De novo chromosome level assembly of a plant genome from long read sequence data.

Recent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. Chromosome level genome assemblies using sequence contigs generated from long read sequencing have involved the use of proximity analysis (Hi-C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (HiFi; PacBio) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii, a genome that has been used as a model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig (six with telomere repeats at both ends) and the other six assembled from two to four main contigs. The small number of chromosome breaks appears to be the result of highly repetitive regions including ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now appears possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed.

Evaluation of In-Field Inoculation Methods for Characterization of Macadamia Germplasm to Husk Spot Incidence and Severity.

Husk spot, a fungal disease of macadamia pericarps (Pseudocercospora macadamiae), induces premature abscission in several major commercial cultivars. Breeding for resistance to husk spot is a priority of the Australian macadamia industry. Due to the large tree size of macadamia and high numbers of progeny in breeding populations, inoculating for resistance screening is laborious and time consuming. Previously utilized methods included direct applications of P. macadamiae suspensions and the hanging of bags of diseased husks above developing fruit in tree canopies. In this study, both methods were modified to allow for efficient application in large-scale breeding populations, and their efficacy was evaluated. Two quantities of diseased husk per bag, 'large' (75 g) and 'small' (30 g), and two concentrations of sprayed P. macadamiae suspensions, 'stock' (5 × 105 propagules/ml) and 'dilute' (2.5 × 105 propagules/ml), were tested across two fruiting seasons. Treatments were compared against a control (sterile water) in commercial cultivars A38 and A4. Husk spot incidence and severity produced by small bags were significantly affected by season. A significant season effect was less common for other treatments. All four treatments infected over 50% of target fruit in each season, but the highest husk spot incidence across both seasons (≥85%) was produced from large bags. Overall, the large bags were the most reliable method for infection of target fruit. Results also demonstrate the importance of considering the effect of season when selecting husk spot inoculation methods.

Genomic selection and genetic gain for nut yield in an Australian macadamia breeding population.

BACKGROUND: Improving yield prediction and selection efficiency is critical for tree breeding. This is vital for macadamia trees with the time from crossing to production of new cultivars being almost a quarter of a century. Genomic selection (GS) is a useful tool in plant breeding, particularly with perennial trees, contributing to an increased rate of genetic gain and reducing the length of the breeding cycle. We investigated the potential of using GS methods to increase genetic gain and accelerate selection efficiency in the Australian macadamia breeding program with comparison to traditional breeding methods. This study evaluated the prediction accuracy of GS in a macadamia breeding population of 295 full-sib progeny from 32 families (29 parents, reciprocals combined), along with a subset of parents. Historical yield data for tree ages 5 to 8 years were used in the study, along with a set of 4113 SNP markers. The traits of focus were average nut yield from tree ages 5 to 8 years and yield stability, measured as the standard deviation of yield over these 4 years. GBLUP GS models were used to obtain genomic estimated breeding values for each genotype, with a five-fold cross-validation method and two techniques: prediction across related populations and prediction across unrelated populations. RESULTS: Narrow-sense heritability of yield and yield stability was low (h2 = 0.30 and 0.04, respectively). Prediction accuracy for yield was 0.57 for predictions across related populations and 0.14 when predicted across unrelated populations. Accuracy of prediction of yield stability was high (r = 0.79) for predictions across related populations. Predicted genetic gain of yield using GS in related populations was 474 g/year, more than double that of traditional breeding methods (226 g/year), due to the halving of generation length from 8 to 4 years. CONCLUSIONS: The results of this study indicate that the incorporation of GS for yield into the Australian macadamia breeding program may accelerate genetic gain due to reduction in generation length, though the cost of genotyping appears to be a constraint at present.

Genome-wide association studies for yield component traits in a macadamia breeding population.

BACKGROUND: Breeding for new macadamia cultivars with high nut yield is expensive in terms of time, labour and cost. Most trees set nuts after four to five years, and candidate varieties for breeding are evaluated for at least eight years for various traits. Genome-wide association studies (GWAS) are promising methods to reduce evaluation and selection cycles by identifying genetic markers linked with key traits, potentially enabling early selection through marker-assisted selection. This study used 295 progeny from 32 full-sib families and 29 parents (18 phenotyped) which were planted across four sites, with each tree genotyped for 4113 SNPs. ASReml-R was used to perform association analyses with linear mixed models including a genomic relationship matrix to account for population structure. Traits investigated were: nut weight (NW), kernel weight (KW), kernel recovery (KR), percentage of whole kernels (WK), tree trunk circumference (TC), percentage of racemes that survived from flowering through to nut set, and number of nuts per raceme. RESULTS: Seven SNPs were significantly associated with NW (at a genome-wide false discovery rate of < 0.05), and four with WK. Multiple regression, as well as mapping of markers to genome assembly scaffolds suggested that some SNPs were detecting the same QTL. There were 44 significant SNPs identified for TC although multiple regression suggested detection of 16 separate QTLs. CONCLUSIONS: These findings have important implications for macadamia breeding, and highlight the difficulties of heterozygous populations with rapid LD decay. By coupling validated marker-trait associations detected through GWAS with MAS, genetic gain could be increased by reducing the selection time for economically important nut characteristics. Genomic selection may be a more appropriate method to predict complex traits like tree size and yield.

Scion control of miRNA abundance and tree maturity in grafted avocado.

BACKGROUND: Grafting is the common propagation method for avocado and primarily benefits orchard production by reducing the time to tree productivity. It also allows use of scions and rootstocks specifically selected for improved productivity and commercial acceptance. Rootstocks in avocado may be propagated from mature tree cuttings ('mature'), or from seed ('juvenile'). While the use of mature scion material hastens early bearing/maturity and economic return, the molecular factors involved in the role of the scion and/or rootstock in early bearing/reduced juvenility of the grafted tree are still unknown. RESULTS: Here, we utilized juvenility and flowering associated miRNAs; miR156 and miR172 and their putative target genes to screen pre-graft and post-graft material in different combinations from avocado. The abundance of mature miR156, miR172 and the miR156 target gene SPL4, showed a strong correlation to the maturity of the scion and rootstock material in avocado. Graft transmissibility of miR156 and miR172 has been explored in annual plants. Here, we show that the scion may be responsible for grafted tree maturity involving these factors, while the rootstock maturity does not significantly influence miRNA abundance in the scion. We also demonstrate that the presence of leaves on cutting rootstocks supports graft success and contributes towards intergraft signalling involving the carbohydrate-marker TPS1. CONCLUSION: Here, we suggest that the scion largely controls the molecular 'maturity' of grafted avocado trees, however, leaves on the rootstock not only promote graft success, but can influence miRNA and mRNA abundance in the scion. This constitutes the first study on scion and rootstock contribution towards grafted tree maturity using the miR156-SPL4-miR172 regulatory module as a marker for juvenility and reproductive competence.

Japanese plums (Prunus salicina Lindl.) and phytochemicals--breeding, horticultural practice, postharvest storage, processing and bioactivity.

Previous reviews of plum phytochemical content and health benefits have concentrated on the European plum, Prunus domestica L. However, the potential bioactivity of red- and dark red-fleshed Japanese plums, Prunus salicina Lindl., so-called blood plums, appears to warrant a significant increase in exposure, as indicated in a recent review of the whole Prunus genus. Furthermore, Japanese plums are the predominant plum produced on an international basis. In this review the nutrient and phytochemical content, breeding, horticultural practice, postharvest treatment and processing as well as bioactivity (emphasising in vivo studies) of Japanese plum are considered, with a focus on the anthocyanin content that distinguishes the blood plums.

Discovery of Single Nucleotide Polymorphisms for Resistance to Abnormal Vertical Growth in Macadamia.

Abnormal vertical growth (AVG) syndrome is a serious threat to the Australian macadamia industry as it decreases the yield of nuts by as much as 70% per annum. A lack of information on the cause of AVG has hindered the development of an effective disease management strategy. Discovery of genetic markers associated with disease resistance can be used as tool for rapid selection of elite cultivars, hence helps in efficient disease management. Differences in field susceptibility of macadamia cultivars provide an opportunity for discovery of genetic markers that are associated with host resistance. REML mixed model analysis was performed to estimate the AVG rating of 51 cultivars from multiple origins using phenotypic data from 359 trees planted in four sites. Most of the Hawaiian cultivars were found as susceptible, while selections from the Australian macadamia industry breeding program were predominantly resistant. All the cultivars were genotyped for 13,221 DArTseq-based single nucleotide polymorphism (SNP) markers. A bulked sample analysis was performed using 20 genotypes each at the extremes of AVG phenotypic ratings. Ten SNP markers were predicted to be associated with AVG resistance and two arbitrarily selected SNP markers were validated using PCR and Sanger sequencing. Our findings suggest that AVG resistance in the commercial cultivars may be derived from the genomic introgression of Macadamia tetraphylla through interspecific hybridization. The results may support marker-assisted selection for macadamia germplasm with AVG resistance.

Effect of Storage on the Nutritional Quality of Queen Garnet Plum.

Due to high perishability, plums are harvested at an early stage of maturity to extend postharvest storage life. Storage time and temperature can significantly affect the phytochemical and sugar composition of plums, altering their palatability and nutritional quality. In this study, variations in physiochemical properties (total soluble solids (TSS), titratable acidity (TA), color (chroma and hue angle)), phytochemical composition (total phenolic content (TPC), total anthocyanin content (TAC), and carotenoids), and sugars in three different tissues of the Queen Garnet plum (QGP) during storage at two common domestic storage temperatures (4 and 23 °C) were evaluated. There was an increase (p > 0.05) in TSS and a reduction (p < 0.05) in TA of the outer flesh at 23 °C. Chroma values of all the tissues reduced (p < 0.05) at 23 °C. At 4 °C, chroma values fluctuated between storage days. The TAC of the peel was the highest (p < 0.05) among the different tissues and continued to increase up to 10 days of storage at 23 °C (3-fold increase). At 4 °C, the highest (p < 0.05) TAC (peel) was observed after 14 days of storage (1.2-fold increase). TPC showed similar results. The highest (p < 0.05) TPC was recorded in the peel after 10 days of storage at 23 °C (2.3-fold increase) and after 14 days of storage at 4 °C (1.3-fold increase), respectively. Total carotenoids in the flesh samples at both storage temperatures were reduced (p < 0.05) after 14 days. Total sugars also decreased during storage. The results of the present study clearly showed that common domestic storage conditions can improve the nutritional quality of plums by increasing the content of bioactive anthocyanins and other phenolic compounds. However, the increase in phytochemicals needs to be counterbalanced with the decrease in total sugars and TA potentially affecting the sensory attributes of the plums.

The genome of the endangered Macadamia jansenii displays little diversity but represents an important genetic resource for plant breeding.

Macadamia, a recently domesticated expanding nut crop in the tropical and subtropical regions of the world, is one of the most economically important genera in the diverse and widely adapted Proteaceae family. All four species of Macadamia are rare in the wild with the most recently discovered, M. jansenii, being endangered. The M. jansenii genome has been used as a model for testing sequencing methods using a wide range of long read sequencing techniques. Here, we report a chromosome level genome assembly, generated using a combination of Pacific Biosciences sequencing and Hi-C, comprising 14 pseudo-molecules, with a N50 of 52 Mb and a total genome assembly size of 758 Mb of which 56% is repetitive. Completeness assessment revealed that the assembly covered -97.1% of the conserved single copy genes. Annotation predicted 31,591 protein coding genes and allowed the characterization of genes encoding biosynthesis of cyanogenic glycosides, fatty acid metabolism, and anti-microbial proteins. Re-sequencing of seven other genotypes confirmed low diversity and low heterozygosity within this endangered species. Important morphological characteristics of this species such as small tree size and high kernel recovery suggest that M. jansenii is an important source of these commercial traits for breeding. As a member of a small group of families that are sister to the core eudicots, this high-quality genome also provides a key resource for evolutionary and comparative genomics studies.

Improvements in the sequencing and assembly of plant genomes.

Advances in DNA sequencing have made it easier to sequence and assemble plant genomes. Here, we extend an earlier study, and compare recent methods for long read sequencing and assembly. Updated Oxford Nanopore Technology software improved assemblies. Using more accurate sequences produced by repeated sequencing of the same molecule (Pacific Biosciences HiFi) resulted in less fragmented assembly of sequencing reads. Using data for increased genome coverage resulted in longer contigs, but reduced total assembly length and improved genome completeness. The original model species, Macadamia jansenii, was also compared with three other Macadamia species, as well as avocado (Persea americana) and jojoba (Simmondsia chinensis). In these angiosperms, increasing sequence data volumes caused a linear increase in contig size, decreased assembly length and further improved already high completeness. Differences in genome size and sequence complexity influenced the success of assembly. Advances in long read sequencing technology continue to improve plant genome sequencing and assembly. However, results were improved by greater genome coverage, with the amount needed to achieve a particular level of assembly being species dependent.

Genetic Structure of Wild Germplasm of Macadamia: Species Assignment, Diversity and Phylogeographic Relationships.

Macadamia is an Australian native rainforest tree that has been domesticated and traded internationally for its premium nuts. Common cultivars rely upon a limited gene pool that has exploited only two of the four species. Introducing a more diverse germplasm will broaden the genetic base for future crop improvement and better adaptation for changing environments. This study investigated the genetic structure of 302 accessions of wild germplasm using 2872 SNP and 8415 silicoDArT markers. Structure analysis and principal coordinate analysis (PCoA) assigned the 302 accessions into four distinct groups: (i) Macadamia integrifolia, (ii) M. tetraphylla, and (iii) M. jansenii and M. ternifolia, and (iv) admixtures or hybrids. Assignment of the four species matched well with previous characterisations, except for one M. integrifolia and four M. tetraphylla accessions. Using SNP markers, 94 previously unidentified accessions were assigned into the four distinct groups. Finally, 287 accessions were identified as pure examples of one of the four species and 15 as hybrids of M. integrifolia and M. tetraphylla. The admixed accessions showed the highest genetic diversity followed by M. integrifolia, while M. ternifolia and M. jansenii accessions were the least diverse. Mantel test analysis showed a significant correlation between genetic and geographic distance for M. integrifolia (r = 0.51, p = 0.05) and a positive but not significant correlation for M. tetraphylla (r = 0.45, p = 0.06). This study provides a population genetics overview of macadamia germplasm as a background for a conservation strategy and provides directions for future macadamia breeding.

Maximising recombination across macadamia populations to generate linkage maps for genome anchoring.

The Proteaceae genus Macadamia has a recent history of domestication as a commercial nut crop. We aimed to establish the first sequence-based haploid-correlated reference genetic linkage maps for this primarily outcrossing perennial tree crop, with marker density suitable for genome anchoring. Four first generation populations were used to maximise the segregation patterns available within full-sib, biparental and self-pollinated progeny. This allowed us to combine segregation data from overlapping subsets of >4,000 informative sequence-tagged markers to increase the effective coverage of the karyotype represented by the recombinant crossover events detected. All maps had 14 linkage groups, corresponding to the Macadamia haploid chromosome number, and enabled the anchoring and orientation of sequence scaffolds to construct a pseudo-chromosomal genome assembly for macadamia. Comparison of individual maps indicated a high level of congruence, with minor discrepancies satisfactorily resolved within the integrated maps. The combined set of maps significantly improved marker density and the proportion (70%) of the genome sequence assembly anchored. Overall, increasing our understanding of the genetic landscape and genome for this nut crop represents a substantial advance in macadamia genetics and genomics. The set of maps, large number of sequence-based markers and the reconstructed genome provide a toolkit to underpin future breeding that should help to extend the macadamia industry as well as provide resources for the long term conservation of natural populations in eastern Australia of this unique genus.

Chromosome-Scale Assembly and Annotation of the Macadamia Genome (Macadamia integrifolia HAES 741).

Macadamia integrifolia is a representative of the large basal eudicot family Proteaceae and the main progenitor species of the Australian native nut crop macadamia. Since its commercialisation in Hawaii fewer than 100 years ago, global production has expanded rapidly. However, genomic resources are limited in comparison to other horticultural crops. The first draft assembly of M. integrifolia had good coverage of the functional gene space but its high fragmentation has restricted its use in comparative genomics and association studies. Here we have generated an improved assembly of cultivar HAES 741 (4,094 scaffolds, 745 Mb, N50 413 kb) using a combination of Illumina paired and PacBio long read sequences. Scaffolds were anchored to 14 pseudo-chromosomes using seven genetic linkage maps. This assembly has improved contiguity and coverage, with >120 Gb of additional sequence. Following annotation, 34,274 protein-coding genes were predicted, representing 90% of the expected gene content. Our results indicate that the macadamia genome is repetitive and heterozygous. The total repeat content was 55% and genome-wide heterozygosity, estimated by read mapping, was 0.98% or an average of one SNP per 102 bp. This is the first chromosome-scale genome assembly for macadamia and the Proteaceae. It is expected to be a valuable resource for breeding, gene discovery, conservation and evolutionary genomics.

Comparison of long-read methods for sequencing and assembly of a plant genome.

BACKGROUND: Sequencing technologies have advanced to the point where it is possible to generate high-accuracy, haplotype-resolved, chromosome-scale assemblies. Several long-read sequencing technologies are available, and a growing number of algorithms have been developed to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology, as well as the most appropriate software for assembly and polishing. It is thus important to benchmark different approaches applied to the same sample. RESULTS: Here, we report a comparison of 3 long-read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION), and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of Pacific Biosciences and Nanopore reads. Results obtained from combining long-read technologies or short-read and long-read technologies are also presented. The assemblies were compared for contiguity, base accuracy, and completeness, as well as sequencing costs and DNA material requirements. CONCLUSIONS: The 3 long-read technologies produced highly contiguous and complete genome assemblies of M. jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput, and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

Ultra-high-throughput DArTseq-based silicoDArT and SNP markers for genomic studies in macadamia.

Macadamia (Macadamia integrifolia, M. tetraphylla and hybrids) is an Australian native nut crop and has a significant economic value in the food industries worldwide. Long juvenility along with traditional breeding strategies impede quick genetic improvement of this crop. The existing cultivars constitute only second to fourth generation of the wild germplasm in the rainforest. The utilisation of molecular markers for genomic selection and genome-wide association studies may accelerate genetic gains. Identification of a robust, reproducible, and cost-effective marker system is instrumental in increasing the efficiency of genomic studies. This study is the first to report the potential of two ultra-high-throughput diversity array technology (DArT) markers (silicoDArT and SNP) in macadamia. Both markers were used to identify the genetic diversity and population structure in 80 macadamia cultivars. Parentage analysis of 25 scions in a rootstock trial was conducted to confirm plant identity where recorded identities did not corroborate with phenotypic field observations. A total of 22,280 silicoDArT and 7,332 SNP markers were reported, of which 11,526 silicoDArT and 3,956 SNP markers were used for analyses after screening with quality control parameters including >95% call rate, >95% reproducibility, and >0.05 one ratio. The average polymorphic information content (PIC) values of silicoDArT and SNP markers were 0.29 and 0.21, respectively. Genetic variance among the cultivars ranged from 0.003 to 0.738 in silicoDArT and 0.004 to 0.412 in SNP markers. Four distinct population groups were identified from SNP data analysis. Most of the accessions used in this study were descended from two or more populations. Cluster analysis clearly separated genotypes of distinct origins, such as the Hawaii Agricultural Experiment Station and Hidden Valley Plantation accessions. Two wild accessions of Macadamia jansenii and M. ternifolia were found to be distantly related to the cultivars. Wild germplasm individuals and their hybrids with cv. '660' formed separate clusters, suggesting that crossing between wild and cultivated genepools can extend genetic diversity. DArTseq-based SNP markers were successfully utilized to confirm the genetic identity of 25 scions in a rootstock trial. Our study suggests that DArT platforms are a robust system for the facilitation of genomic studies with regard to macadamia.