New Zealand has a long history of interest in the genus Actinidia, being the country which commercialized the fruit of Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson var. deliciosa, as kiwifruit, and which recently released Actinidia chinensis Planch. var. chinensis 'Hort16A', the gold-fleshed kiwifruit, as an alternative cultivar. While New Zealand was instrumental in bringing these fruits to commercial attention, the genus is native to China and neighbouring countries where more than 60 species are known. This germplasm is relatively unexplored in terms of horticultural development of new and novel cultivars and offers a huge range of fruit characters and 'eating attributes', and plants suited to a wide range of climatic conditions. The diversity of flavours, fragrances, colours, and health factors are also of interest in genomic studies, offering the possibility of defining chemical pathways and identifying gene function.
species present challenges to research and breeding. All known species in the genus are dioecious. Female plants bear flowers that are hermaphroditic in appearance but produce only empty pollen grains, while male plants have flowers that are unisexual with numerous stamens surrounding a rudimentary pistil whose growth is suppressed before style elongation or ovule initiation. Full dioecism is shown by about 4% of seed plants, and a second group display a variety of sub-dioecious conditions [1
]. Genetic studies have shown that dioecy has evolved many times in plants, and have demonstrated a variety of sex-determining systems [2
]. In Actinidia
, bulk segregant analysis with random amplified polymorphic DNA (RAPD) markers supported the hypothesis that sex-determining genes were localized in a pair of chromosomes that function like an XX/XY system with male heterogamety [4
]. The small size (<1 μm) of the chromosomes has made cytological studies difficult with the techniques available, and sex-determining chromosomes have not been positively identified. He et al.
] using an improved chromosome binding technique, analyzed the karyotypes of diploid A. chinensis
at the primary differentiation stage and reported that the sex chromosomes could not be identified from karyotypes of somatic cells. However, when they examined the pachytene stage of pollen mother cell meiosis, all 29 pairs of homologous chromosomes of pistillate and staminate plants paired tightly, except for a pair of nucleolar (SAT-) chromosomes in staminate plants. The two SAT-chromosomes were similar in length and shape, but in staminate plants the SAT region, about 15% of the total nucleolar chromosomal length, did not pair. He et al
] suggested the SAT region of nucleolar chromosomes may be the region of sex determination. They also suggested that sex chromosomes were probably at an early stage of differentiation in Actinidia
The DNA content of the 2C genome of A. chinensis
measured by flow cytometry was reported to be 1.3 – 1.4 pg [8
], which corresponds to about 1.3 × 109
bp per genome. The genus contains species that form a polyploid series from diploid to octoploid [9
As kiwifruit is a relatively new crop, knowledge of its genetic make-up is limited, so the development of a comprehensive genetic map and the use of molecular markers have the potential to improve efficiency in breeding new cultivars. A map will also help to simplify genomic studies to identify and isolate genes. Genetic linkage maps based on the recombination values of molecular markers have been constructed in an increasing number of plants (tomato [10
], rice [12
], barley, [13
], lotus [14
], cotton [16
], grape [17
]) and are proving valuable tools for plant breeding. The construction of a genetic map in an obligate outbreeding species, such as A. chinensis
, is more complex than one derived from inbred or homozygous parents. Maps in outbreeding species have been developed by utilising the two-way pseudo-testcross procedure [18
], where the mapping population is the F1
progeny of a cross between unrelated, highly heterozygous individuals. Constructing the linkage map is complicated, as the two-way pseudo-test cross may segregate for up to four alleles at any locus, with one or both parents heterozygous at any given locus. The linkage phase of the markers will often be unknown, and can be different for the two parents, which can lead to inaccuracies in the estimation of recombination frequencies [20
]. The recombination frequencies can, however, be separately estimated for each parent so that two maps are developed, and these maps can be integrated using markers that are heterozygous in both parents. Two low density linkage maps have been reported in Actinidia
. Testolin et al.
] used the progeny of an interspecific cross between A. chinensis
and A. callosa
to construct, at a LOD score ≥ 2.0, a female map of 203 loci over 38 linkage groups and a male map of 143 loci over 30 linkage groups.
While marker systems such as restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), random amplified polymorphic DNA (RAPDs), or single nucleotide polymorphisms (SNPs) have been developed to facilitate genetic mapping and gene discovery, the marker system of choice in many plant species is microsatellites (simple sequence repeats or SSRs). Microsatellites are arrays of short tandem repeat motifs of 1 to 5 base pairs in length which are characterized by their abundance, their distribution in both non-coding and coding regions of eukaryotic genomes, reproducibility, Mendelian mode of inheritance and co-dominant nature [23
]. They are recognised as highly informative genetic markers because of their inherent variability. This hypervariability is due to the high mutation rate within the nucleotide sequences of the microsatellites, and increases with increasing number of tandem repeats. In humans, heterozygosities generally exceed 0.5 and range as high as 0.9, with as many as 50 alleles per locus [24
], and mutation rates, though variable among loci, exceed rates for non-microsatellite loci by up to four orders of magnitude [25
]. Similar hypervariability within microsatellite loci has been reported for birds, insects and plants, and loci may be polymorphic even in species where low levels of genetic diversity make alternate marker systems less useful [27
The time-consuming and expensive process of developing enriched genomic libraries and the subsequent sequencing and seeking of the simple sequence repeats is now often replaced with data mining of expressed sequence tag (EST) libraries to give a rapid, efficient and low-cost alternative for identifying microsatellites in plant species. Microsatellites have been found to occur regularly in ESTs [29
]. The frequency of occurrence of microsatellites of suitable length (20 nucleotides or more) varied in five cereals examined from 1.5% for maize to 4.7% for rice [30
]. This percentage would be sufficient to yield numerous markers from plant species in which large numbers of ESTs have been developed. In Actinidia
the frequency of occurrence and level of polymorphism of EST-derived di-nucleotide microsatellites were sampled and found to be numerous in both the 5' and 3' ends of the genes represented, and highly polymorphic (93.5%) in the mapping population [31
The construction of a single map for a cross in an outbreeding species, rather than two separate maps for the parents, depends on the availability of markers that are heterozygous in both parents. These markers form allelic bridges [19
]. Dominant markers such as RAPDs or AFLPs are generally of very limited use in combining parental maps, therefore, when the homologous linkage groups of the parents of a mapping population are required to be integrated, co-dominant markers such as microsatellites or RFLPs are the markers of choice, and allow the construction of either separate parental maps, or an integrated map for the cross [21
Here we present comprehensive genetic linkage maps of female and male informative markers mapped in a cross in the outbreeding species A. chinensis, and also an integrated map of the cross, achieved through the use of co-dominant microsatellite markers. The twenty nine linkage groups are defined, and the position of sex-determining loci identified. Genetic linkage maps in Actinidia have been developed to supply markers for breeding novel cultivars, to provide tools for comparative and quantitative trait mapping, and to investigate the evolution and function of genetic control mechanisms.