Genetic diversity analysis of the Amaranthus genus using genomic tools

Abstract

The benefits of traditional leafy vegetables and global concerns about biodiversity loss have led researchers to renew their interest in nutritious underutilized crops. Amaranthus species are highly promising nutritious leafy vegetable food sources in Africa and have appreciable levels of essential micronutrients. Uninformative morphological characters and poorly resolved marker dendrograms have led to interest in alternative forms of molecular identifications in the Amaranthus genus. A draft genome sequence was assembled for a leafy amaranth accession, A. tricolor. Approximately 1.5 billion sequence reads were generated using the Illumina HiScan and MiSeq high-throughput sequencing platforms, composed of both paired end and long mate pair libraries. The de novo assembled genome had 44x coverage of the estimated 402 Mb genome size, had an N50 scaffold size of 51 Kb and a 54% repetitive sequence fraction. Additionally, the RNA-Seq transcriptomic analysis of leaf, shoot and root material was used to assemble a draft transcriptome (125 350 contigs, contig N50 of 1 036 bp) and subsequently used as evidence to infer gene models on the genomic scaffolds. Inadequate phylogenies, which revealed poor inter and intra species resolution, were obtained for a diverse set of Amaranthus species (14 unknown and 45 previously identified accessions) using chloroplast barcoding genes (MatK, rbcL and ITS). However, using the sequence data from the draft genome assembly, it was possible to extract, assemble and annotate a full-length chloroplast sequence for A. tricolor with approximately 200x coverage. Furthermore, shallow sequencing of the remaining Amaranthus species enabled the reference guided assembly of all the chloroplasts. The phylogenomic analysis of the Amaranthus species revealed classifications of unknown accessions and shed light on the possible domestication events of modern day cultivated grain amaranth. Amaranthus tricolor grouped together with additional leafy amaranth, A. blitum, A. viridis and A. graecizans, representing the Amaranthus subgenus Albersia. The genome also revealed 671 microsatellites with the potential as SSR markers and further validation revealed six robust SSR markers for inter- and intraspecies genotyping. Allele scores obtained from both gel based capillary electrophoresis (ABI) and next generation sequencing (MiSeq) technologies were highly comparable. Downstream analysis of SSR markers screened with NGS revealed informative genetic relationships between known and unknown Amaranthus accessions. The draft genome sequence proved to be extremely useful in generating molecular tools for this important plant genus. In combination, these genomic tools can be further utilized in future to assist in selecting and exploiting genotypes for breeding programs and contribute to global food security.Globale bekommernisse oor biodiversiteitsverlies en verborge voordele van blaargroentes het navorsers gelei om hul belangstelling in die ontwikkeling en instandhouding van voedsame, onderbenutte gewasse te vernuwe. Amaranthus-spesies is hoogs belowende blaargroente-tipes in veral Afrika, met aansienlike hoë vlakke van noodsaaklike mikronutriënte. 'n Konsepgenoom-volgorde is saamgestel vir 'n blaaragtige amarant spesie, Amaranthus tricolor. Ongeveer 1.5 biljoen volgorde fragmente is gegenereer deur die Illumina HiScan- en MiSeq-platforms, deur kortmaat-paar en langmaat-paar volgordes te gebruik. Die de novo saamgestelde genoom het 44x dekking van die geraamde 402 Mb genoomgrootte (N50 van 51 Kb) geopenbaar en besit 'n 54% herhalende sekwensie-fraksie. Daarbenewens is die RNA-Seq transkriptomiese analise van blaar-, skiet- en wortelmateriaal gebruik om 'n transkripsoom (125 350 fragmente, N50 van 1 036 bp) op te stel, en daarna gebruik as bewys om geen-modelle op die genomiese fragmente af te lei. Onvoldoende filogenieë is verkry vir Amaranthus spesies met chloroplast “barcoding” gene (MatK, rbcL en ITS), wat swak inter- en intraspesie-resolusie geopenbaar het. Met behulp van die volgorde data uit die konsep genoom, was dit egter moontlik om 'n vol-lengte chloroplast volgorde vir A. tricolor te onttrek (met ongeveer 200x volgorde dekking) en te annoteer. Deur die A. tricolor volgorde as verwysing te gebruik, kon die res van die spesies se chloroplaste saamgestel word deur middel van lae vlak volgorde bepalings. Filogenomiese analise van die Amaranthus-spesies het die klassifikasies van onbekende spesies moontlik gemaak, en verder lig gewerp op die moontlike domestikasie van huidige gekweekte amarant. Amaranthus tricolor groepeer met ander blaar amaranth, A. blitum, A. viridis en A. graecizans, wat die subgenus Albersia verteenwoordig. Daarbenewens het die genoom 671 potensiële SSR lokusse onthul, en verdere validering van die merker sub-stel het 6 robuuste SSR merkers vir inter- en intraspesie genotipering beskikbaar gestel Resultate vir alleel inligting verkry uit gel-gebaseerde kapillêre elektroforese (ABI) en volgende generasie volgorde bepalings (NGS) tegnologieë was hoogs vergelykbaar. Die NGS analise van SSR-merkers, het informatiewe genetiese verwantskappe tussen Amaranthus-toetredings ge-openbaar. Die genoom bepaling vir A. tricolor het gelei na die suksesvolle ontwerp van molekulêre gereedskap vir hieropvolgende molekulêre analises in die Amaranthus genus. Hierdie genomiese/ molekulêre instrumente kan toekomstig gebruik word om hulp te verleen met die kies en implimentering van genotipes vir voornemende teelprogramme en moontlik uiteindelik bydra tot wêreldwye voedselsekuriteit.