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.

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.

The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications.

Background: Genomic methods for identifying causative variants for trait loci applicable to a wide range of germplasm are required for plant biologists and breeders to understand the genetic control of trait variation. Results: We implemented Cas9-targeted sequencing for fine-mapping in apple, a method combining CRISPR-Cas9 targeted cleavage of a region of interest, followed by enrichment and long-read sequencing using the Oxford Nanopore Technology (ONT). We demonstrated the capability of this methodology to specifically cleave and enrich a plant genomic locus spanning 8 kb. The repeated mini-satellite motif located upstream of the Malus × domestica (apple) MYB10 transcription factor gene, causing red fruit colouration when present in a heterozygous state, was our exemplar to demonstrate the efficiency of this method: it contains a genomic region with a long structural variant normally ignored by short-read sequencing technologies Cleavage specificity of the guide RNAs was demonstrated using polymerase chain reaction products, before using them to specify cleavage of high molecular weight apple DNA. An enriched library was subsequently prepared and sequenced using an ONT MinION flow cell (R.9.4.1). Of the 7,056 ONT reads base-called using both Albacore2 (v2.3.4) and Guppy (v3.2.4), with a median length of 9.78 and 9.89 kb, respectively, 85.35 and 91.38%, aligned to the reference apple genome. Of the aligned reads, 2.98 and 3.04% were on-target with read depths of 180 × and 196 × for Albacore2 and Guppy, respectively, and only five genomic loci were off-target with read depth greater than 25 × , which demonstrated the efficiency of the enrichment method and specificity of the CRISPR-Cas9 cleavage. Conclusions: We demonstrated that this method can isolate and resolve single-nucleotide and structural variants at the haplotype level in plant genomic regions. The combination of CRISPR-Cas9 target enrichment and ONT sequencing provides a more efficient technology for fine-mapping loci than genome-walking approaches.

We assembled the genome of Leptospermum scoparium ‘Crimson Glory’ using a combination of Illumina paired‐end sequencing, high‐throughput chromosome conformation capture (Hi‐C) and high density genetic mapping. As ‘Crimson Glory’ is a variety of mānuka, this is the first genome assembly for a plant species culturally recognised as a treasure (taonga) by the indigenous Māori of Aotearoa New Zealand. The mānuka genome spans a total of 297 Mbp organised in 11 pseudo‐chromosomes that are syntenic with the Eucalyptus genome. A large proportion of the genome assembly corresponds to fungal and bacterial sequences, indicating the presence of an associated microbiome. A total of 31,220 protein‐coding gene models were detected throughout the genome, including genes involved in biosynthesis of biologically active phenylpropanoids, triketones and terpenes, as well as genes involved in biotic resistance. The mānuka genome sequence will help shed new light on the genetic control of unique characters such as nectar and foliage biochemical composition, flowering time and disease resistance.

Angiosperm sex chromosomes, where present, are generally recently evolved. The key step in initiating the development of sex chromosomes from autosomes is the establishment of a sex-determining locus within a region of non-recombination. To better understand early sex chromosome evolution, it is important to determine the process by which recombination is suppressed around the sex determining genes. We have used the dioecious angiosperm kiwifruit Actinidia chinensis var. chinensis, which has an active-Y sex chromosome system, to study recombination rates around the sex locus, to better understand key events in the development of sex chromosomes. We have confirmed the sex-determining region (SDR) in A. chinensis var. chinensis, using a combination of high density genetic mapping and fluorescent in situ hybridisation (FISH) of Bacterial Artificial Chromosomes (BACs) linked to the sex markers onto pachytene chromosomes. The SDR is a subtelomeric non-recombining region adjacent to the nucleolar organiser region (NOR). A region of restricted recombination of around 6 Mbp in size in both male and female maps spans the SDR and covers around a third of chromosome 25. As recombination is suppressed over a similar region between X chromosomes and between and X and Y chromosomes, we propose that recombination is suppressed in this region because of the proximity of the NOR and the centromere, with both the NOR and centromere suppressing recombination, and this predates suppressed recombination due to differences between X and Y chromosomes. Such regions of suppressed recombination in the genome provide an opportunity for the evolution of sex chromosomes, if a sex-determining locus develops there or translocates into this region.