Paul M. Datson

Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red‐fleshed kiwifruit (Actinidia chinensis)

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.

An R2R3 MYB transcription factor determines red petal colour in an Actinidia(kiwifruit) hybrid population

BackgroundRed colour in kiwifruit results from the presence of anthocyanin pigments. Their expression, however, is complex, and varies among genotypes, species, tissues and environments. An understanding of the biosynthesis, physiology and genetics of the anthocyanins involved, and the control of their expression in different tissues, is required. A complex, the MBW complex, consisting of R2R3-MYB and bHLH transcription factors together with a WD-repeat protein, activates anthocyanin 3-O-galactosyltransferase (F3GT1) to produce anthocyanins. We examined the expression and genetic control of anthocyanins in flowers of Actinidia hybrid families segregating for red and white petal colour.ResultsFour inter-related backcross families between Actinidia chinensis Planch. var. chinensis and Actinidia eriantha Benth. were identified that segregated 1:1 for red or white petal colour. Flower pigments consisted of five known anthocyanins (two delphinidin-based and three cyanidin-based) and three unknowns. Intensity and hue differed in red petals from pale pink to deep magenta, and while intensity of colour increased with total concentration of anthocyanin, no association was found between any particular anthocyanin data and hue. Real time qPCR demonstrated that an R2R3 MYB, MYB110a, was expressed at significant levels in red-petalled progeny, but not in individuals with white petals.A microsatellite marker was developed that identified alleles that segregated with red petal colour, but not with ovary, stamen filament, or fruit flesh colour in these families. The marker mapped to chromosome 10 in Actinidia.The white petal phenotype was complemented by syringing Agrobacterium tumefaciens carrying Actinidia 35S::MYB110a into the petal tissue. Red pigments developed in white petals both with, and without, co-transformation with Actinidia bHLH partners. MYB110a was shown to directly activate Actinidia F3GT1 in transient assays.ConclusionsThe transcription factor, MYB110a, regulates anthocyanin production in petals in this hybrid population, but not in other flower tissues or mature fruit. The identification of delphinidin-based anthocyanins in these flowers provides candidates for colour enhancement in novel fruits.

Meiotic chromosome pairing behaviour of natural tetraploids and induced autotetraploids of Actinidia chinensis

Key messageNon-preferential chromosome pairing was identified in tetraploidActinidia chinensisand a higher mean multivalent frequency in pollen mother cells was found in colchine-induced tetraploids ofA. chinensiscompared with naturally occurring tetraploids.AbstractDiploid and tetraploid Actinidia chinensis are used for the development of kiwifruit cultivars. Diploid germplasm can be exploited in a tetraploid breeding programme via unreduced (2n) gametes and chemical-induced chromosome doubling of diploid cultivars and selections. Meiotic chromosome behaviour in diploid A. chinensis ‘Hort16A’ and colchicine-induced tetraploids from ‘Hort16A’ was analysed and compared with that in a diploid male and tetraploid males of A. chinensis raised from seeds sourced from the wild in China. Both naturally occurring and induced tetraploids formed multivalents, but colchicine-induced tetraploids showed a higher mean multivalent frequency in the pollen mother cells. Lagging chromosomes at anaphase I and II were observed at low frequencies in the colchicine-induced tetraploids. To investigate whether preferential or non-preferential chromosome pairing occurs in tetraploid A. chinensis, the inheritance of microsatellite alleles was analysed in the tetraploid progeny of crosses between A. chinensis (4x) and A. arguta (4x). The frequencies of inherited microsatellite allelic combinations in the hybrids suggested that non-preferential chromosome pairing had occurred in the tetraploid A. chinensis parent.

Meiotic chromosome pairing in Actinidia chinensis var. deliciosa

Polyploids are defined as either autopolyploids or allopolyploids, depending on their mode of origin and/or chromosome pairing behaviour. Autopolyploids have chromosome sets that are the result of the duplication or combination of related genomes (e.g., AAAA), while allopolyploids result from the combination of sets of chromosomes from two or more different taxa (e.g., AABB, AABBCC). Allopolyploids are expected to show preferential pairing of homologous chromosomes from within each parental sub-genome, leading to disomic inheritance. In contrast, autopolyploids are expected to show random pairing of chromosomes (non-preferential pairing), potentially leading to polysomic inheritance. The two main cultivated taxa of Actinidia (kiwifruit) are A. chinensis (2x and 4x) and A. chinensis var. deliciosa (6x). There is debate whether A. chinensis var. deliciosa is an autopolyploid derived solely from A. chinensis or whether it is an allopolyploid derived from A. chinensis and one or two other Actinidia taxa. To investigate whether preferential or non-preferential chromosome pairing occurs in A. chinensis var. deliciosa, the inheritance of microsatellite alleles was analysed in the tetraploid progeny of a cross between A. chinensis var. deliciosa and the distantly related Actinidia eriantha Benth. (2x). The frequencies of inherited microsatellite allelic combinations in the hybrids suggested that non-preferential chromosome pairing had occurred in the A. chinensis var. deliciosa parent. Meiotic chromosome analysis showed predominantly bivalent formation in A. chinensis var. deliciosa, but a low frequency of quadrivalent chromosome formations was observed (1 observed in 20 pollen mother cells).

Rapid radiations of both kiwifruit hybrid lineages and their parents shed light on a two-layer mode of species diversification

Reticulate speciation caused by interspecific hybridization is now recognized as an important mechanism in the creation of biological diversity. However, depicting the patterns of phylogenetic networks for lineages that have undergone interspecific gene flow is challenging. Here we sequenced 25 taxa representing natural diversity in the genus Actinidia with an average mapping depth of 26× on the reference genome to reconstruct their reticulate history. We found evidence, including significant gene tree discordance, cytonuclear conflicts, and changes in genome-wide heterozygosity across taxa, collectively supporting extensive reticulation in the genus. Furthermore, at least two separate parental species pairs were involved in the repeated origin of the hybrid lineages, in some of which a further phase of syngameon was triggered. On the basis of the elucidated hybridization relationships, we obtained a highly resolved backbone phylogeny consisting of taxa exhibiting no evidence of hybrid origin. The backbone taxa have distinct demographic histories and are the product of recent rounds of rapid radiations via sorting of ancestral variation under variable climatic and ecological conditions. Our results suggest a mode for consecutive plant diversification through two layers of radiations, consisting of the rapid evolution of backbone lineages and the formation of hybrid swarms derived from these lineages.

Characterisation, evolutionary trends and mapping of putative resistance and defence genes in Actinidia (kiwifruit)

A virulent strain of Pseudomonas syringae actinidae has invaded kiwifruit (genus Actinidia) orchards in New Zealand, making the study of Actinidia resistance genes critical. Examination of expressed sequence tag (EST) libraries constructed in various species of Actinidia showed that, based on homologies to Arabidopsis, many families of resistance genes were represented. The mapping of these genes to establish their chromosomal positions on the Actinidia chinensis genetic linkage map was facilitated by designing 71 primer pairs to the EST sequences. The genetic markers mapped across 22 of the linkage groups, both as individuals and as members of a cluster. The clusters were generally homologous in nature, though heterologous examples were present. A sample of genes representing both the basal defence and the resistance-gene mediated defence pathways, here termed collectively ‘resistance genes’, was characterised. Chromosomal sites of resistance gene markers representing a range of such genes have been identified and can be considered during the selection of parents in breeding programmes.

Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome.

Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al. method: some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS) method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145) of BamH I sites shared with the reference genome, compared to only 14% (11,513) by stdGBS.

THE CONTROL OF KIWIFRUIT RED FLESH COLOUR

Red-fleshed fruit occur in a few taxa of the genus Actinidia. In such taxa, anthocyanins can accumulate in different parts of the fruit including the skin, the whole pericarp or only the inner pericarp. Differences in the relative amounts of cyanidin- and delphinidin-based anthocyanins account for the different shades of red observed. Red-fleshed fruit of A. chinensis accumulate mainly cyanidin 3-Oxylo-galactoside and cyanidin 3-O-galactoside, which usually are restricted to the inner pericarp of the fruit, creating a characteristic and colourful pattern in mature fruit. In order to understand the process of anthocyanin synthesis in Actinidia fruit, we have studied the expression of the anthocyanin biosynthetic genes identified in our Actinidia EST database. Of the different genes of the pathway, only the expression of one glycosyltransferase (F3GT1), confirmed functionally to be cyanidin 3-O-galactosyltransferase, was strongly correlated with red pigmentation of the fruit. However, inheritance of the gene could not be associated with the segregation of the red phenotype of the fruit, suggesting that other factors are able to regulate the biosynthetic pathway. We show that the F3GT1 gene is regulated by a MYB-bHLH mechanism; allelic variation on these genes is likely to account for the expression of the different phenotype.

Genome-Based Breeding

High-throughput genotyping enables genomic selection (GS) to be used as a tool for breeding kiwifruit. Genomic selection is a marker-assisted selection method in which genome-wide marker data are used to make phenotypic predictions. Kiwifruit breeding could benefit from GS either to select males for use as parents based on their genomic breeding values, for faster culling of seedlings, or to accelerate seedling selection into advanced trials. Methods to obtain high-coverage genotypes from kiwifruit for GS include genotyping-by-sequencing (GBS), restriction-associated DNA (RAD) sequencing, capture-based techniques and single nucleotide polymorphism (SNP) arrays. Genomic selection has been successfully applied to many diploid taxa, and a pilot study in diploid Actinidia chinensis var. chinensis has used GBS data. To apply GS to the autopolyploid kiwifruit populations successfully, improvements in detecting and analysing the genotype information are being made.

Hybridization amongst New Zealand Schoenus (Cyperaceae)

Artificial pollinations were made between five New Zealand species of Schoenus (Cyperaceae) to determine whether they could form interspecific hybrids. No hybrids were produced and the site of the interspecific incompatibility was investigated using fluorescence microscopy. The site of pollen inhibition varied in the different cross combinations. No relationship was observed between pollen size and subsequent pollen germination and tube growth in the style. The results do not support the claim that S. concinnus and S. nitens form hybrids in nature.