classification of amaranthus hybridus

J. The resultant VCF files were merged and based on the presence or absence of SNPs, a binary matrix was constructed from which a phylogenetic tree was obtained as mentioned above. This was supported by the solid stem characteristics of A. cruentus for A.cru_Suvarna as reported by Malligawad and Patil (2010). Integrating Hi-C links with assembly graphs for chromosome-scale assembly. Enhancements to the ADMIXTURE algorithm for individual ancestry estimation. Proc. The details of the sequencing are presented in Supplementary Table S1. The public and in house generated data were mapped to A.hyp.V.2.1 and AhKP reference using bowtie2 (Langmead and Salzberg, 2012). Li, H. (2020). SPD: classification, characterization and writing of the manuscript. The plant was used for food and medicine by several Native American groups and in traditional African medicine. Amaranthus hybridus f. aciculatus Thell. (accessed 23 Septemper 2015).∗, Soderlund, C., Bomhoff, M., and Nelson, W. M. (2011). We also improved AhK593 using simulated mate pairs from the reference genome of the Plainsman accession (Lightfoot et al., 2017), to build scaffolds of contigs from AhK593 to an L50 of 56 (AhK56) and subsequently using raw HiC data of the Plainsman accession from public sources to obtain a scaffold-level assembly with an L50 of 20 (AhK20) using SALSA (Ghurye et al., 2019). Furthermore, the classification of another accession, Suvarna, adapted to the local environment and selected for yield and other desirable traits, is clearly Amaranthus cruentus. Also, the browser can be queried using the accessions of Arabidopsis to visualize the expression profile of the corresponding orthologs on AhKP. In a review article, synteny has been used to filter, organize and process local similarities between genome sequences of related organisms to build a coherent global chromosomal context (Batzoglou, 2005). In that, the genome of the landrace AhKP, reported here, is closest to accession A.hyp_Nepal_PI619259 and to A.hyp_India_PI481125. PLoS One 12:e0180528. – Australian amaranth P Farm Sci. The draft genomes of four species including bushbaby, African elephant, rabbit and guinea pig from the “Mammal 24 - 2X” project were built using both human and canine references (Gnerre et al., 2009). (A) Gene expression profile of 12 genes involved in lysine biosynthesis pathway across different developmental stages (15, 20, 25, and 30 days) of different tissues (Rt, root; St, stem; L, leaf; Bud, Flwr, Flower; YSe, young seed; and MSe, mature seed). The average insert size was around 200 bp for PE libraries and 1.75, 3, 5, and 10 kb for four MP libraries. The clusters were generated in cBot and paired-end sequenced on Illumina HiSeq 2500 platform. The evolution of herbicide‐resistant weeds is one of the most important concerns of global agriculture. doi: 10.1371/journal.pone.0180528, Vij, S., Kuhl, H., Kuznetsova, I. S., Komissarov, A., Yurchenko, A. Of these only 20,548 positions could be found covered in all whole-genome sequencing data across all samples studied here. PLoS Comput. (2012). Sci. Possible aliases, alternative names and misspellings for Amaranthus hybridus. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. We selected this landrace because it is well adapted for cultivation in India during the last century and is currently a candidate for TILLING-based crop improvement. 12:e1006500. thus retroflexus, Amaranthus graecizans, Amaranthus dubius, and Amaranthus hybridus. Grain amaranth, also known as Ramdana (The God’s grain) or Rajgira or Rajeera, has been in continuous cultivation at least since last century in India. Bioinformatics 26, 841–842. Since this landrace is more closely similar to all other landraces and accessions for A. hypochondriacus from India and South Asia (Supplementary Table S1), AhKP offers a better reference for the improvement of grain amaranth crops in South Asia. Fingerprints for two grain amaranthus varieties KBGA1 and Suvarna using RAPD and legume based SSR markers. Amaranthus hecticus Willd. Phytochemical Studies of Rajigara Amaranthus Species for its Potential use in Medicine. The draft genome and transcriptome of Amaranthus hypochondriacus: a C4 dicot producing high-lysine edible pseudo-cereal. The placement of A.hyp_K_white on the phylogenetic tree of grain amaranths of known accessions clearly suggests that A.hyp_K_white is genetically distal from Plainsman and is most closely related to the accession PI619259 from Nepal (Ramdana). GBS raw data of 95 accessions were downloaded from Wu and Blair (2017) of which A. palmeri was excluded from the analysis because of the reported high level of missing data. A., Van Heusden, P., et al. slim amaranth smooth pigweed This plant can be weedy or invasive according to the authoritative sources noted below.This plant may be known by one or more common names in different places, and some are listed above. Amaranthus hybridus subsp. Abstract. 39:e68. The successful integration of WGS and GBS data attempted here, is apparent from the clustering of variants from WGS and GBS data for the same accession together as marked with red arrow in Figure 4A. Since then, the chromosome-level genome of a different cultivar with an accession of PI558499 (Plainsman), has been deciphered using state-of-the-art technologies including Bionano, HiC, and long PacBio reads (Lightfoot et al., 2017). The normalization is validated by clustering of both WGS and GBS data from A.hyp_Plainsman_PI558499 and A.hyp_Mexico_PI511731 close to each other (Figure 4A, red arrow). (2015). Leaves glabrous or with sparse hairs on the main veins below… 1849. Here, to understand/translate the high-lysine phenotype and to validate the gene structure obtained from our annotation efforts, the transcriptomes have been mapped to AhKP reference and the expression profiles of the predicted genes have been generated across the developmental stages. In this report, Suvarna is undoubtedly classified as A. cruentus based on 20,548 reported SNPs and roughly 6 million variants covered in WGS data (Figure 3). doi: 10.1371/journal.pcbi.1007273, Gnerre, S., Lander, E. S., Lindblad-Toh, K., and Jaffe, D. B. Acad. & Godr. Only 20,548 SNPs were commonly covered in all 13 WGS datasets and were used during classification. (2016). ADMIXTURE analysis shown in Figure 4B, also suggests that there is significant gene flow between A. caudatus and A. quitensis. Further, we generated synteny of the AhK20 against A.hyp.V.2.1 using Symap (Soderlund et al., 2011) based on which AhK20 was improved to the final AhKP assembly. Interestingly, this germplasm includes seeds from many amaranth landraces from South Asia including India. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Such an approach can simply be extended for reference-guided improvement of draft genomes of a plant using simulated mate pairs of varying insert sizes from an existing assembly of a different variety/cultivar of the same species. Figure 1A shows the synteny of the scaffolds from the assembly AhK20 on A.hyp.V.2.1 and Figures 1B–D show synteny of AhKP on to A.hyp.V.2.1 (Lightfoot et al., 2017) in various representations. We also downloaded WGS data from NCBI for seven other accessions including A.cau_Bolivia_PI642741, A.cru_Mexico_PI 477913, A.hyp_India_PI481125, A.hyp_Plainsman_PI558499, A.hyp_Nepal_PI619259, A.hyp_Pakistan_PI540446, A.hyp_ Mexico_PI511731, and A.hyb_Greece_PI605351. Also, the two landraces under A. hypochondriacus, A.hyp_K_white and A.hyp_K_red, cluster apart within the same clade validating our observation that the seeds of these two varieties faithfully produce plants with inflorescence unique to the respective phenotype. It is extremely variable, and many other Amaranthus species are believed to be natural hybridizations or derive from A. hybridus. Giving chromosomal context to genes and other genetic elements is one of the most sought-after goals in genome assembly. “Variability in ‘Plainsman’ grain amaranth,” in Perspectives on New Crops and New Uses, ed. doi: 10.1186/gb-2009-10-8-r88. Assisted assembly: how to improve a de novo genome assembly by using related species. These regions were used to restrict the variant calling from whole-genome data to only the regions covered in GBS. doi: 10.1093/bib/6.1.6, Berardini, T. Z., Reiser, L., Li, D., Mezheritsky, Y., Muller, R., Strait, E., et al. Amaranthus is part of the Amaranthaceae that is part of the larger grouping of the Carophyllales. Amaranthus shows a wide variety of morphological diversity among and even within certain species. Classification Browse Images ... Amaranthus hybridus L., Sp. KRR: assembly and analysis of other landraces. Amaranthus hybridus was originally a pioneer plant in eastern North America. doi: 10.1086/519795, Quinlan, A. R., and Hall, I. M. (2010). 990. Suvarna (R 104-1 -1), a pureline released by University of Agricultural Sciences (UAS), Bangalore-1992 from the material “Rodale Plus” received from Rodale Institute (Rathod, 2017) has previously been classified as A. hypochondriacus in Meera et al., 2014. Genesis 53, 474–485. (A) Shows a classification of 94 accessions from GBS data and 13 from WGS data after normalization of the two sequencing approaches using 271,305 SNPs. ER: forinitiating translational work and validating transcripts. One way to do this would be to identify and use the alleles found from GBS data with the respective alleles found from WGS. Plant Genome 2, 260–270. The quality, quantity, and size distribution of the libraries were evaluated using Qubit (Invitrogen) and TapeStation (Agilent) (Sunil et al., 2014). Hojas acuminadas o agudas hacia el ápice con … Genome annotation and curation using MAKER and MAKER-P. Curr. It took about 500 years after the Columbian exchange and intense efforts by the United States before this grain received the much-deserved global attention. RepeatModeler2: automated genomic discovery of transposable element families. A.cru_Suvarna, yet another landrace sequenced and reported here is clearly classified as A. cruentus. Food Sci. PGS: for guidance throughout the project. The database is built using HTML5, bootstrap and JavaScript. 8:1960. doi: 10.3389/fpls.2017.01960, Zheng, X., Levine, D., Shen, J., Gogarten, S. M., Laurie, C., and Weir, B. S. (2012). This crop was declared “The Future Crop” by the United States in the 1980s based on a decade of intense research in the 1980s (National Research Council, Amaranth: Modern Prospects for an Ancient Crop, National Academy Press, Washington, DC, 1986). More recently, a high-quality chromosome-level assembly of A. hypochondriacus (PI558499, Plainsman) was reported. doi: 10.1093/nar/gkr123, Sunil, M., Hariharan, A. K., and Nayak, S. (2014). Also, the tree generated prior to the normalization method has been presented in Supplementary Figure S3 for highlighting the extent of improvement upon normalization. Toward this end, our lab had initiated sequencing the genome of Amaranthus (A.) It grows in many different places, including disturbed habitats. Beck Homonyms Amaranthus hybridus L. Amaranthus hybridus Vell. (A) Synteny of assemblies with respect to the Plainsman reference A.hyp.V.2.1 against AhK20. doi: 10.1111/j.1750-3841.2012.02645.x, Chakraborty, M., Baldwin-Brown, J. G., Long, A. D., and Emerson, J. J. Brenan Amaranthus hybridus var. Common Name(s): Smooth Amaranth, Slim Amaranth, Slender Pigweed, Slender Pigweed - Green Form, Slender Pigweed - Red Form, Green Amaranth, Green Pigweed, Pigweed, Smooth Pigweed The taxonomy-based classification seems reproducible with green being A. hypochondriacus, light blue being A. cruentus, dark blue being A. caudatus and yellow being Amaranthus quitensis. In this case, one could use synteny between species at protein levels to build chromosomes. … 11:579529. doi: 10.3389/fpls.2020.579529. doi: 10.1101/856591, Ghurye, J., Rhie, A., and Walenz, B. P. (2019). Bioinformatics 65:e57. Suvarna, an accession/landrace from India, often classified as A. hypochondriacus in the literature, clearly clusters with A. cruentus and shows high similarity to the accession A.cru_Mexico_PI477913, also classified as A. cruentus. Genet. https://en.wikipedia.org/w/index.php?title=Amaranthus_hybridus&oldid=971234259, Plants used in traditional African medicine, Plants used in traditional Native American medicine, Creative Commons Attribution-ShareAlike License, This page was last edited on 4 August 2020, at 22:31. The NCBI taxonomy database is not an authoritative source for nomenclature or classification - please consult the relevant scientific literature for the most reliable information. It is of interest to decorate this phylogenetic tree with landraces of importance to India and elsewhere. A whole-genome assembly of the domestic cow. This section is divided into three parts. Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M. A. R., Bender, D., et al. (2009). This complicates taxonomyand Amaranthus has generally been consider… Briefly, for PE libraries, the fragmented DNA was end-repaired, 3′-adenylated, ligated with Illumina adapters, and PCR enriched with Illumina sequencing indexes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). (A) Phylogenetic tree using the 20,548 SNPs reported for grain amaranths (green: A. hypochondriacus, blue: A. caudatus, and purple: A. cruentus, dashed arrows: ornamental, solid arrows: landraces with green star for A.hyp_K_white and red star for A.hyp_Plainsman variety, (B) Classification using 5,545,132 SNPs from the mapping of short reads onto AhKP as reference, (C) Classification using 6,383,490 SNPs from the mapping of short reads to A.hyp.V2.1 as reference. Also, assisted assembly of closely related species significantly improved the contiguity of low coverage mammalian assemblies (Gnerre et al., 2009). paniculatus Dostal, Kvetevna CSSR 444. Manley BS, Wilson HP, Hines TE, 1996. The first section describes the results from our efforts to assemble a near-chromosome level assembly for a landrace A.hyp_K_white using contigs from previously reported draft genome of A.hyp_K_white (Sunil et al., 2014), low-coverage PacBio reads and a high-quality reference genome of Plainsman (Lightfoot et al., 2017). SR: library preparation and aiding writing of manuscript. Polysaccharides extracted from traditional Chinese medicines may be effective substances with antioxidant activity. It has been naturalized in many places of warmer climate. Scaffolds from AhK20 are further stitched based on synteny to A.hyp.V.2.1 (Lightfoot et al., 2017) to get the final chromosome-level assembly AhKP for the accession A.hyp_K_white. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. SS: for overseeing the project and writing of the manuscript. Interestingly, the only A. hybridus accession from Greece (A.hyb_Greece_PI605351) included in the classification is not in the same clade as Plainsman but, instead, clusters along with all accessions from A. cruentus (near blue arrow in Figure 4A). This platform is a single-molecule, real-time (SMRT) sequencing machine that uses a sequencing-by-synthesis method to generate good quality very long reads. 77, R93–R104. This vegetable is known as an important source of iron. JBrowse: a next-generation genome browser. The size selection for all the libraries were done using solid-phase reversible immobilization (SPRI) beads (Agencourt AMPure XP Beads) from Beckman Coulter. (2012). Before long-read sequencing became commonplace, experimentally generated mate-pair reads of increasing insert sizes were routinely used to generate scaffolds from contigs. J. Mol. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. The authors wish to acknowledge GKVK for providing us with seeds for Suvarna and Dr. Xingbo Wu of Dr. Blair’s lab for providing us with raw GBS data from 94 accession. Amaranthus hybridus var. Amaranthus hybridus grows from a short taproot and can be up to 2.5 m in height. A, and Hubley, R (2008–2015). However, according to our classification with 271,305 SNPs, by combining GBS and WGS data, we reproduce the observation made by Wu and Blair (2017). Gandhinagar: INFLIBNET. We hypothesize that the only component showing light blue that is common between Suvarna and A.hyp_K_white in the ADMIXTURE with K = 5 and 6 (Figure 3B) holds the genotype responsible for inflorescence within this haplo-block. Genome Biol. DNA was sheared using Adaptive Focused Acoustic technology (Covaris, Inc.) to generate fragments of desired insert size. (2020). (2019). Amaranthus hybridus was originally a pioneer plant in eastern North America. The landrace A.hyp_K_white is currently being used to identify mutations in targeted loci for a given desirable phenotype from a germplasm collection using eco-TILLING and to discover novel mutations that result in desirable traits like determinate growth, enhanced seed yield, seed lysine content and oil content using TILLING-based approaches. doi: 10.1093/bioinformatics/btp352, Lightfoot, D. J., Jarvis, D. E., Ramaraj, T., Lee, R., Jellen, E. N., and Maughan, P. J. doi: 10.1093/dnares/dsu021, Sunil, M., Hariharan, N., Dixit, S., Choudhary, B., and Srinivasan, S. (2017). It has been reported to have been found in every state except Wyoming, Utah, and Alaska. Pl. These plants are grown as ornamentals or for their grain, they do not exist in the wild, and are not weeds. More recently, our group demonstrated that two draft genomes of the same species could be used to mutually improve scaffolding of the genome of Anopheles stephensi to the point that a set of low-resolution physical markers was sufficient to build the chromosomes (Chida et al., 2020). Amaranthus caudatus Baker & Clarke, in Dyer Amaranthus chlorostachys Willd. 19, 1630–1638. As a second approach, the phylogenetic tree shown in Figure 4A and generated using AhKP as reference (Figure 4A) combines variants called for the 94 accessions using both raw genotyping-by-sequencing (GBS) data from public sources (Wu and Blair, 2017) and whole-genome sequencing (WGS) data for listed accessions in Supplementary Table S2. SNAP (Korf, 2004) and Augustus were also used to predict gene models and used in the subsequent rounds of MAKER (Campbell et al., 2014). Seeds of A.hyp_K_white, A.hyp_K_red were obtained from local market in Karnataka, India, A.cru_ornamental, A.cau_ornamental from Park seeds and A.cru_Suvarna from Gandhi Krishi Vigyana Kendra (GKVK), Bengaluru, Karnataka, India. View all Smooth pigweed (Amaranthus hybridus) and livid amaranth (A. lividus) response to several imidazolinone and sulfonylurea herbicides. A detailed summary of this method of normalization has been explained in Figure 7. (B) Comparison of CDS sizes (nucleotides) of lysine biosynthesis pathway genes predicted in AhKP with Arabidopsis. U.S.A. 108, 10249–10254. 14, 155–167. The merged SNP file was processed using PLINK (Purcell et al., 2007) and ADMIXTURE (v1.3) was used to analyze the population structure (Alexander and Lange, 2011). Figure 6. Sci. Table 1 shows the assembly statistics. hybridus S. Watson Amaranthus eugenii Sennen Amaranthus flavescens Moq. 13(2): 259. This is because DNA diverges faster even between very closely related species. The database is also integrated with graphical visualization for gene expression data of 16 developmental stages with query search options. 5, 511–516. The analysis of the Hybridus complex within the A. Amaranthus subgenus revealed insights on the history of cultivated grain amaranths. 1a, g), origin of flower bud from For improving the agronomic traits of grain amaranth and for the transfer of desirable traits to dicot crops, a reference genome of a local landrace is necessary. Maker pipeline includes de novo assembled amaranth transcriptome with 125581 scaffolds, repeat elements predicted by RepeatModeler and Arabidopsis proteome (TAIR10) (Berardini et al., 2015). For a small number of landraces, it is not trivial to generate the sequences of the same 10% by reproducing the protocol/reagents, which challenges the placement of additional varieties on the phylogenetic tree generated by GBS. GBSX: a toolkit for experimental design and demultiplexing genotyping by sequencing experiments. Figure 2. Avaliable at: https://hort.purdue.edu/newcrop/proceedings1999/v4-184.html (accessed October 4, 2020). batalleri (Sennen) J.L.Carretero (synonym) Amaranthus hybridus subvar multispiculatus (Sennen) J.L.Carretero (synonym) Amaranthus incurvatus Tim. However, A.hyp_Plainsman_PI599488 clusters in a distal clade with A.hyp_Mexico_PI511731 in both Figures 3B, 4A as expected because Plainsman is a cross between A. hypochondriacus from Mexico and A. hybridus from Pakistan (Guillen-Portal et al., 1999). The flowchart below (Figure 6) shows the pipeline used to obtain the final assembly. Lh3/Wgsim. For example, mate-pair libraries from one Arabidopsis thaliana strain were shared across many strains to build super-scaffolds for all individuals (Schneeberger et al., 2011). We then selected K =4–6 as the optimal number subpopulation “K” based on the lowest cross-validation (c.v) error value plot as reported by ADMIXTURE(v1.3) where we see a dip in the c.v value at K = 4 and 6 (Supplementary Figure S4). 81, 559–575. doi: 10.1073/pnas.1220349110, Kolmogorov, M., Yuan, J., Lin, Y., and Pevzner, P. A. P: Species Amaranthus lineatus R. Br. 44:e147. (synonym) In the first approach we have attempted to create a classification tree with WGS data for landraces sequenced here and only those accessions for which WGS data was available (NCBI Project accession SRP061623). It has been reported to have been found in every state except Wyoming, Utah, and Alaska. Methods 9, 357–359. ex Gren. BASIONYM: Amaranthus incurvatus Grenier & Godron 1846. Users can access the Jbrowse by clicking on the Genome browser button or using the tools menu. (2009). bellardii Moq. The assembly statistics of the initial assembly was sufficient for reference-based scaffolding using both the simulated mate-pairs from the reference genome and raw HiC data for Plainsman from public resources (Lightfoot et al., 2017). We used multiple assembly tools adapted/developed recently to handle error-prone long reads and merged these assemblies with the contigs from our previously reported draft assembly. These two assemblies were then merged using Quickmerge (Chakraborty et al., 2016) to improve the L50 to 623. Besides, a C0t analysis shown in Supplementary Figure S2 suggests distinct dissociation time for simple repeat between these two accessions. RepeatModeler Open-1.0. This suggests that A. caudatus is a major clade under A. quitensis. (2017) from 16 developmental stages including leave, stem and root from 15,20,25 and 30 day plant, bud, flower, young seed and mature seed were mapped to AhKP reference using bowtie2 (Langmead and Salzberg, 2012). Figure 4 validates taxonomy-based classification (color-coded accessions) of the majority of the accessions and landraces. SyMAP v3.4: a turnkey synteny system with application to plant genomes. Nucleic Acids Res. VASCAN was last updated on 2020-09-04; Comment nous citer. In traditional medicinal system different parts of the plant Amaranthus hybridus (A. hybridus) 10:R42. These include A.hyp_K_white (Figure 2A), A.hyp_K_red (Figure 2B), two ornamental varieties A.cau_ornamental (love-lies-bleeding, Figure 2C) and A.cru_ornamental (Autumn touch, Figure 2D) and A.cru_Suvarna (Figure 2E). hypochondriacus (A.hyp_K_white) and had reported a draft genome in 2014. Recently, a chromosome level genome of Lates calcarifer was assembled from a draft genome using long-read sequencing, transcriptome data, optical/genetic mapping and synteny to two closely related seabasses (Vij et al., 2016). It is believed that these landraces, which are in contiguous cultivation in distal geographical locations in India, have already adapted to diverse environmental conditions prevalent in Nepal, as well as in East and South India. At K = 6 other unique components within A. hypochondriacus gets resolved. Fast gapped-read alignment with Bowtie 2. Basic local alignment search tool. 15:74. doi: 10.1186/s12915-017-0412-4, Love, M. I., Huber, W., and Anders, S. (2014). Plant Sci. The accession PI490752 originally classified as A. hypochondriacus now classifies under A. quitensis. No use, distribution or reproduction is permitted which does not comply with these terms. Weed Technology, 10(4):835-841; 22 ref. Amaranthus hybridus L. (Amaranthaceae) commonly known as ‘Cheera’ in Malayalam, is an erect branched annual herb distributed throughout tropical and temperate regions of India as a common weed in the agricultural fields and wastelands. This was further improved by merging the Illumina assembly from our previously reported draft genome of the same landrace A.hyp_K_white (Sunil et al., 2014), to get a contig-level assembly with an L50 of 593 (AhK593). BEDTools: a flexible suite of utilities for comparing genomic features. This practice went into oblivion after the Columbian exchange. The complex includes the three cultivated grain amaranths and their wild relatives and was well separated from other species in the subgenus. Based on the number of variants called for these and other accessions using both AhKP and A.hyp.V.2.1 as references suggest that all the landraces of Amaranthus hypochondriacus sequenced here are different from the A.hyp_Plainsman variety (Supplementary Table S2). Copyright © 2020 Deb, Jayaprasad, Ravi, Rao, Whadgar, Hariharan, Dixit, Sunil, Choudhary, Stevanato, Ramireddy and Srinivasan. A. patulus Bertoloni is a spreading form of A. hybridus. Schneeberger, K., Ossowski, S., and Ott, F. (2011). Diversity in grain amaranths and relatives distinguished by genotyping by sequencing (GBS). Amaranthus hybridus can be consumed in various ways, namely made vegetables, directly consumed when raw, or made into … We believe that this is the first demonstration of generating a cost-effective de novo assembly for a landrace utilizing low coverage PacBio reads in conjunction with the genome and HiC data from another accession. For MP libraries, the fragmented DNA was end-repaired, followed by end labeling using the biotin-dNTP mix, size selected and later, circularized using circularization ligase. Protoc. Abstract—The hybridus species complex of the genus Amaranthus is a group of weedy and cultivated plants from the New World that are considered difficult to identify. SW: development of genome browser. RepeatMasker analysis classified 50.5% (196421031 bp) of the AhKP genome as repetitive sequences. (2016). incurvatus (Timoroy ex Gren. In this context, the draft assembly of a landrace from India was sequenced and reported (Sunil et al., 2014). (2017). boiRxiv [Preprint]. In this article, we report a de novo assembly of a landrace (A.hyp_K_white) and demonstrate that, in the presence of a reference genome for a distal variety, a chromosome-level assembly can be generated at a reasonable cost. In the third and last section, results from classification of the landraces/varieties sequenced here with sequencing data for many accessions from public resources including GBS and WGS technologies are discussed. For genomics-based crop improvement of local landraces, it is critical to classify these with respect to accession from the germplasm collection. U.S.A. 110, 1785–1790. The two assemblies obtained were then merged together using Quickmerge (Chakraborty et al., 2016). 1753. doi: 10.1101/2020.06.27.174920, Flynn, J. M., Hubley, R., and Goubert, C. (2019). A draft genome for the same landrace was reported by our group in 2014 (Sunil et al., 2014). The Illumina data of all the plants with accessions listed in Supplementary Table S2 were downloaded from NCBI SRA (SRP061623). (2015). Genome Res. The many faces of sequence alignment. 1950. A. hybridus with accession of PI605351 clusters in the same clade as Suvarna with another accessions (PI477913) from A. cruentus. For example, there is a component (Figure 4B, pink) only present in all A. hypochondriacus from India, which is missing in Plainsman. It is interesting to note that A.cau_ornamental clusters close to A.cau_Bolivia_PI642741. Also, the read depth considered during variant calling was restricted using samtools mpileup to 10 to match the depth of GBS data (Li et al., 2009). It is among the species consumed as Quelite quintonilli in Mexican food markets. Genome Biol. (2017). The reads were demultiplexed using GBSX (Herten et al., 2015) using the provided barcode sequences. Li, H., Handsaker, B., and Wysoker, A. The Arabidopsis information resource: making and mining the “gold standard” annotated reference plant genome. While a large number of landraces, adapted to local environments for small scale cultivation exist, their origins and relations to the large germplasm, collection at GRIN-Global are not established. Figure 1. McLean KS, Roy KW, 1991. This is also obvious from the stem solidness as reported by Malligawad and Patil (2010) and as shown in Figure 2E. *Correspondence: Subhashini Srinivasan, ssubha@ibab.ac.in; Bibha Choudhary, vibha@ibab.ac.in, †These authors have contributed equally to this work, Front. Users can access the database from the link given in the data availability section. Front. Single-molecule sequencing and Hi-C-based proximity-guided assembly of amaranth (Amaranthus hypochondriacus) chromosomes provide insights into genome evolution. Amaranthaceae – Plant Classification Group – Famine Foods - Plants that are not normally considered as crops are consumed in times of famine - Purdue University, West Lafayette, Indiana Amaranthus catechu Moq. The SNPs were merged and used for phylogenetic classification. Here, a chromosome level assembly (AhKP) of a landrace, A.hyp_K_white, under contiguous cultivation in India for over several centuries is reported. Also, we normalized the variants from GBS and WGS data for various accessions enabling decoration of the phylogenetic tree including many accessions with the landraces of interest from India. Reference-assisted chromosome assembly. At K = 4 and 5 there is no resolution between species except A. hypochondriacus. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. J. Hum. Genomic DNA was extracted from fresh leaves using the DNeasy Mini Plant DNA Extraction kit (Qiagen) following the manufacturer’s protocol and quantified using fluorometry (Qubit 2.0, Invitrogen). Library preparation was done for Illumina whole genome sequencing in-house and outsourced for PacBio RSII sequencing. Amaranthus spinosus is an Annual herb with multi-branched, smooth, herbaceous annual growing to 2 ft. doi: 10.1073/pnas.1107739108, Skinner, M. E., Uzilov, A. V., Stein, L. D., Mungall, C. J., and Holmes, I. H. (2009). doi: 10.1002/0471250953.bi0411s48, Caselato-Sousa, V. M., and Amaya-Farfán, J. (2007). Differential genomic arrangements in Caryophyllales through deep transcriptome sequencing of A. hypochondriacus. The raw PacBio data was assembled using Canu (Koren et al., 2017) and Flye (Kolmogorov et al., 2019) independently. It grows in many different places, including disturbed habitats. Impact Factor 4.402 | CiteScore 7.8More on impact ›, Amaranthus: Naturally Stress-Resistant Resources for Improved Agriculture and Human Health Population genetic diversity was analyzed for four Amaranth species (A. hypochondriacus, A. caudatus, A. cruentus, and A. quitensis). doi: 10.1371/journal.pgen.1006500, Wu, X., and Blair, M. W. (2017). At K = 6, we also see pink components uniformly present in all the A. hypochondriacus from the Indian subcontinent with the exception of accessions A.hyp_Mexico_PI511721(GBS), A.hyp_Mexico_PI511731(GBS). In Argentina this weed first evolved multiple resistance (to 2 herbicide sites of action) in 2014 and infests Corn (maize), and Soybean. Amaranth is added as an ingredient in pasta, bread, instant drinks, baby’s food, etc. Bioinformatics 25, 2078–2079. (2009). Further DESeq2 (Love et al., 2014) was used to get normalized read counts of all the predicted genes using the MAKER (Campbell et al., 2014) gene annotation pipeline. The red arrow indicates the position of A.hyp_Plainsman_PI558499 WGS sample next to its corresponding GBS, green arrow indicates position of A.hyp_K_white and blue arrow indicates the position of A.cru_Suvarna in the phylogenetic tree. Here, we present the most contiguous draft assemblies of these three species to date. Assembly of long, error-prone reads using repeat graphs. Amaranthus hybridus subsp. This plant originated in America, but it has spread all over the world. However, we report a new observation that PI649506 is a A. hypochondriacus originally annotated as A. cruentus. For the 27,658 SNP positions 150 base pair sequences were downloaded from public sources and coordinates for all 27,658 positions on A.hyp.V.2.1 were extracted by BLAST alignment. Received: 20 August 2020; Accepted: 12 October 2020;Published: 11 November 2020. PLINK: a tool set for whole-genome association and population-based linkage analyses. Whole-genome PacBio sequencing was done by Molecular Biology and Genomic Core, Washington University using P5/C3 chemistry on the Pacific Biosciences RSII platform. State of knowledge on amaranth grain: a comprehensive review. Bioinformatics 28, 3326–3328. We have devised a method to normalize for the same during the variant calling (see section “Materials and Methods”). incurvatus Amaranthus hybridus Linnaeus, subsp. Amaranthus hybridus L. is a competitive weed for summer crops in South America.In this article, we intend to unravel the molecular mechanisms by which an A. hybridus population from Argentina has become … Amaranthus powellii: bracteoles mostly 4.5--6 mm long, mostly 2--3 times as long as the sepals (vs. A. hybridus, with bracteoles mostly 2.5--4 mm long, 1.2--2 times as long as … Post demultiplexing, the reads were mapped to AhKP using bowtie2 (Langmead and Salzberg, 2012) and SNP calling was done using the method described in the above section. Besides, the seed sizes shown in Figure 2F also validate classification for Suvarna as A. cruentus with relatively bigger seed size. Smooth Pigweed (Amaranthus hybridus (syn: quitensis)) is a dicot weed in the Amaranthaceae family. Amaranthus … The files were then merged, and the genotype matrix was created using a custom script. Figure 5. Predicting genes in single genomes with AUGUSTUS. & Godr.) In a previous report, our lab had sequenced and reported developmental transcriptome of A.hyp_K_white from several tissues (Sunil et al., 2014, 2017). Weeds as a source of Colletotrichum capsici causing anthracnose on tomato fruit … Avaliable at: https://github.com/lh3/wgsim (accessed June 4, 2020). 3 Amaranthus hypocondriacus Linnaeus 1753. Interestingly, A.hyp_Mexico_ PI511721 clusters with A.hyp_Plainsman_PI558499, which does not show any pink components. Workflow used in the assembly of AhKP. 2017 (Wu and Blair, 2017) WGS (Lightfoot et al., 2017)2. The variants from WGS data from all the plants in Figure 2 were compared with those from the Plainsman accession and a handful of other accessions from public resources including A. hypochondriacus, Amaranthus caudatus, Amaranthus cruentus, and Amaranthus hybridus (Lightfoot et al., 2017). Nucleic Acids Res. We utilized a combination of Pacific Biosciences long-read sequencing and chromatin contact … Figure 3 shows classification using both the 20,548 out of 27,658 reported SNPs covered in all samples (Figure 3A) and ∼6 million variants called from mapping WGS reads to AhKP and A.hyp.V2.1 reference, respectively, (Figures 3B,C). At this stage, the scaffolds were long enough to allow the use of HiC data generated for Plainsman to obtain high-resolution assembly (AhK20). Correction: chromosomal-level assembly of the asian seabass genome using long sequence reads and multi-layered scaffolding. Also, A.hyp_K_white is closest to A.hyp_Nepal_PI619259 and A.hyp_India_PI481125 with A.hyp_K_red relatively distal from A.hyp_K_white. BC: for overseeing the experimental component of the project. 21, 585–602. (2013). Similarly, the malarial strain, Plasmodium falciparum HB3, was improved using the reference of P. falciparum 3D7 combined with an assisted assembly approach that significantly improved the contiguity of the former (Gnerre et al., 2009). The tree generated using the 20,548 SNPs (Figure 3B) is independent of any reference and hence, can be considered unbiased. Amaranthus berchtholdii Moq. Proc. chlorostachys (Willd.) Genome Res. The Amaranth database is running on EC2 instances of Amazon cloud service (AWS). Background. Malligawad, L. H., and Patil, V. C. (2010). Interestingly, grain amaranths, domesticated around 8000 years ago, enjoyed equal status as corn during the Aztec and Inca civilizations (Caselato-Sousa and Amaya-Farfán, 2012). Amaranthus hybridus, commonly called smooth amaranth, smooth pigweed, red amaranth, or slim amaranth, is a species of annual flowering plant.It is a weedy species found now over much of North America and … Plant Sci. Whole-genome sequencing from a few landraces and ornamental varieties generated in-house and several other accessions from public sources are clustered using 20,548 SNPs out of the 27,658 SNPs reported for grain amaranths (Figure 3A). (synonym) Amaranthus hybridus var. The version described in this paper is version JPXE02000000. While GBS is a cost-effective technology for classifying large number of accessions, it covers only 10% of the genome, which depends on the sample preparation protocol and reagents used. Guillen-Portal, F. R., Baltensperger, D. D., Nelson, L. A., and D’Croz-Mason, N. (1999). BMC Biol. A brief comparison of major agronomic traits between A.hyp_K_white and Plainsman (Supplementary Figure S1) is shown in Supplementary Table S3. doi: 10.1002/cpbi.57, Kim, J., Larkin, D. M., and Cai, Q. erythrostachys de Candolle, Prodr. 1961. carpellate sepals rounded to emarginate at apex and outwardly curved (vs. A. hybridus, with carpellate sepals acute to subacute at apex and erect). 37, 540–546. A near-chromosome level genome assembly of Anopheles stephensi. Amaranthus batalleri Sennen Amaranthus bellardii Moq. Front. This high-quality assembly has now allowed placement of about one hundred accessions from the germplasm on a phylogenetic tree (Wu and Blair, 2017) allowing for both establishing genotype-to-phenotype relationships and to place various landraces with very distinct phenotype on the tree for further characterization. J. Here, we report a chromosome-level assembly of A.hyp_K_white (AhKP) using low-coverage PacBio reads, contigs from the reported draft genome of A.hyp_K_white, raw HiC data and reference genome of Plainsman (A.hyp.V.2.1). Vegetable Ama-ranthus can be easily distinguished by inflorescence features like mostly or exclusively axillary glomerules or short spikes (Fig. Front Plant Sci. A decade-long research conducted by the Rodale Institute during the 1980s enabled the creation of more than 800 species/varieties, which are currently maintained in a germplasm (GRIN-Global). It is a glabrous or glabrescent plant. 27, 722–736. Amaranthus hybridus L. is an annual, erect or less commonly ascending herb that is a member of the Amaranthaceae family (Akubugwo et al., 2007; Das, 2016).This plant is often used as a vegetable to treat intestinal bleeding, diarrhea and excessive menstruation (Olusola & Anslem, 2010).The Amaranthus … Although easily controlled and not particularly competitive, it is recognized as a harmful weed of North American crops. The phylogenetic tree was constructed from the genotype matrix using the clustering algorithm hclust and SNPRelate under R and Bioconductor package (Zheng et al., 2012). Biol. The sequence alignment/map format and SAMtools. Since GBS only covers 10% of the genome, there is a need to normalize the variants from WGS data for comparison. doi: 10.1038/nmeth.1923. Cladus: Amaranthaceae s.str. Herten, K., Hestand, M. S., Vermeesch, J. R., and Van Houdt, J. K. J. Species Amaranthus hybridus L. – slim amaranth P: Species Amaranthus hybridus × hypochondriacus [unnamed hybrid] – Plainsman amaranth P: Species Amaranthus hypochondriacus L. – Prince-of-Wales feather P: Species Amaranthus interruptus R. Br. Further, the regions covered in sequencing were identified using bedtools genomecov (Quinlan and Hall, 2010) from bam files, and the regions, which were not covered in sequencing in any of the samples, were removed from the genotype matrix. More recently, using genotyping-by-sequencing (GBS), 94 accessions for grain amaranths have been classified (Wu and Blair, 2017). Figure 4. NH: transcriptome analysis. ... Amaranthus caudatus, Amaranthus cruentus, and Amaranthus hybridus (Lightfoot et al., 2017). Unfortunately, despite India being one of the few countries where multiple landraces of grain amaranths are under continuous cultivation for more than a century, they have received little attention and failed to reach the status of a staple crop. The editor and reviewers' affiliations are the latest provided on their Loop research profiles and may not reflect their situation at the time of review. PacBio reads using RSII technologies sequenced in 2013 with an average length of 7.5 kb with a coverage of 25X for A.hyp_K_white were assembled using state-of-the-art tools CANU (Koren et al., 2017)and FLYE (Kolmogorov et al., 2019) to obtain an assembly with L50 values of 1395 and 944, respectively, as shown in the flowchart in section “Materials and Methods” (Figure 6). The utility of mate-pairs from one strain to build the scaffolds for the other require DNA level similarity, which is often not the case even for closely related species. Amaranthus hybridus Taxonomy ID: 3565 (for references in articles please use NCBI:txid3565) current name. However, this produced skewed classification because of variation in the depth of sequencing between GBS and WGS while calling variants. The database consists of a landing page, genome browser and NCBI BLAST (Altschul et al., 1990) tool. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JPXE00000000. Natl. Bos taurus. Download this complete Project material titled; Assessment Of Vehicular Emissions On Air Quality And Proximate Composition Of Amaranthus Hybridus And Mangifera Indica In Zaria, Nigeria with abstract, chapters 1-5, references, and questionnaire. doi: 10.1093/bioinformatics/bts606, Zimin, A. V., Delcher, A. L., Florea, L., Kelley, D. R., Schatz, M. C., Puiu, D., et al. Our reclassification of A.hyp_PI490752 as A. quitensis, which was originally annotated as A. hypochondriacus, seconds the observation made by Wu and Blair (2017). doi: 10.1101/2020.04.27.063040, Deb, S., Suvrath, J., Ravi, S., Whadgar, S., Hariharan, N., Sunil, M., et al. doi: 10.1186/gb-2009-10-4-r42, Keywords: grain amaranths plainsman, A. hypochondriacus, A. cruentus, A. caudatus, genotyping by sequencing, whole genome sequencing, global-germplasm resources information network (GRIN), Citation: Deb S, Jayaprasad S, Ravi S, Rao KR, Whadgar S, Hariharan N, Dixit S, Sunil M, Choudhary B, Stevanato P, Ramireddy E and Srinivasan S (2020) Classification of Grain Amaranths Using Chromosome-Level Genome Assembly of Ramdana, A. hypochondriacus. Amaranthus hybridus, commonly called green amaranth,[2] slim amaranth,[3] smooth amaranth, smooth pigweed, or red amaranth, is a species of annual flowering plant. Plainsman reference: Phytozome1 A. hypochondriacus genome V.2.1(A.hyp.V.2.1) (Lightfoot et al., 2017) GBS: Blair et al. Classification of grain amaranths using chromosome-level genome assembly of ramdana, A. hypochondriacus. The largest genus, Amaranthus, contains about 70 species of herbs, including the ornamentals love-lies-bleeding, prince’s feather (A. hybridus), and Joseph’s coat (A. tricolor). Whole Genome libraries for A.hyp_K_white, A.hyp_K_red, A.cru_ornamental, A.cau_ornamental and A.cru_Suvarna were prepared using the TruSeq DNA Sample Preparation Kit (Illumina) by following the manufacturer’s low throughput protocol. It is also found in many provinces of Canada, and in parts of Mexico, the West Indies, Central America, and South America. One microgram and 10 μg of the DNA were used for the preparation of Paired-End (PE) and Mate-Pair (MP) libraries, respectively. Meera, N., Lohithaswa, H. C., Murthy, N., and Hittalmani, S. (2014). BMC Bioinformatics 5:59. doi: 10.1186/1471-2105-5-59, Langmead, B., and Salzberg, S. L. (2012). Biotechnol. Acad. The genus also contains many weedy plants known as pigweed, especially rough pigweed (A. retroflexus), prostrate pigweed (A. graecizans), and white … (1990). Amaranthus hybridus is a herbal plant that is grown to be consumed leaves. Amaranthus tuberculatus, Amaranthus hybridus, and Amaranthus palmeri are agronomically important weed species. (2004). Figure 3. In fact, using independent mapping data and conserved synteny between the cattle and human genomes, 91% of the cattle genome was placed onto 30 chromosomes (Zimin et al., 2009). How to cite us (B) block plot, (C) circular and (D) dot plot showing synteny of chromosomes of AhKP assembly on to Plainsman (A.hyp.V2.1). It is a weedy species found now over much of North America and introduced into Europe and Eurasia. Gigascience 1:18. doi: 10.1186/2047-217X-1-18. doi: 10.1002/dvg.22877, Campbell, M. S., Holt, C., Moore, B., and Yandell, M. (2014). 15:550. doi: 10.1186/s13059-014-0550-8, Luo, R., Liu, B., Xie, Y., Li, Z., Huang, W., Yuan, J., et al. This database is made from a framework provided by Meghagen LLC. Genome Biol. In this article, we intend to unravel the molecular mechanisms by which an A. hybridus population from Argentina has become resistant to extraordinarily high levels of glyphosate. Amaranthus paniculatus Linnaeus 1763. A. doi: 10.3835/plantgenome2009.08.0022. incurvatus (Grenier & Godron) Brenan, Watsonia 4: 268. Annotation using the MAKER (Campbell et al., 2014) annotation pipeline predicted 18,858 gene models which has been validated for the 12 genes from lysine biosynthesis pathway by comparing it to Arabidopsis gene model as shown in Figure 5. From the mapped reads, variants were called using samtools (v1.9) mpileup (Li et al., 2009) and bcftools (v1.9) (Li et al., 2009). Effect of plant density and planting geometry on growth and yield of grain amaranths. doi: 10.1093/bioinformatics/btq033. We generated WGS data with coverage of ∼50–150X using the Illumina platform for selected landraces and ornamental varieties. Amaranthus hybridus … ADMIXTURE analysis shown in Figure 4B suggests that there is significant gene flow between A. caudatus and A. quitensis. This landrace was selected for its aggressive growth, and its yield compared to a few other landraces including one with red inflorescence cultivated in India. Plant Sci., 11 November 2020 Interestingly, similar to Figure 2, A.hyp_K_white and A.hyp_K_red, both landraces from India cluster closely together along with accessions A.hyp_Nepal_PI619259 and A.hyp_India_PI481125. A separate VCF file was made for all the 13 datasets as listed in Supplementary Table S2 with the respective alleles at these positions. At K = 4, a significant component of A. quitensis is found in all four species. Stems and leaves are smooth and hairless, sometimes shiny in appearance. A classification based on hundreds of thousands of SNPs validated taxonomy-based classification for a majority of the accessions providing the opportunity for reclassification of a few. (2009). Avaliable at: http://www.repeatmasker.org. To combine WGS data and GBS data, we created GBS like data from whole-genome reads. Figure 2 shows the photographs of fully-grown plants sequenced and reported here. However, at K = 5 and 6 components of A. quitensis is profound only in A. caudatus. The scaffolds from this step are further improved with simulated mate pairs using wgsim (Li, 2020) from Plainsman with SSPACE. MS: PacBio data, developmental transcriptome and taxonomic classification of landraces. Maughan, P. J., Yourstone, S. M., Jellen, E. N., and Udall, J. Amaranthus chlorostachys var. Brief Bioinformatics 6, 6–22. hypocondriacus Grain amaranth is yet to reach an agronomic status in India. SJ: assembly of AhKP. However, at K = 6 there is resolution in components for all four species with green for A. hypochondriacus, dark blue for A. cruentus and major yellow representing components of A. caudatus and A. quitensis. A classification based on hundreds of thousands of SNPs validated taxonomy-based classification for a majority of the accessions providing the opportunity for reclassification of a few. (2009). The circular DNA was sheared again as explained earlier, and the biotinylated fragments were purified using streptavidin beads (DynabeadsTM M-280 Streptavidin, Invitrogen), the fragments were end-repaired, 3′-adenylated and ligated with Illumina adapters. This was further improved by merging the Illumina assembly from the draft genome reported elsewhere and polished using the Illumina reads. Amaranthus hybridus L. is a competitive weed for summer crops in South America. Gene finding in novel genomes. This assembly was improved using HiC data from Painsman (Lightfoot et al., 2017) to obtain an assembly with a L50 of 99 (column 3, Table 1). J. Janick (Alexandria, VA: ASHS Press), 184–189. Figure 7. Amaranthus hybridus var. Also, morphological features like stem solidness and total height at maturity as mentioned by Malligawad and Patil (2010) and seed size (Figure 2F) supports this classification. We have attempted to classify the landraces reported here using multiple approaches. Classification schemes have varied between a single species approach, Amaranthus hybridus s.l., and a five Figure 3A shows classification using the 27,658 SNPs reported for grain amaranth by Maughan et al. DNA Res. Articles, Campo Experimental Valle de México, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Mexico, Centro de Investigaciones y Estudios Avanzados, Instituto Politécnico Nacional de México (CINVESTAV), Mexico, Michoacana University of San Nicolás de Hidalgo, Mexico. bioRxiv [Preprint]. A high-performance computing toolset for relatedness and principal component analysis of SNP data. Reference-guided assembly of four diverse Arabidopsis thaliana genomes. It has been naturalized in many places of warmer climate. BMC Bioinformatics 16:73. doi: 10.1186/s12859-015-0514-3, Hoff, K. J., and Stanke, M. (2019). A. hybridus is the wild progenitor of several cultivated amaranths: A. caudatus, A. cruentus (q.v. doi: 10.1101/gr.215087.116, Korf, I. While the genomes of hundreds of organisms at the draft stage allow deciphering the majority of the proteomes, draft genomes lack chromosomal context under which they evolve and transcribe, which is necessary for a full understanding of biology. 10:R88. It is also found in many provinces of Canada, and in parts of Mexico, the West Indies, Central America, and South America. 15:e1007273. Stems angled or with longitudinal lines or ridges, green or brown, leaves alternate, broadly lance late to ovate, discolors, conspicuously veined beneath, up to 7 cm long, 4 cm wide. Amaranthus hybridus f. aciculatus Thell. Anuales, tallos erectos, glabros abajo, tornándose subglabros o escasamente pubescentes hacia arriba con tricomas de hasta 1 mm de largo, muy delgados e irregularmente doblados; monoicas. doi: 10.1038/s41587-019-0072-8, Koren, S., Walenz, B. P., Berlin, K., Miller, J. R., Bergman, N. H., and Phillippy, A. M. (2017). Nat. The assembly reported here is obtained using relatively low coverage of long reads from PacBio RSII technologies in conjunction with a high-quality reference for another distal accession of the same species A.hyp_Plainsman_PI558499. Tools, such as SOAPdenovo, use the known insert size between the mate-pair reads to connect contigs into longer scaffolds by filling the gaps with unidentified nucleotides (Ns) (Luo et al., 2012). However, natural selection puts sufficient selection pressure on protein sequences for maintaining functional contiguity required during evolution.

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