Caihong Zhong

High-density interspecific genetic maps of kiwifruit and the identification of sex-specific markers

Kiwifruit (Actinidia chinensis Planchon) is an important specialty fruit crop that suffers from narrow genetic diversity stemming from recent global commercialization and limited cultivar improvement. Here, we present high-density RAD-seq-based genetic maps using an interspecific F1 cross between Actinidia rufa ‘MT570001’ and A. chinensis ‘Guihai No4’. The A. rufa (maternal) map consists of 2,426 single-nucleotide polymorphism (SNP) markers with a total length of 2,651 cM in 29 linkage groups (LGs) corresponding to the 29 chromosomes. The A. chinensis (paternal) map consists of 4,214 SNP markers over 3,142 cM in 29 LGs. Using these maps, we were able to anchor an additional 440 scaffolds from the kiwifruit draft genome assembly. Kiwifruit is functionally dioecious, which presents unique challenges for breeding and production. Three sex-specific simple sequence repeats (SSR) markers can be used to accurately sex type male and female kiwifruit in breeding programmes. The sex-determination region (SDR) in kiwifruit was narrowed to a 1-Mb subtelomeric region on chromosome 25. Localizing the SDR will expedite the discovery of genes controlling carpel abortion in males and pollen sterility in females.

Correlation between ploidy level and fruit characters of the main kiwifruit cultivars in China: implication for selection and improvement

Abstract To understand the relationship between ploidy levels and fruit characters in kiwifruit, the ploidy of 30 important Chinese cultivars is determined by flow cytometry and eight fruit characters are described following the UPOV descriptors. The provenance of the cultivars is noted. The type of fruit hairs, fruit flesh colour and fruit weight are found to be related to the ploidy. Cultivars at different ploidies vary in fruit hairs, ranging from downy to hispid. Diploid and tetraploid cultivars have mostly yellow or golden yellow fruit flesh, whereas hexaploid cultivars have mostly green fruit flesh. Partial correlation analysis indicates a positive correlation between ploidy and fruit weight. However, fruit shape, fruit skin colour, soluble solids content, vitamin C content and total acid content vary and are not significantly correlated to ploidy. Cultivars are predominantly selected from the Xuefeng mountains, Mufu mountains, Qinling mountains and Daba mountains, centres of morphological polymorphism and ploidy variation of Actinidia chinensis var. chinensis and A. chinensis var. deliciosa.

Maternal inheritance of mitochondrial genomes and complex inheritance of chloroplast genomes in Actinidia Lind.: evidences from interspecific crosses

The inheritance pattern of chloroplast and mitochondria is a critical determinant in studying plant phylogenetics, biogeography and hybridization. To better understand chloroplast and mitochondrial inheritance patterns in Actinidia (traditionally called kiwifruit), we performed 11 artificial interspecific crosses and studied the ploidy levels, morphology, and sequence polymorphisms of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of parents and progenies. Sequence analysis showed that the mtDNA haplotypes of F1 hybrids entirely matched those of the female parents, indicating strictly maternal inheritance of Actinidia mtDNA. However, the cpDNA haplotypes of F1 hybrids, which were predominantly derived from the male parent (9 crosses), could also originate from the mother (1 cross) or both parents (1 cross), demonstrating paternal, maternal, and biparental inheritance of Actinidia cpDNA. The inheritance patterns of the cpDNA in Actinidia hybrids differed according to the species and genotypes chosen to be the parents, rather than the ploidy levels of the parent selected. The multiple inheritance modes of Actinidia cpDNA contradicted the strictly paternal inheritance patterns observed in previous studies, and provided new insights into the use of cpDNA markers in studies of phylogenetics, biogeography and introgression in Actinidia and other angiosperms.

Origin and Evolution of the Kiwifruit Canker Pandemic.

Recurring epidemics of kiwifruit (Actinidia spp.) bleeding canker disease are caused by Pseudomonas syringae pv. actinidiae (Psa). In order to strengthen understanding of population structure, phylogeography, and evolutionary dynamics, we isolated Pseudomonas from cultivated and wild kiwifruit across six provinces in China. Based on the analysis of 80 sequenced Psa genomes, we show that China is the origin of the pandemic lineage but that strain diversity in China is confined to just a single clade. In contrast, Korea and Japan harbor strains from multiple clades. Distinct independent transmission events marked introduction of the pandemic lineage into New Zealand, Chile, Europe, Korea, and Japan. Despite high similarity within the core genome and minimal impact of within-clade recombination, we observed extensive variation even within the single clade from which the global pandemic arose.

Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit

Summary Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.

Geographical distribution and morphological diversity of red-fleshed kiwifruit germplasm (Actinidia chinensis Planchon) in China

The fruit flesh color of kiwifruit (Actinidia chinensis) is generally green or yellow when ripened. Developing kiwifruit cultivars with new fruit flesh color such as red-fleshed color has stimulated much interest for kiwifruit breeders and researchers recently due to its potentially importance for meeting the increasing and changing markets and consumers. In the present study, the whole geographical distribution and morphological variation of wild red-fleshed kiwifruit in China were investigated. In total of 56 accessions of red-fleshed kiwifruit were found across 19 separated localities, representing different ecological and climatic conditions throughout South and Central China. Characterization of nine horticulturally important fruit traits of all accessions showed that there are extensive variations in fruit shape, fruit hairs and as to red intensity, spread of the red pigments and background flesh color. The results presented here have updated the current knowledge on the natural distribution and ecological adaptation of wild red-fleshed kiwifruit resource, which should be valuable for kiwifruit agriculture such as the determination of planting areas of red-fleshed kiwifruit cultivars. The accessions with extensive morphological variation can also contribute to kiwifruit breeding in future such as developing new cultivars with red color flesh.

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.

Analysis of volatile compounds responsible for kiwifruit aroma by desiccated headspace gas chromatography–mass spectrometry

A new method for desiccated headspace (DHS) sampling of aqueous sample to GC-MS for the analysis of volatile compounds responsible for kiwifruit aroma in different kiwifruit cultivars has been developed based on the complete hydrate formation between the sample solvent (water) with anhydrous salt (calcium chloride) at an elevated temperature (above the boiling point of the aqueous sample) in a non-contact format, which overcame the water-effect challenge to directly introduce aqueous sample into GC-MS analysis. By means of DHS, the volatile compounds in three different kiwifruit cultivars were analyzed and compared under the optimized operating conditions, mainly time and temperature for headspace equilibration, column temperature program for GC-MS measurement. As a result, 20 peaks of volatile compounds responsible for kiwifruit aroma were detected and remarkable differences were found in the relative contents of three major volatile compounds among the three different kiwifruit cultivars, i.e., acetaldehyde, ethanol and furfural. The DHS sampling technique used in the present method can make the GC-MS analysis of volatile compounds in the aqueous sample within complex matrix possible without contaminating the GC-MS instrument. In terms of the analysis of volatile compounds in kiwifruit, the present method enabled a direct measurement on the filtrate of the aqueous kiwifruit pulp, without intermediate trap phase for the extraction of analytes, which will be more reliable and simpler as compared with any other headspace method in use. Thus, DHS coupled with GC-MS will be a new valuable tool available for the kiwifruit related research and organoleptic quality control.

Whole transcriptome sequencing of Pseudomonas syringae pv. actinidiae- infected kiwifruit plants reveals species-specific interaction between long non-coding RNA and coding genes

An outbreak of kiwifruit bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) beginning in 2008 caused disaster to the kiwifruit industry. However the mechanisms of interaction between kiwifruit and Psa are unknown. Long noncoding RNAs (lncRNAs) are known to regulate many biological processes, but comprehensive repertoires of kiwifruit lncRNAs and their effects on the interaction between kiwifruit and Psa are unknown. Here, based on in-depth transcriptomic analysis of four kiwifruit materials at three stages of infection with Psa, we identified 14,845 transcripts from 12,280 loci as putative lncRNAs. Hierarchical clustering analysis of differentially-expressed transcripts reveals that both protein-coding and lncRNA transcripts are expressed species-specifically. Comparing differentially-expressed transcripts from different species, variations in pattern-triggered immunity (PTI) were the main causes of species-specific responses to infection by Psa. Using weighted gene co-expression network analysis, we identified species-specific expressed key lncRNAs which were closely related to plant immune response and signal transduction. Our results illustrate that different kiwifruit species employ multiple different plant immunity layers to fight against Psa infection, which causes distinct responses. We also discovered that lncRNAs might affect kiwifruit responses to Psa infection, indicating that both protein-coding regions and noncoding regions can affect kiwifruit response to Psa infection.

Volatile fingerprints and biomarkers of three representative kiwifruit cultivars obtained by headspace solid-phase microextraction gas chromatography mass spectrometry and chemometrics

Volatile aroma of kiwifruit is a mixture of complicated and time-dependent compounds, and thereby the study of these compounds required distinguished analytical techniques as well as robust data analysis techniques. In this work, we report on the volatile fingerprints and biomarkers of three representative kiwifruit cultivars with commercial importance using headspace solid-phase microextraction gas chromatography-mass spectrometric (HS-SPME-GC-MS) coupled with multivariate analysis. As a result, 95 volatiles have been analyzed from the fingerprints, and ultimately six of which were identified as volatile biomarkers of the kiwifruit cultivars studied, which are formic acid octyl ester, 2-Methylbicyclo[4.3.0]non-1(6)-ene, 1-ethoxy-2,4-hexadiene, and 2-methyl-5-(1-methylethyl)-bicyclo[3.1.0]hex-2-ene for Jintao (A. chinensis), and 1-methoxy-2-methyl-benzene and (E,E)-2,4-heptadienal for Cuiyu (A. deliciosa), respectively. Since the samples of each cultivar were in various maturities, these compounds could be taken as the maturity-independent volatile biomarkers for the kiwifruit cultivars, which would be valuable for marker-assisted flavour breeding in the kiwifruit production.