Heterozygous Genome Research Articles

The haplotype-resolved chromosome pairs of a heterozygous diploid African cassava cultivar reveal novel pan-genome and allele-specific transcriptome features.

BackgroundCassava (Manihot esculenta) is an important clonally propagated food crop in tropical and subtropical regions worldwide. Genetic gain by molecular breeding has been limited, partially because cassava is a highly heterozygous crop with a repetitive and difficult-to-assemble genome.FindingsHere we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads. We present 2 chromosome-scale haploid genomes phased with Hi-C technology for the diploid African cassava variety TME204. With consensus accuracy >QV46, contig N50 >18 Mb, BUSCO completeness of 99%, and 35k phased gene loci, it is the most accurate, continuous, complete, and haplotype-resolved cassava genome assembly so far. Ab initio gene prediction with RNA-seq data and Iso-Seq transcripts identified abundant novel gene loci, with enriched functionality related to chromatin organization, meristem development, and cell responses. During tissue development, differentially expressed transcripts of different haplotype origins were enriched for different functionality. In each tissue, 20–30% of transcripts showed allele-specific expression (ASE) differences. ASE bias was often tissue specific and inconsistent across different tissues. Direction-shifting was observed in <2% of the ASE transcripts. Despite high gene synteny, the HiFi genome assembly revealed extensive chromosome rearrangements and abundant intra-genomic and inter-genomic divergent sequences, with large structural variations mostly related to LTR retrotransposons. We use the reference-quality assemblies to build a cassava pan-genome and demonstrate its importance in representing the genetic diversity of cassava for downstream reference-guided omics analysis and breeding.ConclusionsThe phased and annotated chromosome pairs allow a systematic view of the heterozygous diploid genome organization in cassava with improved accuracy, completeness, and haplotype resolution. They will be a valuable resource for cassava breeding and research. Our study may also provide insights into developing cost-effective and efficient strategies for resolving complex genomes with high resolution, accuracy, and continuity.

Gene prediction for leaf margin phenotype and fruit flesh color in pineapple (Ananas comosus) using haplotype-resolved genome sequencing.

Pineapple (Ananas comosus (L.) Merr.) is one of the most economically important tropical fruit species. The major aim of the breeding programs in several countries, including Japan, is quality improvement, mainly for the fresh market. ‘Yugafu’, a Japanese cultivar with distinctive pipe-type leaf margin phenotype and white flesh color, is popular for fresh consumption. Therefore, genome sequencing of ‘Yugafu’ is expected to assist pineapple breeding. Here, we developed a haplotype-resolved assembly for the heterozygous genome of ‘Yugafu’ using long-read sequencing technology and obtained a pair of 25 pseudomolecule sequences inherited from the parental accessions ‘Cream pineapple’ and ‘HI101’. The causative genes for leaf margin and fruit flesh color were identified. Fine mapping revealed a 162-kb region on CLG23 for the leaf margin phenotype. In this region, 20 kb of inverted repeat was specifically observed in the ‘Cream pineapple’ derived allele, and the WUSCHEL-related homeobox 3 (AcWOX3) gene was predicted as the key gene for leaf margin morphogenesis. Dominantly repressed AcWOX3 via RNAi was suggested to be the cause of the pipe-type leaf margin phenotype. Quantitative trait locus (QTL) analysis revealed that the terminal region of CLG08 contributed to white flesh and low carotenoid content. Carotenoid cleaved dioxygenase 4 (AcCCD4), a key gene for carotenoid degradation underlying this QTL, was predicted as the key gene for white flesh color through expression analysis. These findings could assist in modern pineapple breeding and facilitate marker-assisted selection for important traits.

\u2018Nebbiolo\u2019 genome assembly allows surveying the occurrence and functional implications of genomic structural variations in grapevines (Vitis vinifera L.)

Background‘Nebbiolo’ is a grapevine cultivar typical of north-western Italy, appreciated for producing high-quality red wines. Grapevine cultivars are characterized by possessing highly heterozygous genomes, including a great incidence of genomic rearrangements larger than 50 bp, so called structural variations (SVs). Even though abundant, SVs are an under-explored source of genetic variation mainly due to methodological limitations at their detection.ResultsWe employed a multiple platform approach to produce long-range genomic data for two different ‘Nebbiolo’ clones, namely: optical mapping, long-reads and linked-reads. We performed a haplotype-resolved de novo assembly for cultivar ‘Nebbiolo’ (clone CVT 71) and used an ab-initio strategy to annotate it. The annotated assembly enhanced our ability to detect SVs, enabling the study of genomic regions not present in the grapevines’ reference genome and accounting for their functional implications. We performed variant calling analyses at three different organizational levels: i) between haplotypes of clone CVT 71 (primary assembly vs haplotigs), ii) between ‘Nebbiolo’ and ‘Cabernet Sauvignon’ assemblies and iii) between clones CVT 71 and CVT 185, representing different ‘Nebbiolo’ biotypes. The cumulative size of non-redundant merged SVs indicated a total of 79.6 Mbp for the first comparison and 136.1 Mbp for the second one, while no SVs were detected for the third comparison. Interestingly, SVs differentiating cultivars and haplotypes affected similar numbers of coding genes.ConclusionsOur results suggest that SVs accumulation rate and their functional implications in ‘Nebbiolo’ genome are highly-dependent on the organizational level under study. SVs are abundant when comparing ‘Nebbiolo’ to a different cultivar or the two haplotypes of the same individual, while they turned absent between the two analysed clones.

Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), anallogamous forage crop species.

Relative to other crops, red clover (Trifolium pratense L.) has various favorable traits making it an ideal forage crop. Conventional breeding has improved varieties, but modern genomic methods could accelerate progress and facilitate gene discovery. Existing short-read-based genome assemblies of the ∼420megabase pair (Mbp) genome are fragmented into >135,000 contigs, with numerous order and orientation errors within scaffolds, probably associated with the plant's biology, which displays gametophytic self-incompatibility resulting in inherent high heterozygosity. Here, we present a high-quality long-read-based assembly of red clover with a more than 500-fold reduction in contigs, improved per-base quality, and increased contig N50 by three orders of magnitude. The 413.5Mbp assembly is nearly 20% longer than the 350Mbp short-read assembly, closer to the predicted genome size. We also present quality measures and full-length isoform RNA transcript sequences for assessing accuracy and future genome annotation. The assembly accurately represents the seven main linkage groups in an allogamous (outcrossing), highly heterozygous plant genome.

Haploidy in somatic cells is induced by mature oocytes in mice

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research).

Potato (Solanum tuberosum) is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strategies and designs to obtain successful gene editing. In the present study, in-depth sequencing of the glucan water dikinase (GWD)1 and the downy mildew resistant 6 (DMR6-1) genes in the potato cultivars Saturna and Wotan, respectively, revealed both indels and a 1.3–2.8 higher SNP prevalence when compared to the heterozygous diploid RH genome sequence as expected for a tetraploid compared to a diploid. This complicates guide RNA (gRNA) and diagnostic PCR designs. High editing efficiencies at the cell pool (protoplast) level are pivotal for achieving full allelic knock-out in tetraploids and for reducing the downstream cumbersome and delicate ex-plant regeneration. Here, CRISPR/Cas ribonucleoprotein particles (RNP) were delivered transiently to protoplasts by polyethylene glycol (PEG) mediated transformation. For each of GWD1 and DMR6-1, 6–10 gRNAs were designed to target regions comprising the 5′ and the 3′ end of the two genes. Similar to other studies including several organisms, editing efficiency of the individual RNPs/gRNAs varied significantly, and some generated specific indel patterns. While RNPs targeting the 5′ end of GWD1 yielded significantly higher editing when compared to targeting the 3′ end, editing efficiencies in the 5′ and 3′ end of DMR6-1 appeared to be somewhat similar. Simultaneous targeting of either the 5′ or the 3′ end with two RNPs (multiplexing) yielded a clear positive synergistic effect on the total editing when targeting the 3′ end of the GWD1 gene only. Multiplexing of the two genes, residing on different chromosomes, yielded no or slightly negative effects on the individual RNP/gRNA editing efficiencies when compared to editing efficiencies obtained in the single RNP/gRNA transformations. These initial findings may instigate larger studies needed for facilitating and optimizing precision breeding in plants.

Two divergent haplotypes from a highly heterozygous lychee genome suggest independent domestication events for early and late-maturing cultivars

Lychee is an exotic tropical fruit with a distinct flavor. The genome of cultivar ‘Feizixiao’ was assembled into 15 pseudochromosomes, totaling ~470 Mb. High heterozygosity (2.27%) resulted in two complete haplotypic assemblies. A total of 13,517 allelic genes (42.4%) were differentially expressed in diverse tissues. Analyses of 72 resequenced lychee accessions revealed two independent domestication events. The extremely early maturing cultivars preferentially aligned to one haplotype were domesticated from a wild population in Yunnan, whereas the late-maturing cultivars that mapped mostly to the second haplotype were domesticated independently from a wild population in Hainan. Early maturing cultivars were probably developed in Guangdong via hybridization between extremely early maturing cultivar and late-maturing cultivar individuals. Variable deletions of a 3.7 kb region encompassed by a pair of CONSTANS-like genes probably regulate fruit maturation differences among lychee cultivars. These genomic resources provide insights into the natural history of lychee domestication and will accelerate the improvement of lychee and related crops.

Chromosomal-level genome of macadamia (&lt;i&gt;Macadamia integrifolia&lt;/i&gt;)

Macadamia from the family Proteaceae is a plant native to Australia and has long been favoured by people for its crispy and high nutritional and medicinal value. Here, the genome of GUIRE 1 (GR1), a highly heterozygous superior cultivar of macadamia nut, was sequenced and assembled using nanopore sequencing, and a 807-Mb genome (contig N50, 1.9 Mb; scaffold N50, 54.70 Mb) and 14 chromosomes were obtained. A total of 453 Mb (about 55.95%) repetitive sequences and 37,657 protein-coding genes were obtained by gene annotation and homologous protein comparison. Proteaceae diverged from Nelumbonaceae nearly 115.37 million years ago and from Rubiaceae about 140 million years ago. A genome-wide duplication (WGD) event occurred in macadamia 41 million years ago based on the WGD analysis. The functional enrichment analysis of <italic>M. integrifolia</italic>-specific gene families revealed their roles in signal transduction, protein phosphorylation, protein binding, and defense response. Here, a highly heterozygous genome of <italic>M. integrifolia</italic> was unlocked to provide a database for breeding and molecular mechanism research.

From model to alfalfa: Gene editing to obtain semidwarf and prostrate growth habits

Alfalfa (Medicago sativa L.) is a nutritious forage crop with wide ecological adaptability. The molecular breeding of alfalfa is restricted by its heterozygous tetraploid genome and the difficult genetic manipulation process. Under time and resource constraints, we applied a more convenient approach. We investigated two MtGA3ox genes, MtGA3ox1 and MtGA3ox2, of Medicago truncatula, a diploid legume model species, finding that MtGA3ox1 plays a major role in GA-regulated plant architecture. Mutation of neither gene affected nitrogenase activity. These results suggest that MtGA3ox1 can be used in semidwarf and prostrate alfalfa breeding. Based on the M. truncatula MtGA3ox1 sequence, MsGA3ox1 was cloned from alfalfa, and two knockout targets were designed. An efficient CRISPR/Cas9-based genome editing protocol was used to generate msga3ox1 mutants in alfalfa. We obtained three lines that carried mutations in all four alleles in the T0 generation. Fifteen clonal plants were vegetatively propagated from each transgenic line using shoot cuttings. The plant height and internode length of msga3ox1 null mutants were significantly decreased. The number of total lateral branches, leaf/stem ratio and crude protein content of aerial plant parts of msga3ox1 mutants were significantly increased. Thus, we obtained semi-dwarf and prostrate alfalfa by gene editing.

The Tetragnatha kauaiensis Genome Sheds Light on the Origins of Genomic Novelty in Spiders.

Spiders (Araneae) have a diverse spectrum of morphologies, behaviors, and physiologies. Attempts to understand the genomic-basis of this diversity are often hindered by their large, heterozygous, and AT-rich genomes with high repeat content resulting in highly fragmented, poor-quality assemblies. As a result, the key attributes of spider genomes, including gene family evolution, repeat content, and gene function, remain poorly understood. Here, we used Illumina and Dovetail Chicago technologies to sequence the genome of the long-jawed spider Tetragnatha kauaiensis, producing an assembly distributed along 3,925 scaffolds with an N50 of ∼2 Mb. Using comparative genomics tools, we explore genome evolution across available spider assemblies. Our findings suggest that the previously reported and vast genome size variation in spiders is linked to the different representation and number of transposable elements. Using statistical tools to uncover gene-family level evolution, we find expansions associated with the sensory perception of taste, immunity, and metabolism. In addition, we report strikingly different histories of chemosensory, venom, and silk gene families, with the first two evolving much earlier, affected by the ancestral whole genome duplication in Arachnopulmonata (∼450 Ma) and exhibiting higher numbers. Together, our findings reveal that spider genomes are highly variable and that genomic novelty may have been driven by the burst of an ancient whole genome duplication, followed by gene family and transposable element expansion.

Large structural variations in the haplotype-resolved African cassava genome.

SummaryCassava (Manihot esculenta Crantz, 2n = 36) is a global food security crop. It has a highly heterozygous genome, high genetic load, and genotype‐dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi‐C to resolve each haplotype of the genome of a farmer‐preferred cassava line, TME7 (Oko‐iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON‐Phase and Hi‐C read data. The ultralong‐range information from Hi‐C sequencing was also used for scaffolding. Comparison of the two phases revealed >5000 large haplotype‐specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele‐specific expression was further explored. RNA‐sequencing data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy‐to‐use web‐based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype‐specific structural variation, gene hemizygosity, and allele‐specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.

Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato

Genetic gain in potato is hampered by the heterozygous tetraploid genome of cultivated potato. Converting potato into a diploid inbred-line based F1-hybrid crop provides a promising route towards increased genetic gain. The introduction of a dominant S-locus inhibitor (Sli) gene into diploid potato germplasm allows efficient generation of self-fertilized seeds and thus the development of potato inbred lines. Little is known about the structure and function of the Sli locus. Here we describe the mapping of Sli to a 12.6 kb interval on chromosome 12 using a recombinant screen approach. One of two candidate genes present in this interval shows a unique sequence that is exclusively present in self-compatible lines. We describe an expression vector that converts self-incompatible genotypes into self-compatible and a CRISPR-Cas9 vector that converts SC genotypes into SI. The Sli gene encodes an F-box protein that is specifically expressed in pollen from self-compatible plants. A 533 bp insertion in the promotor of that gene leads to a gain of function mutation, which overcomes self-pollen rejection.

Four chromosome scale genomes and a pan-genome annotation to accelerate pecan tree breeding

Genome-enabled biotechnologies have the potential to accelerate breeding efforts in long-lived perennial crop species. Despite the transformative potential of molecular tools in pecan and other outcrossing tree species, highly heterozygous genomes, significant presence–absence gene content variation, and histories of interspecific hybridization have constrained breeding efforts. To overcome these challenges, here, we present diploid genome assemblies and annotations of four outbred pecan genotypes, including a PacBio HiFi chromosome-scale assembly of both haplotypes of the ‘Pawnee’ cultivar. Comparative analysis and pan-genome integration reveal substantial and likely adaptive interspecific genomic introgressions, including an over-retained haplotype introgressed from bitternut hickory into pecan breeding pedigrees. Further, by leveraging our pan-genome presence–absence and functional annotation database among genomes and within the two outbred haplotypes of the ‘Lakota’ genome, we identify candidate genes for pest and pathogen resistance. Combined, these analyses and resources highlight significant progress towards functional and quantitative genomics in highly diverse and outbred crops.

Genome analysis of Candida subhashii reveals its hybrid nature and dual mitochondrial genome conformations.

Candida subhashii belongs to the CUG-Ser clade, a group of phylogenetically closely related yeast species that includes some human opportunistic pathogens, such as Candida albicans. Despite being present in the environment, C. subhashii was initially described as the causative agent of a case of peritonitis. Considering the relevance of whole-genome sequencing and analysis for our understanding of genome evolution and pathogenicity, we sequenced, assembled and annotated the genome of C. subhashii type strain. Our results show that C. subhashii presents a highly heterozygous genome and other signatures that point to a hybrid ancestry. The presence of functional pathways for assimilation of hydroxyaromatic compounds goes in line with the affiliation of this yeast with soil microbial communities involved in lignin decomposition. Furthermore, we observed that different clones of this strain may present circular or linear mitochondrial DNA. Re-sequencing and comparison of strains with differential mitochondrial genome topology revealed five candidate genes potentially associated with this conformational change: MSK1, SSZ1, ALG5, MRPL9 and OYE32.

Panicum virgatum (Poaceae)

Switchgrass (Panicum virgatum L.) is a promising biofuel crop and one of the dominant species in North American tallgrass prairies. It grows rapidly, reaching up to 2.7 m, and planted fields remain productive for 15 years. The large, highly repetitive, and highly heterozygous genome of the generally tetraploid species have always been challenging for sequencing and assembly. Using PacBio long-read sequencing and bacterial artificial chromosome clone technology, researchers recently published a highly continuous genome assembly (v5) of a lowland switchgrass genotype AP13, which consisted of 626 contigs with a contig N50 of 5.5 Mb and a total of 97.2% bases that were assembled in the chromosomes.

Allele-defined genome reveals biallelic differentiation during cassava evolution

Allele-defined genome reveals biallelic differentiation during cassava evolution

Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads.

Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

Benchmarking Oxford Nanopore read assemblers for high-quality molluscan genomes.

Choosing the optimum assembly approach is essential to achieving a high-quality genome assembly suitable for comparative and evolutionary genomic investigations. Significant recent progress in long-read sequencing technologies such as PacBio and Oxford Nanopore Technologies (ONT) has also brought about a large variety of assemblers. Although these have been extensively tested on model species such as Homo sapiens and Drosophila melanogaster, such benchmarking has not been done in Mollusca, which lacks widely adopted model species. Molluscan genomes are notoriously rich in repeats and are often highly heterozygous, making their assembly challenging. Here, we benchmarked 10 assemblers based on ONT raw reads from two published molluscan genomes of differing properties, the gastropod Chrysomallon squamiferum (356.6 Mb, 1.59% heterozygosity) and the bivalve Mytilus coruscus (1593 Mb, 1.94% heterozygosity). By optimizing the assembly pipeline, we greatly improved both genomes from previously published versions. Our results suggested that 40-50X of ONT reads are sufficient for high-quality genomes, with Flye being the recommended assembler for compact and less heterozygous genomes exemplified by C. squamiferum, while NextDenovo excelled for more repetitive and heterozygous molluscan genomes exemplified by M. coruscus. A phylogenomic analysis using the two updated genomes with 32 other published high-quality lophotrochozoan genomes resulted in maximum support across all nodes, and we show that improved genome quality also leads to more complete matrices for phylogenomic inferences. Our benchmarking will ensure efficiency in future assemblies for molluscs and perhaps also for other marine phyla with few genomes available. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

High-quality, haplotype-phased de novo assembly of the highly heterozygous fig genome, a major genetic resource for fig breeding

High-quality, haplotype-phased de novo assembly of the highly heterozygous fig genome, a major genetic resource for fig breeding

A draft phased assembly of the diploid Cascade hop (Humulus lupulus) genome.

Hop (Humulus lupulus L. var Lupulus) is a diploid, dioecious plant with a history of cultivation spanning more than one thousand years. Hop cones are valued for their use in brewing and contain compounds of therapeutic interest including xanthohumol. Efforts to determine how biochemical pathways responsible for desirable traits are regulated have been challenged by the large (2.8 Gb), repetitive, and heterozygous genome of hop. We present a draft haplotype-phased assembly of the Cascade cultivar genome. Our draft assembly and annotation of the Cascade genome is the most extensive representation of the hop genome to date. PacBio long-read sequences from hop were assembled with FALCON and partially phased with FALCON-Unzip. Comparative analysis of haplotype sequences provides insight into selective pressures that have driven evolution in hop. We discovered genes with greater sequence divergence enriched for stress-response, growth, and flowering functions in the draft phased assembly. With improved resolution of long terminal retrotransposons (LTRs) due to long-read sequencing, we found that hop is over 70% repetitive. We identified a homolog of cannabidiolic acid synthase (CBDAS) that is expressed in multiple tissues. The approaches we developed to analyze the draft phased assembly serve to deepen our understanding of the genomic landscape of hop and may have broader applicability to the study of other large, complex genomes.