R Genes and RGCs in the Citrus Genome – Features and Distributions

Clustering of R genes and RGCs

Clustering of citrus R genes was first observed in genetic mapping of Ctv and Tyr1 (Deng et al., 2000; Ling et al., 2000). Initially, a RAPD marker amplifi ed with random primer OP07 was identifi ed linked to the Ctv locus (Gmitter et al., 1996). In a separate effort toward mapping citrus nem- atode resistance with a separate population, this random primer yielded a DNA frag- ment linked to Tyr1 and apparently of the same size as the marker associated with Ctv. When the Ctv -linked RAPD fragment was cloned and converted into SCAR marker SCO07, its high specifi city allowed the con- fi rmation that the two RAPD markers iden- tifi ed in different mapping populations for two resistance traits were the same and that Ctv and Tyr1 are closely linked in coupling phase. This relationship between Ctv and Tyr1 has been confi rmed further in subse- quent mapping with a number of specifi c PCR markers derived from RGCs or BAC end sequences. The genetic distance between the two loci was estimated to be approximately 12 cM, using populations of fewer than 100 progeny individuals (Deng et al., 2000; Ling et al., 2000). A more pre- cise estimation is being attempted using a large population (X. Xiang, Q. Zheng, S. Huang, C. Chen, L. W. Duncan, Z. Deng,

K.D. Bowman and F.G. Gmitter, JR, unpub- lished results unpublished). The quantita-

tive inheritance of citrus nematode resist- ance may hinder pinpointing of Tyr1 on to a linkage map; consequently, the exact genetic location of Tyr1 will be not as well defi ned as that of Ctv, which creates diffi - culties for a precise estimation of the genetic distance.

Physical evidence of R gene and RGC clustering has also become available recently from BAC and BAC contig analy- sis. BAC clones contain genomic DNA inserts in the range of 35–300 kb, therefore they are ideal for examining gene or sequence clustering on a much fi ner scale compared with genetic mapping. In charac- terizing BACs identifi ed with NBS–LRR class RGCs, Deng et al. (2001a) found that more than 40% of the 29 BAC contain 2–4 copies of NBS–LRR class sequences within each BAC. Deng and Gmitter (2003) analysed 35 BACs identifi ed with receptor- like kinase class RGCs and recognized that approximately 50% of the clones each carry 2–3 copies of Xa21 -like sequences. The average size of these BACs is approximately 115 kb. Considering the close relationship between RGCs and R genes in plants, this clustering feature of RGCs in citrus BACs may serve as a good indication of possible R gene distribution in the citrus genome.

Sequencing of overlapping BAC clones at the Ctv locus and its immediate vicinity has revealed the existence of fi ve copies of CC–NBS–LRR type complete gene sequences, two copies of pseudogene sequences and nine resistance gene frag- ments (Yang et al., 2003). Similar gene con- tents have been predicted from sequencing BACs covering the Ctv locus but from a dif- ferent source (Z. Deng, S. Huang, C. Chen and F.G. Gmitter, Jr, unpublished). These data, therefore, have provided the most direct evidence of R gene or RGC clustering around a functional resistance gene locus in citrus.

It was first recognized that the Ctv–Tyr1 region may contain a major clus- ter of resistance genes in 2000 (Deng et al., 2000), based on the available data at that time (Fang et al., 1998; Deng et al., 2000; Ling et al., 2000). As more genetic mapping,

RGC cloning and BAC sequences accumu- late, this region seems to contain more R genes or RGCs than previously thought. An up-to-date count of R genes and R gene-like sequences includes up to 12 copies of NBS–LRR sequences and 3–4 copies of LRR sequences. Though the functions of most of these sequences remain to be determined, the discovery of this cluster may have important implications for disease resist- ance breeding.

Transposable elements and R genes

One surprising feature revealed in BAC clone sequencing was the rich presence of transposable elements around R gene sequences. Five copia -like and three gypsy - like retrotransposons, and two mutator-like transposons have been identifi ed within the 282 kb DNA at the Ctv locus (Yang et al., 2003). In addition, six small DNA fragments similar to parts of other transposable ele- ments are present within the 282 kb. The total length of these elements together is greater than 65 kb, i.e. accounting for approximately 23% of the nucleotides. In a sense, R gene sequences are surrounded by transposable elements at the Ctv locus.

Similar phenomena have been observed in other NBS–LRR sequence-rich BACs (C. Chen and F.G. Gmitter, Jr, unpub- lished; Z. Deng, S. Huang, C. Chen and F.G. Gmitter, Jr, unpublished). It seems that the NBS–LRR class R gene sequences are quite often associated with retrotransposable ele- ments in citrus. The presence of transpos- able elements with other classes of R gene sequences has not yet been characterized in citrus. It has been proposed that trans- posons might have participated in diversifi - cation of the Xa21 gene in rice for R gene evolution (Song et al., 1997). Arabidopsis has a small genome and a relatively low portion of transposable elements. In many plants with large genomes, transposable elements often contribute to most of their DNA contents. Compared with these plants, the citrus genome is quite small and has been expected to contain a relatively small

percentage of its DNA content. It remains to be determined whether transposable ele- ments contribute to R gene evolution in citrus and if they are widely distributed around other R genes in citrus.