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

Abstract

Background Cassava (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. Findings Here 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. Conclusions The 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.

Mutations in DNA polymerase δ subunit 1 mediate CMD2-type resistance to Cassava Mosaic Geminiviruses

“…1b, Supplementary Data 1). These data co-localize the CMD2/LCR locus with the previously identified CMD2 locus 9 , placing it on Chromosome12 between 5 and 13 Mb of the TME204 haplotype 1 assembly 23 (Fig. 2b).…”

“…Genotyping by Sequencing and in silico bulk segregant analysis: Approximately 1,300 individual F 1 progeny and the parental lines from the NASE14xTME204-LCR population generated in Kenya were characterised with genotyping-by-sequencing (GBS) at UW-Madison Biotechnology Center following their standard ApeKI restriction enzyme protocol. Reads of 100bp were demultiplexed 56 and mapped 57 to the TME204 hap1 assembly 23 . SNPs were called using GATK4 58,59 and quality filtered SNPs that were heterozygous in both parents retained using vcftools v0.1.14 60 .…”

“…The marker order in both TME7 and TME204 22,23 assemblies is different than in the AM560-2 v6.1 assembly 27 , suggesting a translocation or assembly error in the region which may have complicated previous efforts to find CMD2 (Fig. 2f).…”

“…We also pursued fine-mapping to pinpoint the CMD2/LCR genomic location. The recently released haplotype resolved genome assemblies of CMD2-resistant African cultivars TME7 22 and TME204 23 were leveraged to perform in silico bulk segregant analysis (BSA) (based on Takagi et al (2013) 24 and Mansfeld and Grumet (2018) 25 ) to map CMD2 resistance. First, F 1 progeny were screened in the field in Uganda and genotyped with GBS (Fig.…”

“…We identified 64 (∼6.57 cM) recombinants between markers M1 and M8 and further screened those individuals using three additional markers (M3, M5, M7). This allowed the identification of recombinants which narrowed the CMD2/LCR locus to 190 kb, between M3 (8,965,853 bp) and M7 (9,155,913 bp) in the TME204-hap1 assembly 23 (Fig. 2e,f).…”