Molecular breeding of flower load related traits in dioecious autotetraploid Actinidia arguta.

Flowering plants exhibit a wide range of sexual reproduction systems, with the majority being hermaphroditic. However, some plants, such as Actinidia arguta (kiwiberry), have evolved into dioecious species with distinct female and male vines. In this study, we investigated the flower load and growth habits of female kiwiberry genotypes to identify the genetic basis of high yield with low maintenance requirements. Owing to the different selection approaches between female and male genotypes, we further extended our study to male kiwiberry genotypes. By combining both investigations, we present a novel breeding tool for dioecious crops. A population of A. arguta seedlings was phenotyped for flower load traits, in particular the proportion of non-floral shoots, proportion of floral shoots, and average number of flowers per floral shoot. Quantitative trait locus (QTL) mapping was used to analyse the genetic basis of these traits. We identified putative QTLs on chromosome 3 associated with flower-load traits. A pleiotropic effect of the male-specific region of the Y chromosome (MSY) on chromosome 3 affecting flower load-related traits between female and male vines was observed in an A. arguta breeding population. Furthermore, we utilized Genomic Best Linear Unbiased Prediction (GBLUP) to predict breeding values for the quantitative traits by leveraging genomic data. This approach allowed us to identify and select superior genotypes. Our findings contribute to the understanding of flowering and fruiting dynamics in Actinidia species, providing insights for kiwiberry breeding programs aiming to improve yield through the utilization of genomic methods and trait mapping. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01476-7.

Implementation of different relationship estimate methodologies in breeding value prediction in kiwiberry (Actinidia arguta).

In dioecious crops such as Actinidia arguta (kiwiberries), some of the main challenges when breeding for fruit characteristics are the selection of potential male parents and the long juvenile period. Currently, breeding values of male parents are estimated through progeny tests, which makes the breeding of new kiwiberry cultivars time-consuming and costly. The application of best linear unbiased prediction (BLUP) would allow direct estimation of sex-related traits and speed up kiwiberry breeding. In this study, we used a linear mixed model approach to estimate narrow sense heritability for one vine-related trait and five fruit-related traits for two incomplete factorial crossing designs. We obtained BLUPs for all genotypes, taking into consideration whether the relationship was pedigree-based or marker-based. Owing to the high cost of genome sequencing, it is important to understand the effects of different sources of relationship matrices on estimating breeding values across a breeding population. Because of the increasing implementation of genomic selection in crop breeding, we compared the effects of incorporating different sources of information in building relationship matrices and ploidy levels on the accuracy of BLUPs' heritability and predictive ability. As kiwiberries are autotetraploids, multivalent chromosome formation and occasionally double reduction can occur during meiosis, and this can affect the accuracy of prediction. This study innovates the breeding programme of autotetraploid kiwiberries. We demonstrate that the accuracy of BLUPs of male siblings, without phenotypic observations, strongly improved when a tetraploid marker-based relationship matrix was used rather than parental BLUPs and female siblings with phenotypic observations. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01419-8.

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.

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

UNLABELLED: 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.

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

Non-preferential chromosome pairing was identified in tetraploid Actinidia chinensis and a higher mean multivalent frequency in pollen mother cells was found in colchine-induced tetraploids of A. chinensis compared with naturally occurring tetraploids. Diploid 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.

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

BACKGROUND: Red 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. RESULTS: Four 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. CONCLUSIONS: The 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.

Induced polyploidy dramatically increases the size and alters the shape of fruit in Actinidia chinensis.

BACKGROUND AND AIMS: Some otherwise promising selections of Actinidia chinensis (kiwifruit) have fruit that are too small for successful commercialization. We have therefore made the first detailed study in diploid kiwifruit of the effects of chromosome doubling induced by colchicine on fruit size, shape and crop loading. METHODS: Flow cytometric analysis of young leaves and chromosome analysis of flower buds and root tips was used to confirm the stability of induced autotetraploids. Fruit weight, size and crop load were measured in the third year after planting in the field and for three consecutive years. DNA fingerprinting was used to confirm the origin of the material. KEY RESULTS: There was a very significant increase in fruit size in induced autotetraploids of different genotypes of A. chinensis. With the commercially important diploid cultivar 'Hort16A', most regenerants, Type A plants, had fruit which were much the same shape as fruit of the diploid but, at the same fruit load, were much larger and heavier. Some regenerants, Type B plants, produced fruit similar to 'fasciated' fruit. Fruit of the autotetraploids induced from three female red-fleshed A. chinensis selections were also 50-60 % larger than fruit of their diploid progenitors. The main increase in fruit dimensions was in their diameters. These improved fruit characteristics were stable over several seasons. CONCLUSIONS: Chromosome doubling has been shown to increase significantly fruit size in autotetraploid A. chinensis, highlighting the considerable potential of this technique to produce new cultivars with fruit of adequate size. Other variants with differently shaped fruit were also produced but the genetic basis of this variation remains to be elucidated. Autoploids of other Actinidia species with commercial potential may also show improved fruit characteristics, opening up many new possibilities for commercial development.

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.

A gene-rich linkage map in the dioecious species Actinidia chinensis (kiwifruit) reveals putative X/Y sex-determining chromosomes.

BACKGROUND: The genus Actinidia (kiwifruit) consists of woody, scrambling vines, native to China, and only recently propagated as a commercial crop. All species described are dioecious, but the genetic mechanism for sex-determination is unknown, as is the genetic basis for many of the cluster of characteristics making up the unique fruit. It is, however, an important crop in the New Zealand economy, and a classical breeding program would benefit greatly by knowledge of the trait alleles carried by both female and male parents. The application of marker assisted selection (MAS) in seedling populations would also aid the accurate and efficient development of novel fruit types for the market. RESULTS: Gene-rich female, male and consensus linkage maps of the diploid species A. chinensis have been constructed with 644 microsatellite markers. The maps consist of twenty-nine linkage groups corresponding to the haploid number n = 29. We found that sex-linked sequence characterized amplified region (SCAR) markers and the 'Flower-sex' phenotype consistently mapped to a single linkage group, in a subtelomeric region, in a section of inconsistent marker order. The region also contained markers of expressed genes, some of unknown function. Recombination, assessed by allelic distribution and marker order stability, was, in the remainder of the linkage group, in accordance with other linkage groups. Fully informative markers to other genes in this linkage group identified the comparative linkage group in the female map, where recombination ratios determining marker order were similar to the autosomes. CONCLUSION: We have created genetic linkage maps that define the 29 linkage groups of the haploid genome, and have revealed the position and extent of the sex-determining locus in A. chinensis. As all Actinidia species are dioecious, we suggest that the sex-determining loci of other Actinidia species will be similar to that region defined in our maps. As the extent of the non-recombining region is limited, our result supports the suggestion that the subtelomeric region of an autosome is in the early stages of developing the characteristics of a sex chromosome. The maps provide a reference of genetic information in Actinidia for use in genetic analysis and breeding programs.

Ribosomal DNA locus evolution in Nemesia: transposition rather than structural rearrangement as the key mechanism?

We investigated chromosome evolution in Nemesia using fluorescent in-situ hybridization (FISH) to identify the locations of 5S and 45S (18-26S) ribosomal genes. Although there was conservation between Nemesia species in chromosome number, size and centromere position, there was large variation in both number and position of ribosomal genes in different Nemesia species (21 different arrangements of 45S and 5S rRNA genes were observed in the 29 Nemesia taxa studied). Nemesia species contained between one and three pairs of 5S arrays and between two and four pairs of 45S arrays. These were either sub-terminally or interstitially located and 45S and 5S arrays were often located on the same chromosome pair. Comparison of the positions of rDNA arrays with meiotic chromosome behaviour in interspecific hybrids of Nemesia suggests that some of the changes in the positions of rDNA have not affected the surrounding chromosome regions, indicating that rDNA has changed position by transposition. Chromosome evolution is frequently thought to occur via structural rearrangements such as inversions and translocations. We suggest that, in Nemesia, transposition of rDNA genes may be equally if not more important in chromosome evolution.