Markers for Specific Genes

Markers for several genes have been identi- fi ed in citrus. In many cases, the approach used was bulked segregant analysis (Michelmore et al., 1991) rather than com- plete mapping. In this strategy, a popula- tion in which the trait segregates is

identifi ed, and equal amounts of DNA from 6–8 individuals with contrasting pheno- types for the trait are combined to form two bulks. For example, with a disease resist- ance gene, one bulk is composed of resist- ant individuals, and one of susceptible individuals. These two samples are then screened for a large number of PCR-based markers. Only if a marker is closely linked to the trait gene is it likely that one bulk will have a specifi c marker band that is absent in all individuals of the other bulk. The basis of this approach is that a 1/8 dilu- tion of template DNA still allows detection of a band. Candidate markers are then tested on individual plant samples to deter- mine whether the marker–trait association is robust. It is effi cient to analyse two bulks of each phenotypic class to reduce the fre- quency of false positives. Often many can- didate markers are discarded based on analysis of these single-individual samples. For the remaining markers, the analysis is then extended to a larger population of 100 or more individuals to confi rm the associa- tion and estimate a map distance between the trait and marker. For traits determined by major genes, bulked segregant analysis is

quite effective in citrus. However, because the individual plants are not homozygous, only some linked markers are informative for dominant traits. For example, in an F2- type segregation, a dominant marker allele

(M) linked in repulsion phase to a trait allele (t) will not be discovered because the dominant phenotypic class (genotypes TT and Tt) will have marker genotypes mm and Mm. The recessive phenotypic class (tt) will have marker genotype MM and so the marker band will be observed in both trait classes.

Cheng and Roose (1995) located mark- ers for dwarfi ng by the rootstock Flying Dragon trifoliate orange, but these are not needed in breeding because the dwarfi ng trait appears associated with visible mark- ers, curved thorns and ‘zigzag’ stem growth. Additional research (M. L. Roose, unpub- lished) indicates that the dwarfi ng trait is not transmitted to progeny of citrus ´ Flying Dragon crosses. Markers for Ctv, the citrus tristeza virus resistance gene, in trifoliate orange have been developed by several groups (Gmitter et al., 1996; Mestre et al., 1997, Fang et al., 1998b), and markers very close to the resistance are available. Another gene for which markers have been identifi ed is acitric, a pummelo gene that reduces citric acid in fruit when heterozy- gous and nearly eliminates it when homozygous (Fang et al., 1998a). Markers for genes involved in nucellar embryony (Garcia et al., 1999), citrus nematode toler- ance (Ling et al., 2000) and salinity toler- ance (Tozlu et al., 1999b) have also been located. There is considerable ongoing work in this area, and it is likely that mark- ers for many additional traits will be reported in the future. Such markers are useful to the breeder in that they allow MAS as described below.