markers that were very closely linked to the MS-cd1 gene. Moreover, through comparative ge‐ nomics with SRAP sequences, they identified a collinear region on chromosome A10 in *B. rapa* corresponding to a collinear genomic region of chromosome 5 in Arabidopsis, which could lead to cloning of this gene in the future.

SRAP technology has also been used to tag quantitative traits using the same approaches as described previously in qualitative traits. In alfalfa, Castonguay et al., (2010) used SRAP to identify polymorphic genetic loci that controlled superior tolerance to freezing. Through BSA analysis, they found four SRAP markers that were associated with freezing tolerance and the frequency of their occurrence reflected changes in response to selection. In another report, SRAP was used to tag a major QTL controlling cadmium accumulation in oat (Tan‐ huanpaa et al 2007). The concentration of toxic cadmium in oat grains is often over the ac‐ cepted limit and must be reduced. SRAP, RAPD and retrotransposon-microsatellite amplified polymorphism (REMAP) markers were used to perform BSA analysis in an F*2* population and four molecular markers were identified to be associated with cadmium con‐ centration in oat grains. All these four markers were located on the same linkage group, sug‐ gesting that this mapped QTL had major effect on grain cadmium concentration in oat.
