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Ctv contig






Fig. 13.3. BAC contigs of the CTV resistance gene region (Ctv contig) and its allelic susceptibility gene region (ctv contig).The insert sizes of these clones vary between 70 and 175 kb. Clones from the Bam HI, Hin dIII and transformation-competent artifi cial chromosome (TAC) libraries were named with the pre-fi x BC, HBC and TAC, respectively. The genetic map of the Ctv locus region was integrated with BAC contigs and is shown above the contigs.

 

 


sitic pest that causes severe damage to citrus roots and results in decreased pro- duction (Duncan and Cohn 1990). Selecting citrus nematode-resistant rootstocks is an important objective for citrus breeding. The traditional screening and selection approach is very labour intensive and inef- ficient. Molecular linkage markers have made it possible to understand the quanti- tative inheritance of citrus nematode resist- ance. Once their linkage with the nematode resistance was identifi ed and mapped, it became possible to perform early selection in citrus-resistant rootstock breeding pro- grammes, through the MAS process. It was found that most biotypes of Poncirus are resistant to citrus nematode (Cameron et al., 1954) and that the resistance is inher- ited in some fertile intergeneric hybrids from the trifoliate orange (Swingle and Reece 1967). An intergeneric backcross family from (Clementine mandarin (C. retic-


ulata) ´ Hamlin orange (C. sinensis), desig- nated LB6-2) ´ (Swingle citrumelo (C. para- disi ´ P. trifoliata)) was used to identify associated molecular markers and to evalu- ate the mode of inheritance of citrus nema- tode resistance.

It was critical to determine the pheno- type of citrus nematode resistance and sus- ceptibility segregating among individuals of this family. The individuals, including par- ents and hybrids, were inoculated at the same time to monitor the nematode popula- tion development with live citrus nema- todes directly collected from the heavily infected roots of fi eld citrus trees. The num- bers of citrus nematode females, eggs and juveniles were estimated by counting from nematode extractions prepared from root samples (Baines et al., 1968). Nematode extracts were taken three times from each replicate, and the mean number of female nematodes per gram of fresh root (females/g


 

 

 

Fig. 13.4. The phenotypic distribution (mean and standard deviation from six replicates) of 62 hybrids in response to citrus nematode inoculation. The infestation levels of the resistant (Swingle citrumelo) and susceptible parent (LB6-2) are indicated (Ling et al., 2000).

 


 

of root) was calculated for each hybrid. All the hybrids and parental plants were evalu- ated for their response to citrus nematode inoculation by counting the female nema- todes in each of the root samples. Data were subjected to analysis of variance using the MSTATC computer program.

Analysis of variance revealed that there were significant differences among the hybrids for the numbers of females/g of root (P < 0.001). The mean values among the hybrid individuals were contin- uously distributed in a wide range, indicat- ing the quantitative nature of the resis- tant trait (Fig. 13.4; Ling et al., 2000). The most resistant and susceptible individuals were assorted into groups by mean number of females/g of root to perform bulked segregant analysis (BSA) and identify the associated DNA markers, which resulted in a local map with 14 RAPD, SCAR


 

and RGC markers (Fig. 13.5; Ling et al., 2000).

Some 15 specifi c markers were devel- oped in the Tyr1 region and used to evalu- ate their potential in selecting for nematode resistance in a citrus rootstock breeding programme, by applying the markers to sev- eral populations and other individuals with known phenotype (unpublished data). This was done by screening the BAC library described above with NBS–LRR class RGC sequences; over 200 positive clones were identifi ed by two RGCs that mapped in the Tyr1 QTL region. A few of the BAC clones were found to be closely linked with Tyr1, when the primers based on the clones’ insert sequences yielded polymorphic frag- ments that were associated with the pheno- types in the population previously phenotyped by Ling et al. (2000). By apply- ing the primer walking approach, three


 

 

 

Fig. 13.5. The localized linkage map of the citrus nematode resistance gene region, generated by MAPMAKER/QTL. The map distances (cM) were calculated using the Kosambi function and are indicated between markers. The major citrus nematode resistance gene (Tyr1) region corresponded to the highest QTL peak. The LOD score value indicated that the Tyr1 region was most closely associated with marker loci OPO07650 and SCA07650. The co-segregating marker loci are indicated by solid bars.

 


complete NBS–LRR class resistance gene sequences were tagged and identifi ed sepa- rately in three BAC clones. More specifi c markers were developed from these tagged sequences and relatively high density genetic maps were constructed by incorpo- rating the newly developed markers and previously developed markers in the CN (citrus nematode) family. These markers are being applied in a larger mapping popula- tion to enrich the marker linkage map between the Ctv and Tyr1 region. In addi- tion, the utility for MAS for citrus nema- tode resistance is being explored (X. Xiang,

Q. Zheng, S. Huang, C. Chen, L. W. Duncan,

Z. Deng, K. D. Bowman and F. G. Gmitter, Jr, unpublished results).







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