**8. Fine mapping and cloning of clubroot resistance genes**

As the whole genome sequencing and molecular marker development in Brassica species advances [92], those previously identified clubroot resistance loci in Chinese cabbage have been fine mapped and some clubroot resistance genes have been eventually cloned (Table 1). The *Crr3* locating on chromosome R3 was first mapped to a small genetic region between 0.35 cM genetic distance using 888 F2 individual plants [85]. In another report, the clubroot resistance locus *CRa* has been further analyzed to identify the candidate gene [93]. Over 1,600 F2 individual plants were used to select 80 recombinants using two closely linked molecular markers. Further analysis of those recombinants allowed identifying one open reading frame located on chromosome R3, which belongs to a typical resistance gene family and encodes a TIR-NBS-LRR protein [93]. More recently, there are two other independent reports that focused on fine mapping of clubroot resistance loci on chromosome R3. The *CRb* clubroot resistance locus which was described to be effective to *P. brassicae* isolates No. 14, a very aggressive isolate in Japan, has been fine mapped [94]. Using over 2,000 F2 individual plants and F3 progeny testing, 92 F2 recombinants between two closely linked molecular markers were identified. The analysis of these 92 F2 recombinants suggested that the *CRb* clubroot resistance locus might be the same as the *CRa* locus and the *CRa* and *CRb* clubroot resistance loci are different from the clubroot resistance locus *Crr3* [94]. Similarly, gene mapping of five Chinese cabbage cultivars was performed and all these hybrid cultivars were found to contain the same clubroot resistance locus on chromosome R3 [95]. They further fine mapped the clubroot resistance locus in Chinese cabbage to a 187 kilo-base pair (kb) chromosomal region using a large segregating population with over 8,000 individual plants. Molecular markers which are closely linked to the mapped clubroot resistance locus have been developed and those molecular markers can be used in marker-assisted selection to breed Chinese cabbage with clubroot resistance.

Characterization of clubroot resistance genes offers opportunities for further understanding clubroot resistance and interactions of resistance genes and pathogens. Hatakeyama et al. [86] cloned one clubroot resistance gene *Crr1a* on chromosome R8 and confirmed the resistance through plant transformation. Some transgenic *B. rapa* plants are resistant while others are susceptible, suggesting that the *Crr1a* gene might not explain the whole clubroot resistance in the original locus. They also found that *Crr1a* and *Crr1b* were tandem repeats in the same locus and both genes encode typical resistance gene proteins with TIR-NBS-LRR structures.

Based on the previous reports and whole genome sequencing data, clubroot resistance loci on chromosome 3 in *B. rapa* also contain multiple genes that encode TIR-NBS-LRR proteins. The complexity of those clubroot resistance loci needs to be investigated further. When a clubroot resistance locus contains multiple genes encoding the similar proteins, it becomes challenging to know how each individual gene plays a role in the clubroot resistance and how they contribute to the differences of alleles from various resistant sources. It is necessary to further dissect those complex clubroot resistance loci and investigate each individual gene to under‐ stand the functional properties of those loci. Therefore, gene functional analysis for clubroot resistance is still an important research focus in Brassica species.
