Genetic and cytological analyses reveal the recombination landscape of a partially differentiated plant sex chromosome in kiwifruit.

BACKGROUND: Angiosperm sex chromosomes, where present, are generally recently evolved. The key step in initiating the development of sex chromosomes from autosomes is the establishment of a sex-determining locus within a region of non-recombination. To better understand early sex chromosome evolution, it is important to determine the process by which recombination is suppressed around the sex determining genes. We have used the dioecious angiosperm kiwifruit Actinidia chinensis var. chinensis, which has an active-Y sex chromosome system, to study recombination rates around the sex locus, to better understand key events in the development of sex chromosomes. RESULTS: We have confirmed the sex-determining region (SDR) in A. chinensis var. chinensis, using a combination of high density genetic mapping and fluorescent in situ hybridisation (FISH) of Bacterial Artificial Chromosomes (BACs) linked to the sex markers onto pachytene chromosomes. The SDR is a subtelomeric non-recombining region adjacent to the nucleolar organiser region (NOR). A region of restricted recombination of around 6 Mbp in size in both male and female maps spans the SDR and covers around a third of chromosome 25. CONCLUSIONS: As recombination is suppressed over a similar region between X chromosomes and between and X and Y chromosomes, we propose that recombination is suppressed in this region because of the proximity of the NOR and the centromere, with both the NOR and centromere suppressing recombination, and this predates suppressed recombination due to differences between X and Y chromosomes. Such regions of suppressed recombination in the genome provide an opportunity for the evolution of sex chromosomes, if a sex-determining locus develops there or translocates into this region.

Estimation of allele frequencies in polyploids under certain patterns of inheritance.

Allele frequencies have long been studied by biologists interested in evolution and speciation. More recently, with the application of molecular markers in human DNA profiling we have also seen the need for reliable population allele frequency estimates for making probabilistic inferences. There is now interest in applying the same DNA profiling technology to identification of plant varieties. HortResearch maintains a large germplasm of horticultural plant species. It is becoming evident that accurate identification of these accessions through DNA fingerprinting is essential for effective utilisation and maintenance of this germplasm. Microsatellites are the markers of choice for this fingerprinting. However, such markers do not reveal the dosage of alleles in a polyploid. Polyploidy is common amongst horticultural plants. Estimating allele frequencies in a polyploid population is, therefore, complicated because of some marker genotypes being phenotypically indistinguishable. For example, in a tetraploid, with four alleles at a locus showing polysomic inheritance, although 35 genotypes are possible, these will fall into only 15 marker phenotypic classes. Furthermore 'null' individuals are rarely detected in polyploids. Furthermore, some polyploids can be cryptic exhibiting disomy, instead of the polysomic inheritance. We will discuss the implications of these factors and present an EM-type algorithm for estimating allele frequencies of a polyploid population under certain patterns of inheritance. The method will be demonstrated on simulated data. We also discuss the nature of some of the additional problems that may be encountered with estimating allele frequencies in polyploids for which other solutions still need to be developed.

Cross-species amplification of microsatellite loci within the dioecious, polyploid genus Actinidia (Actinidiaceae).

Microsatellite marker transfer across species in the dioecious genus Actinidia (kiwifruit) could offer an efficient and time-effective technique for use during trait transfer for vine and fruit improvement in breeding programmes. We evaluated the cross-species amplification of 20 EST-derived microsatellite markers that were fully informative in an Actinidia chinensis mapping family. We tested all 20 markers on 120 genotypes belonging to 21 species, 5 with varieties and/or chromosome races. These 26 taxa included 16 diploids, 7 tetraploids, 2 hexaploids and 1 octaploid, and represented all four taxonomic sections in the genus. All 20 markers showed some level of cross-species amplification. The most successful marker amplified in all genotypes from all species from all sections of the genus, the least successful amplified fragments only in A. chinensis and A. deliciosa. One species, A. glaucophylla, failed to amplify with all but 2 markers. PIC (Polymorphism information content) values were high, with 14 of 17 markers recording values of 0.90 and above. Sequence data demonstrated the presence of the microsatellite in all the amplified products. Sequence homology was less 5' of the microsatellite and increased toward the start codon of the translated region of the EST from which the marker was derived. The data confirm that EST-derived microsatellite markers from Actinidia species show cross-species amplification with high levels of polymorphism which could make them useful markers in breeding programmes.

EST-derived microsatellites from Actinidia species and their potential for mapping.

To increase the speed and reduce the cost of constructing a genetic map of Actinidia species (kiwifruit), for use in both breeding and functional genomics programmes, we sampled microsatellites from expressed sequence tags (ESTs) to evaluate their frequency of occurrence and level of polymorphism. Perfect dinucleotide repeats were the microsatellites selected, and these were found to be numerous in both the 5' and 3' ends of the genes represented. The microsatellites were of various lengths, the majority being repeats with the pattern (CT)(n)/(GA)(n). One hundred and fifty microsatellites, each with more than 10 dinucleotide repeat units, were chosen as possible markers, and when these were amplified, 93.5% were found to be polymorphic and segregating in a mapping population, with 22.6% amplifying more than one locus. Four marker categories were identified. Fully informative markers made up 27% of the total, 36.2% were female informative, 25.8% were male informative and 10% partly informative. The mapping population was an intraspecific cross in the diploid species Actinidia chinensis, with parents chosen for their diversity in fruit and plant characteristics, and for their geographical separation. Linkage was tested using the software 'Joinmap' and a LOD value of 3. The distribution of the EST-based markers over the linkage groups obtained appeared to be random, taking into consideration the small sample size, that the number of linkage groups (31) exceeded the chromosome number of n=29, and that a number of markers were not assigned to any group. Some microsatellite markers which amplified more than one locus mapped to separate linkage groups. According to our study in A. chinensis, EST-derived microsatellites give large numbers of possible markers very quickly and at reasonable cost. The markers are highly polymorphic, segregate in the mapping population, and increase the value of the genomic map by providing some functional information.