Using full chloroplast genomes of ‘red’ and ‘yellow’ Bixa orellana (achiote) for kmer based identification and phylogenetic inference

- Using full chloroplast genomes of ‘ red ’ and.
- yellow ’ Bixa orellana (achiote) for kmer based identification and phylogenetic inference.
- Background: Full chloroplast genomes provide high resolution taxonomic discrimination between closely related plant species and are quickly replacing single and multi-locus barcoding regions as reference materials of choice for DNA based taxonomic annotation of plants.
- There is a wide range of color variation in pods of Bixa orellana for which genetic loci that distinguish phenotypes have not yet been identified.
- Here we apply whole chloroplast genome sequencing of “ red ” and “ yellow ” individuals of Bixa orellana to provide high quality reference genomes to support kmer database development for use identifying this plant from complex mixtures using shotgun data.
- Results: Fully assembled chloroplast genomes were produced for both red and yellow Bixa orellana accessions (158,918 and 158,823 bp respectively).
- Synteny and gene content was identical to the only other previously reported full chloroplast genome of Bixa orellana (NC_041550).
- We observed a 17 base pair deletion at position in both accessions, relative to NC_041550 and a 6 bp deletion at position and a snp at position 86,493 in red Bixa orellana..
- Conclusions: Our data provide high quality reference genomes of individuals of red and yellow Bixa orellana to support kmer based identity markers for use with shotgun sequencing approaches for rapid, precise identification of Bixa orellana from complex mixtures.
- Kmer based phylogeny of full chloroplast genomes supports monophylly of Bixaceae consistent with alignment based approaches.
- 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
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- Full list of author information is available at the end of the article.
- Complete chloroplast genomes provide numerous single nu- cleotide polymorphisms (snps) that can often discriminate be- tween closely related plant species [1] and even identify biogeographic diversification in the same species [2].
- FDA has created a database of full chloroplast genomes of plants found in foods and dietary supplements (Genometrakr CP) [3], and is beginning the assembly of a concordant database focused primarily on plants used in animal feed, which will include toxin producers, common contaminants and their close relatives.
- Recently, the use of whole chloroplast genomes as references (as opposed to DNA barcodes for.
- Whole chloroplast genomes also have demonstrated utility for phylogenetic reconstruction and inference of broad scale evolutionary relationships.
- An interesting phenomenon in Bixa orellana is the occurrence of mul- tiple variations of seed pod color (from yellow to green to magenta) (Fig.
- 1 Variation in seed pod color of Bixa orellana.
- Stars represent the two samples from the germplasm collection of Bixa orellana from the Facultad de Agronomía - Universidad Nacional de la Amazonía Peruana (UNAP) and the Instituto de Medicina Traditional (IMET), that were used for full chloroplast sequencing.
- to the full chloroplast sequencing for reference materials, might be - the identification of putative regions that dif- ferentiate seed pod color variants.
- 1) from the germplasm collection of the Facultad de Agronomía - Universidad Nacional de la Amazonía Peruana (UNAP), Iquitos and the Instituto de Medicina Tradicio- nal de ES SALUD (IMET) Iquitos, Peru for full chloro- plast sequencing..
- Here, we compare the assembled chloroplast genomes of ‘red’ and ‘yellow’ Bixa orellana individuals with the only other existing published Bixa orellana full chloro- plast accession, comparing gene count, organization and synteny.
- In addition to utility for phylogenic questions and development of identity markers, we demonstrate that chloroplast genomes can be used in conjunction with modern bioinformatic search tools (kmer based) to provide rapid and precise.
- identification of Bixa orellana (or any other plant) in modernized Next Generation Sequencing (NGS) ap- proaches to identification and authentication of ingredi- ents in complex mixtures such as natural products and animal feeds..
- An assembled chloroplast genome was produced for both yellow and red Bixa orellana accessions (Fig.
- The assembled size of the chloroplast was 158,823 and 158,918 for the yellow and red accessions respectively (Table 1).
- The synteny and gene content of the two ac- cessions reported here was identical to that of NC_.
- 2 Chloroplast genomes were annotated with the Verdant online chloroplast assembly tool (http://verdant.iplantcollaborative.org/plastidDB/)..
- The existing reference for Bixa, NC_041550, was added to the Verdant database prior to upload and analysis of our sequences.
- The resulting gff files were downloaded and used to annotate the assembled Bixa chloroplast genomes.
- The overall synteny of Theobroma and Bixa was continuous, and contrasted to Heritiera fomes, exhibited a much smaller inverted re- peat (25,000 for Bixa and Theobroma contrasted to 34, 494 for Heritiera fomes) with an accompanying decrease in gene number (129 and 125 for Bixa and Theobroma versus 130 for Heritiera) due to duplication of genes in single copy portions of the genome.
- The representative Diperocarpaceae genome was more similar in gene structure and content to Bixa and Theobroma than was the Heritiera, with a similar size of the Inverted Repeat (23,911) gene number (130) and relative number of CDS, tRNA and rRNA (86, 36 and 8) (Table 1)..
- MAFFT sequence alignment of the three chloroplast ge- nomes of Bixa allowed us to screen the samples for gen- etic variants.
- The three Bixa ac- cessions grouped together in a discrete monophyletic group no matter which approach was used, reflecting the accuracy of the kmer assembly, and the putative value of the entire chloroplast as a diagnostic marker for the group.
- The Thymelaeaceae were sister to the Bixaceae-Malvaceae clade, with the Dipterocarpaceae Table 1 Gene content and organization..
- 3 Cladograms of phylogenetic trees from: A) bar coding regions, B) five genes, and C) k-mer based phylogeny of Bixa orellana and other Malvales - with Arabidopsis as an outgroup.
- This differs to the APG IV assess- ment (www.mobot.org/MOBOT/research/APweb/) which places Thymelaeaceae basal to both Dipterocarpa- ceae and Bixaceae..
- We combined our two assembled Bixa chloroplast ge- nomes with all the chloroplast genomes for Malvales found within the NCBI Refseq repository (Table S1)..
- There were three refer- ence Bixa chloroplast genomes available (the two pre- sented here and NC_041550).
- We evaluated the accuracy of using whole chloroplast genomes by prepar- ing three separate databases where all combinations of two Bixa chloroplasts genomes were included, and then used the whole genome shotgun/skim data from our two samples (SRR10320715 and SRR10320716) as well as data from available SRA accessions (SRR7941588.
- SRR7941591) as input to test if the kmer software would correctly and unambiguously identify the raw data as that of Bixa orellana.
- We observed a 100% accuracy in assigning Bixa se- quence data to Bixa reference chloroplast genomes (e.g..
- As we work towards a higher resolution understanding of the total ecology and biochemistry of food and the complex intersection it has with mammalian microbiota and states of health and disease, the organization of tools and methods to describe the full spectrum of ingredients in food and animal feeds is crucial.
- Shown here is successful identification of Bixa at and 0.03%.
- Either way, high quality reference databases are crucial to the accurate description of plant materials in foods..
- Kmer based detection and phylogenetic inference A next frontier for DNA based methods is the use of kmers generated from chloroplast genomes to create nu- merous specific targets that can detect the presence of specific species in complex mixtures using metagenomic and/or shotgun data sets of food and food ecologies..
- This approach was evaluated here for the identification of Bixa orellana from plant mixtures.
- The full genomes of Bixa orellana were sequenced, kmers were developed, and mixtures were queried against kmer formatted refer- ence genome databases.
- Bixa orellana was consistently identified even when present at very small percentages of the total mixture with the lowest limit to exhibit reli- able diagnostic capacity being 0.3% (Fig.
- Thus, use of the entire chloroplast genome functioned as a genome scale DNA barcode and readily distinguished Bixa, even in admixture..
- Use of complete chloroplast genomes can also provide reliable insight into phylogenetic relationships among groups.
- An increasing number of published studies have applied a combination of coding genes extracted from diverse chloroplast genomes for use in phylogenetic re- construction [13, 14].
- We demonstrated here that kmer based alignment free distance metrics, D2 and D2*, can be used to estimate genetic distances among a set of rep- resentative Malvales chloroplast genomes (Table S1)..
- We observed with kmer distance metrics and traditional alignment based barcoding (and MLST) phylogenies that Bixaceae was sister to the Malvaceae..
- Biology and chemistry of Bixa oreallana.
- Bixa orellana was identified for complete chloroplast se- quencing because of its importance in many food and feed formulations.
- Arils of Bixa seeds produce bixin and norbixin apocarotenoids which impart bright red, or- ange, and yellow colors used in cheeses, butter, oils,.
- The ‘presscake’ of the seedpod is also used as fodder for animals [4] and in poultry feed to provide rich shades of yellow and orange in egg yolks and chicken flesh..
- Leaves of Bixa orellana also have a long history of use in traditional medicine throughout the Americas [15–17].
- and extracts of the fruit and leaf have demonstrated antibiotic activity against Staphylococcus aureus, Escheri- chia coli and Salmonella typhi.
- 5), of Bixa orellana is the part of the plant where bixin (the monomethyl ester of a dicarboxylic carotenoid (C 25 H 30 O 4.
- the saponified form of the same ca- rotenoid) are produced [4]..
- The major pigment of the fruit is cis-bixin.
- Use of natural color from Bixa orellana in food and textile industries continues to grow in popularity as the use of similar red and yellow colors provided by.
- 5 Seed pods and seed arils of Bixa orellana.
- The structure of the two chloroplast genomes presented here was effectively identical to that of the previously published chloroplast genome of Bixa orellana (NC_.
- Complete synteny was maintained, and the size and gene content of the inverted repeats was the same in all three accessions (Table 1).
- Accession NC_041550.1 exhibited a 17 base gap at 58, 299 in a intergenic spacer between atpB and rbcL rela- tive to the other accessions, ‘red’ Bixa contained a 6 base gap at 75,537 in the intronic region of clpP, in addition to a snp at 86493.
- Individuals were collected from Bixa orellana accessions in the display/research garden of the Instituto de Medi- cina Tradicional de ES SALUD (IMET), (Iquitos, Peru) and the germplasm collection of Bixa orellana in the Facultad de Agronomía - Universidad Nacional de la Amazonía Peruana (UNAP).
- DNA extraction from leaves was performed using DNeasy Plant Mini Kit from Qia- gen® according to the manufacturers specifications.
- DNA was prepared for sequencing using the Nextera™ DNA Flex Library Prep Kit according to the manufacturer’s.
- Following read preparation, genome assembly followed the workflow described in [29] where reads are initially mapped to the nearest relative to enrich for chloroplast derived reads, and then denovo assembled to establish initial contigs, followed by subsequent subdiv- ision and reassembly of contigs in an iterative fashion until the set of contigs can be combined to single complete chromosome.
- Paired, filtered reads were mapped to Bixa orellana (NC_041550) to enrich reads for chloroplast specific se- quences using Geneious R11 (https://www.geneious..
- In parallel, all paired, filtered reads were denovo assembled with SPAdes to generate contigs independently of the refer- ence genome to ensure all changes in structure and syn- teny could be identified.
- Assembled contigs were compared to the reference genome and gaps were filled by extracting sequences ad- jacent to the gaps and mapping the paired filtered reads to those sequences using Bowtie assembler [31] in a ref- erence guided assembly.
- Contigs extended by Bowtie were then subsequently combined with previous contig sets and again compared to the reference genome.
- The existing reference for Bixa, NC_041550, was added to the Verdant database.
- The resulting gff files were downloaded and used to annotate the assembled Bixa chloroplast genomes (Fig.
- We then compared the number, synteny and organization of the set of genes relative to other chloroplast genomes within the Malvales (Theobroma and Heritiera (Malvaceae), Vatica (Dipterocarpaceae) and Daphne (Thymelaeceae.
- Gain or loss of genes was denoted, and movement of genes among subunits of the genome (LSC, IR, SSC) based on expansion of the Inverted Repeat was recorded..
- Representative Chloroplast genome sequences for Mal- vales were downloaded from the GenBank RefSeq library for use in evaluation of the monophylly of the Bixa orel- lana genomes, as well as for inference of the relationship of Bixaceae to Malvales, and in particular with respect to its status as sister group to either Malvaceae or Thyme- laeceae.
- The two Bixa assembled in this study in addition to the existing Bixa reference genome were included in a group of 37 additional Malvales chloroplast genomes (see Table S1) as well as Arabidopsis thaliana from the sister order Brassicales as an outgroup.
- Mixed data was subsequently combined with dif- ferent proportions of Bixa orellana plastome data and analyzed using Genome2-ID portal, a metagenomic soft- ware for plant taxonomic classification using kmers (DNA4 Technologies, LLC).
- To evaluate limit of detec- tion, random selection of Bixa at were added to the mixture..
- to identify, based on this approach, the point at which there was until no longer statistical support for identifi- cation of Bixa from the mixed sample..
- We would like to thank the staff of the Instituto de Medicina Traditional in Iquitos, Peru for their beautiful up-keep of the Medicinal Plants Garden.
- Raw sequence data were deposited in NCBI Sequence Read Archive (SRR10320715 and SRR10320716) as part of the FDA GenomeTrakrCP:.
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