Genome annotation is the process of identifying the location and function of a genome's encoded features. Improving the biological accuracy of annotation is a complex and iterative process requiring researchers to review and incorporate multiple sources of information such as transcriptome alignments, predictive models based on sequence profiles, and comparisons to features found in related organisms. Because rapidly decreasing costs are enabling an ever-growing number of scientists to incorporate sequencing as a routine laboratory technique, there is widespread demand for tools that can assist in the deliberative analytical review of genomic information. To this end, we present Apollo, an open source software package that enables researchers to efficiently inspect and refine the precise structure and role of genomic features in a graphical browser-based platform. Some of Apollo's newer user interface features include support for real-time collaboration, allowing distributed users to simultaneously edit the same encoded features while also instantly seeing the updates made by other researchers on the same region in a manner similar to Google Docs. Its technical architecture enables Apollo to be integrated into multiple existing genomic analysis pipelines and heterogeneous laboratory workflow platforms. Finally, we consider the implications that Apollo and related applications may have on how the results of genome research are published and made accessible.

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Apollo: Democratizing genome annotation

Dioecy, the presence of male and female individuals, has evolved independently in multiple flowering plant lineages1–3. Although theoretical models for the evolution of dioecy, such as the ‘two-mutations’ model, are well established4,5, little is known about the specific genes determining sex and their evolutionary history3. Kiwifruit, a major tree crop consumed worldwide, is a dioecious species. In kiwifruit we previously identified a Y-encoded sex-determinant candidate gene acting as the suppressor of feminization (SuF), named Shy Girl (SyGI)6. Here, we identify a second Y-encoded sex-determinant that we named Friendly Boy (FrBy), which exhibits strong expression in tapetal cells. Gene-editing and complementation analyses in Arabidopsis thaliana and Nicotiana tabacum indicated that FrBy acts for the maintenance of male (M) functions, independently of SyGI, and that these functions are conserved across angiosperm species. We further characterized the genomic architecture of the small (<1 megabase pairs (Mb)) male-specific region of the Y chromosome (MSY), which harbours only two genes expressed extensively in developing gynoecia and androecia, respectively: SyGI and FrBy. Re-sequencing of the genome of a natural hermaphrodite kiwifruit revealed that this individual is genetically male but carries deletion(s) of parts of the Y chromosome, including SyGI. Additionally, expression of FrBy in female kiwifruit resulted in hermaphrodite plants. These results clearly indicate that Y-encoded SyGI and FrBy act independently as the SuF and M factors in kiwifruit, respectively, and provide insight into not only the evolutionary path leading to a two-factor sex-determination system, but also a new breeding approach for dioecious species. A sex determination gene, FrBy, is identified in the dioecious kiwifruit and demonstrated to maintain male functions across angiosperm species. FrBy and the known female suppressor gene, SyGI, support the evolution of dioecy via the ‘two-mutations’ model.

/pdf/two-y-chromosome-encoded-genes-determine-sex-in-kiwifruit-3qosm8mfh4.pdf

Two Y-chromosome-encoded genes determine sex in kiwifruit

Many Actinidia cultivars are characterized by anthocyanin accumulation, specifically in the inner pericarp, but the underlying regulatory mechanism remains elusive. Here we report two interacting transcription factors, AcMYB123 and AcbHLH42, that regulate tissue‐specific anthocyanin biosynthesis in the inner pericarp of Actinidia chinensis cv. Hongyang. Through transcriptome profiling analysis we identified five MYB and three bHLH transcription factors that were upregulated in the inner pericarp. We show that the combinatorial action of two of them, AcMYB123 and AcbHLH42, is required for activating promoters of AcANS and AcF3GT1 that encode the dedicated enzymes for anthocyanin biosynthesis. The presence of anthocyanin in the inner pericarp appears to be tightly associated with elevated expression of AcMYB123 and AcbHLH42. RNA interference repression of AcMYB123, AcbHLH42, AcF3GT1 and AcANS in ‘Hongyang’ fruits resulted in significantly reduced anthocyanin biosynthesis. Using both transient assays in Nicotiana tabacum leaves or Actinidia arguta fruits and stable transformation in Arabidopsis, we demonstrate that co‐expression of AcMYB123 and AcbHLH42 is a prerequisite for anthocyanin production by activating transcription of AcF3GT1 and AcANS or the homologous genes. Phylogenetic analysis suggests that AcMYB123 or AcbHLH42 are closely related to TT2 or TT8, respectively, which determines proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than regulators in dicots. All these experimental results suggest that AcMYB123 and AcbHLH42 are the components involved in spatiotemporal regulation of anthocyanin biosynthesis specifically in the inner pericarp of kiwifruit.

A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang

Actinidia chinensis (kiwifruit) is a perennial horticultural crop species of the Actinidiaceae family with high nutritional and economic value. Two versions of the A. chinensis genomes have been previously assembled, based mainly on relatively short reads. Here, we report an improved chromosome-level reference genome of A. chinensis (v3.0), based mainly on PacBio long reads and Hi-C data. The high-quality assembled genome is 653 Mb long, with 0.76% heterozygosity. At least 43% of the genome consists of repetitive sequences, and the most abundant long terminal repeats were further identified and account for 23.38% of our novel genome. It has clear improvements in contiguity, accuracy, and gene annotation over the two previous versions and contains 40,464 annotated protein-coding genes, of which 94.41% are functionally annotated. Moreover, further analyses of genetic collinearity revealed that the kiwifruit genome has undergone two whole-genome duplications: one affecting all Ericales families near the K-T extinction event and a recent genus-specific duplication. The reference genome presented here will be highly useful for further molecular elucidation of diverse traits and for the breeding of this horticultural crop, as well as evolutionary studies with related taxa.

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A high-quality Actinidia chinensis (kiwifruit) genome.

Annualization of woody perennials has the potential to revolutionize the breeding and production of fruit crops and rapidly improve horticultural species. Kiwifruit (Actinidia chinensis) is a recently domesticated fruit crop with a short history of breeding and tremendous potential for improvement. Previously, multiple kiwifruit CENTRORADIALIS (CEN)-like genes have been identified as potential repressors of flowering. In this study, CRISPR/Cas9- mediated manipulation enabled functional analysis of kiwifruit CEN-like genes AcCEN4 and AcCEN. Mutation of these genes transformed a climbing woody perennial, which develops axillary inflorescences after many years of juvenility, into a compact plant with rapid terminal flower and fruit development. The number of affected genes and alleles and severity of detected mutations correlated with the precocity and change in plant stature, suggesting that a bi-allelic mutation of either AcCEN4 or AcCEN may be sufficient for early flowering, whereas mutations affecting both genes further contributed to precocity and enhanced the compact growth habit. CRISPR/Cas9-mediated mutagenesis of AcCEN4 and AcCEN may be a valuable means to engineer Actinidia amenable for accelerated breeding, indoor farming and cultivation as an annual crop.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13021

Mutagenesis of kiwifruit CENTRORADIALIS-like genes transforms a climbing woody perennial with long juvenility and axillary flowering into a compact plant with rapid terminal flowering.

Most 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. A 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. Our 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.

/pdf/a-manually-annotated-actinidia-chinensis-var-chinensis-1fwuvy7bab.pdf

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Francesco Gambi

Papers

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants