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Summary Stevia rebaudiana Bertoni is popular source of plant‐derived low/no‐calorie natural sweeteners (LNCSs), collectively known as steviol glycosides (SGs). Nevertheless, genetic predisposition for targeted biosynthesis of SGs is complex due to multi‐substrate functionality of key uridine diphosphate glycosyltransferases (UGTs). Here, we created a high‐quality monoploid assembly of 1.34 Gb with N50 value of 110 Mb, 55 551 predicted protein‐coding genes, and ~80% repetitive regions in Rebaudioside‐A (Reb‐A) enriched cultivar of S. rebaudiana. Additionally, a haplotype‐based chromosome assembly consisting of haplotype A and haplotype B with an overall genome size of 2.33Gb was resolved, harbouring 639 634 variants including single nucleotide polymorphisms (SNPs), indels and structural variants (SVs). Furthermore, a lineage‐specific whole genome duplication analysis revealed that gene families encoding UGTs and Cytochrome‐P450 (CYPs) were tandemly duplicated. Additionally, expression analysis revealed five tandemly duplicated gene copies of UGT76G1 having significant correlations with Reb‐A content, and identified key residue (leu200val) in the glycosylation of Reb‐A. Furthermore, missense variations identified in the acceptor region of UGT76G1 in haplotype resolve genome, transcriptional and molecular docking analysis were confirmed with resequencing of 10 diverse stevia genotypes (~25X). Gene regulatory network analysis identified key transcription factors (MYB, bHLH, bZIP and AP2‐ERF) as potential regulators of SG biosynthesis. Overall, this study provides haplotype‐resolved chromosome‐level genome assembly for genome editing and enhancing breeding efforts for targeted biosynthesis of SGs in S. rebaudiana. [ABSTRACT FROM AUTHOR]

Yellowhorn (Xanthoceras sorbifolium), especially its varieties, is the red petal yellowhorn (X. sorbifolium var. purpurea), an important tree species with great ornamental value, and its flower petals change color throughout the flowering period. In this study, we mainly focused on the mechanism of the petal color change with transcriptomics and metabolomics data. A phased chromosome-scale assembly of the red petal yellowhorn genome was generated using the PacBio high-fidelity reads, Illumina short reads, and Phase genomics Proximo Hi-C data. The final de novo assembly yielded 533.67 Mb with a contig N50 of 5.42 Mb, and 27 501 protein-coding genes were predicted. Notably, an alternate haplotig assembly was also obtained. Furthermore, a variation database for the alleles within the genome was constructed. Subsequently, the expression pattern of flower pigmentation-related genes and allelic expression imbalance events were investigated. Apart from 6 genes involved in the anthocyanin biosynthesis pathway regulated by the activation of 15 MYB-bHLH-WD40s, the increased expression of senescence-related gene 1 (SRG1) and 2-oxoglutarate-dependent dioxygenase (DIOX5) might also result in decreasing content of lutein and increasing abundance of (E/Z)-phytoene, cyanidin-3-O-rutinoside, and cyanidin-3-O-sambubioside. These changes in genes and metabolites were most likely related to the petal color change in red petal yellowhorn. This phased chromosome-scale genome assembly provides more accurate genomic information for future molecular breeding and facilitates allele function studies of the red petal yellowhorn.

Summary: Understanding chromosome recombination behavior in polyploidy species is key to advancing genetic discoveries. In blueberry, a tetraploid species, the line of evidences about its genetic behavior still remain poorly understood, owing to the inter‐specific, and inter‐ploidy admixture of its genome and lack of in depth genome‐wide inheritance and comparative structural studies.Here we describe a new high‐quality, phased, chromosome‐scale genome of a diploid blueberry, clone W85. The genome was integrated with cytogenetics and high‐density, genetic maps representing six tetraploid blueberry cultivars, harboring different levels of wild genome admixture, to uncover recombination behavior and structural genome divergence across tetraploid and wild diploid species.Analysis of chromosome inheritance and pairing demonstrated that tetraploid blueberry behaves as an autotetraploid with tetrasomic inheritance. Comparative analysis demonstrated the presence of a reciprocal, heterozygous, translocation spanning one homolog of chr‐6 and one of chr‐10 in the cultivar Draper. The translocation affects pairing and recombination of chromosomes 6 and 10. Besides the translocation detected in Draper, no other structural genomic divergences were detected across tetraploid cultivars and highly inter‐crossable wild diploid species.These findings and resources will facilitate new genetic and comparative genomic studies in Vaccinium and the development of genomic assisted selection strategy for this crop. [ABSTRACT FROM AUTHOR]

In recent years, improved sequencing technology and computational tools have made de novo genome assembly more accessible. Many approaches, however, generate either an unphased or only partially resolved representation of a diploid genome, in which polymorphisms are detected but not assigned to one or the other of the homologous chromosomes. Yet chromosomal phase information is invaluable for the understanding of phenotypic trait inheritance in the cases of compound heterozygosity, allele-specific expression or cis-acting variants. Here we use a combination of tools and sequencing technologies to generate a de novo diploid assembly of the human primary cell line WI-38. First, data from PacBio single molecule sequencing and Bionano Genomics optical mapping were combined to generate an unphased assembly. Next, 10x Genomics linked reads were combined with the hybrid assembly to generate a partially phased assembly. Lastly, we developed and optimized methods to use short-read (Illumina) sequencing of flow cytometry-sorted metaphase chromosomes to provide phase information. The final genome assembly was almost fully (94%) phased with the addition of approximately 2.5-fold coverage of Illumina data from the sequenced metaphase chromosomes. The diploid nature of the final de novo genome assembly improved the resolution of structural variants between the WI-38 genome and the human reference genome. The phased WI-38 sequence data are available for browsing and download at wi38.research.calicolabs.com. Our work shows that assembling a completely phased diploid genome de novo from the DNA of a single individual is now readily achievable.

In recent years, improved sequencing technology and computational tools have made de novo genome assembly more accessible. Many approaches, however, generate either an unphased or only partially resolved representation of a diploid genome, in which polymorphisms are detected but not assigned to one or the other of the homologous chromosomes. Yet chromosomal phase information is invaluable for the understanding of phenotypic trait inheritance in the cases of compound heterozygosity, allele-specific expression or cis-acting variants. Here we use a combination of tools and sequencing technologies to generate a de novo diploid assembly of the human primary cell line WI-38. First, data from PacBio single molecule sequencing and Bionano Genomics optical mapping were combined to generate an unphased assembly. Next, 10x Genomics linked reads were combined with the hybrid assembly to generate a partially phased assembly. Lastly, we developed and optimized methods to use short-read (Illumina) sequencing of flow cytometry-sorted metaphase chromosomes to provide phase information. The final genome assembly was almost fully (94%) phased with the addition of approximately 2.5-fold coverage of Illumina data from the sequenced metaphase chromosomes. The diploid nature of the final de novo genome assembly improved the resolution of structural variants between the WI-38 genome and the human reference genome. The phased WI-38 sequence data are available for browsing and download at wi38.research.calicolabs.com. Our work shows that assembling a completely phased diploid genome de novo from the DNA of a single individual is now readily achievable. [ABSTRACT FROM AUTHOR]

Phasing, and in particular polyploid phasing, have been challenging problems held back by the limited read length of high-throughput short read sequencing methods which can't overcome the distance between heterozygous sites and labor high cost of alternative methods such as the physical separation of chromosomes for example. Recently developed single molecule long-read sequencing methods provide much longer reads which overcome this previous limitation. Here we review the alignment-based methods of polyploid phasing that rely on four main strategies: population inference methods, which leverage the genetic information of several individuals to phase a sample; objective function minimization methods, which minimize a function such as the Minimum Error Correction (MEC); graph partitioning methods, which represent the read data as a graph and split it into k haplotype subgraphs; cluster building methods, which iteratively grow clusters of similar reads into a final set of clusters that represent the haplotypes. We discuss the advantages and limitations of these methods and the metrics used to assess their performance, proposing that accuracy and contiguity are the most meaningful metrics. Finally, we propose the field of alignment-based polyploid phasing would greatly benefit from the use of a well-designed benchmarking dataset with appropriate evaluation metrics. We consider that there are still significant improvements which can be achieved to obtain more accurate and contiguous polyploid phasing results which reflect the complexity of polyploid genome architectures. • We propose a classification of alignment-based polyploid phasing methods into four categories. • The benefits and drawbacks of commonly used benchmarks and performance metrics are discussed. • Indels, polyallelic sites, aneuploidy and heterozygous structural variants are still largely overlooked. • We present the Phasing Toolkit, an open source project to simplify benchmarking and the use of phasing tools. [ABSTRACT FROM AUTHOR]

The generation of haplotype information has recently become very attractive due to its utility for identifying mutations associated with human disease and for the development of personalized medicine. Haplotype information also is crucial for studying recombination mechanisms and genetic diversity, and for analyzing allele-specific gene expression. Classic haplotyping methods require the analysis of hundreds of meiotic progeny. To facilitate haplotyping in the non-meiotic human fungal pathogen Candida albicans, we exploited trisomic heterozygous chromosomes generated via the UAU1 selection strategy. Using this system, we obtained phasing information from allelic biases, detected by SNP/CGH microarray analysis. This strategy has the potential to be applicable to other diploid, asexual Candida species that are important causes of human disease.