BackgroundMost published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) ‘Hongyang’ draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models.ResultsA second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within ‘Hongyang’ The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned ‘Hort16A’ cDNAs and comparing with the predicted protein models for Red5 and both the original ‘Hongyang’ assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised ‘Hongyang’ annotation, respectively, compared with 90.9% to the Red5 models.ConclusionsOur study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4656-3) contains supplementary material, which is available to authorized users.

Background: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs).

MethodsMethod S1: Screening of the expressed candidate sex determinants Developing anthers at stage 1-2, which correspond to the differentiation stage of male or female androecium (see Supplementary Figure S1), were sampled from F1 sibling vines derived from an interspecific cross, A. rufa sel. Fuchu × A. chinensis sel. FCM1, named KE population (15), planted on Kagawa University, Japan (N34.28, E134.13), in 10-22 April in 2016-2017. Total RNA was extracted using the Plant RNA Reagent (Invitrogen) and purified by phenol/chloroform extraction. Two micrograms of total RNA were processed in preparation for Illumina Sequencing, according to a previous report (15). The constructed libraries were sequenced on Illumina's HiSeq 4000 sequencer (50-bp single-end or 150-bp paired-end reads). All Illumina sequencing were conducted at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, and the raw sequencing reads were processed using custom Python scripts developed in the Comai laboratory and available online (http://comailab.genomecenter.ucdavis.edu/index.php/), as previously described (9). Male-specific Ychromosomal sequences in kiwifruit, defined MSY contigs, were comprehensively identified in previous study (15). The mRNA-Seq reads from each 5 male and female individuals from the KE population (Supplemental Table S11) (15) were used to identify the genes substantially expressed in developing anthers. The mRNA-Seq reads were aligned to the hypothetical 61 genes located on the 249 MSY contigs (Akagi et al. 2018), using the Burrows-Wheeler Aligner (BWA) (37) allowing up to ca 3% mismatches. The number of reads mapping to each contigs was recorded from the alignment file produced by the Sequence Alignment/Map (SAM) tool (38) (http://samtools.sourceforge.net/). For Friendly Boy (FrBy), which showed male-specific and anther-enriched expression, the expression patterns were further examined using various plant organs and developing anthers (stage 2a, 2b, 3a, and 3b, see Supplementary Figure S1). Method S2: Expression profiling in kiwifruit antherThe described mRNA-Seq reads from each 5 male and female individuals of the KE population were aligned to the whole CDS sequences sets in A. chinensis (27), using BWA with default parameters. The number of reads mapped to each reference sequences was recorded from the alignment file produced by the Sequence Alignment/Map (SAM) tool (38) (http://samtools.sourceforge.net/). The read counts per gene were generated from the aligned SAM files using a custom R script. Differential expression between male and female individuals was analysed in R (version 3.0.1) using the R package DESeq (Anders and Huber, 2010) (version 1.14; http://bioconductor.org/packages/release/bioc/html/DESeq.html). We conducted DESeq analysis using 5 biological replicates from male and female individuals, with the following parameters: method='per-condition' and sharingMode='maximum'. An FDR threshold of 0.1 was used to identify differentially expressed genes. Method S3: in situ RNA hybridizationRNA in ...

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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.