Reference quality assembly of the 3.5-Gb genome of Capsicum annuum from a single linked-read library

Linked-Read sequencing technology has recently been employed successfully for de novo assembly of human genomes,however,the utility of this technology for complex plant genomes is unproven.We evaluated the technology for this purpose by sequencing the 3.5-gigabase(Gb)diploid pepper(Capsicum annuum)genome with a single Linked-Read library.Plant genomes,including pepper,are characterized by long,highly similar repetitive sequences.Accordingly,significant effort is used to ensure that the sequenced plant is highly homozygous and the resulting assembly is a haploid consensus.With a phased assembly approach,we targeted a heterozygous F1 derived from a wide cross to assess the ability to derive both haplotypes and characterize a pungency gene with a large insertion/deletion.The Supernova software generated a highly ordered,more contiguous sequence assembly than all currently available C.annuum reference genomes.Over 83%of the final assembly was anchored and oriented using four publicly available de novo linkage maps.A comparison of the annotation of conserved eukaryotic genes indicated the completeness of assembly.The validity of the phased assembly is further demonstrated with the complete recovery of both 2.5-Kb insertion/deletion haplotypes of the PUN1 locus in the F1 sample that represents pungent and nonpungent peppers,as well as nearly full recovery of the BUSCO2 gene set within each of the two haplotypes.The most contiguous pepper genome assembly to date has been generated which demonstrates that Linked-Read library technology provides a tool to de novo assemble complex highly repetitive heterozygous plant genomes.This technology can provide an opportunity to cost-effectively develop high-quality genome assemblies for other complex plants and compare structural and gene differences through accurate haplotype reconstruction.

A HapMap leads to a Capsicum annuum SNP infinium array: a new tool for pepper breeding

The Capsicum genus(Pepper)is a part of the Solanacae family.It has been important in many cultures worldwide for its key nutritional components and uses as spices,medicines,ornamentals and vegetables.Worldwide population growth is associated with demand for more nutritionally valuable vegetables while contending with decreasing resources and available land.These conditions require increased efficiency in pepper breeding to deal with these imminent challenges.Through resequencing of inbred lines we have completed a valuable haplotype map(HapMap)for the pepper genome based on single-nucleotide polymorphisms(SNP).The identified SNPs were annotated and classified based on their gene annotation in the pepper draft genome sequence and phenotype of the sequenced inbred lines.A selection of one marker per gene model was utilized to create the PepperSNP16K array,which simultaneously genotyped 16405 SNPs,of which 90.7%were found to be informative.A set of 84 inbred and hybrid lines and a mapping population of 90 interspecific F2 individuals were utilized to validate the array.Diversity analysis of the inbred lines shows a distinct separation of bell versus chile/hot pepper types and separates them into five distinct germplasm groups.The interspecific population created between Tabasco(C.frutescens chile type)and P4(C.annuum blocky type)produced a linkage map with 5546 markers separated into 1361 bins on twelve 12 linkage groups representing 1392.3 cM.This publically available genotyping platform can be used to rapidly assess a large number of markers in a reproducible high-throughput manner for pepper.As a standardized tool for genetic analyses,the PepperSNP16K can be used worldwide to share findings and analyze QTLs for important traits leading to continued improvement of pepper for consumers.Data and information on the array are available through the Solanaceae Genomics Network.