{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,4]],"date-time":"2026-04-04T22:06:07Z","timestamp":1775340367700,"version":"3.50.1"},"reference-count":74,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2021,12,3]],"date-time":"2021-12-03T00:00:00Z","timestamp":1638489600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004837","name":"Spanish Ministry of Science and Innovation","doi-asserted-by":"publisher","award":["PID2019-110847RB-I00"],"award-info":[{"award-number":["PID2019-110847RB-I00"]}],"id":[{"id":"10.13039\/501100004837","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Consejer\u00eda de Transformaci\u00f3n Econ\u00f3mica, Industria, Conocimiento y Universidades, Junta de Andaluc\u00eda","award":["P20_01005"],"award-info":[{"award-number":["P20_01005"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The \u03b1-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR\/Cas was successfully used to knockout \u03b1-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in \u03b1-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.<\/jats:p>","DOI":"10.3390\/ijms222313076","type":"journal-article","created":{"date-parts":[[2021,12,5]],"date-time":"2021-12-05T21:01:45Z","timestamp":1638738105000},"page":"13076","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy \u03b1-Gliadin Gene Family Engineered with CRISPR\/Cas9"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0032-0058","authenticated-orcid":false,"given":"Mar\u00eda H.","family":"Guzm\u00e1n-L\u00f3pez","sequence":"first","affiliation":[{"name":"Department of Plant Breeding, Institute for Sustainable Agriculture-Spanish National Research Council (IAS-CSIC), 14004 C\u00f3rdoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0040-1053","authenticated-orcid":false,"given":"Miriam","family":"Mar\u00edn-Sanz","sequence":"additional","affiliation":[{"name":"Department of Plant Breeding, Institute for Sustainable Agriculture-Spanish National Research Council (IAS-CSIC), 14004 C\u00f3rdoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9858-6171","authenticated-orcid":false,"given":"Susana","family":"S\u00e1nchez-Le\u00f3n","sequence":"additional","affiliation":[{"name":"Department of Plant Breeding, Institute for Sustainable Agriculture-Spanish National Research Council (IAS-CSIC), 14004 C\u00f3rdoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Francisco","family":"Barro","sequence":"additional","affiliation":[{"name":"Department of Plant Breeding, Institute for Sustainable Agriculture-Spanish National Research Council (IAS-CSIC), 14004 C\u00f3rdoba, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,12,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Doudna, J.A., and Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346.","DOI":"10.1126\/science.1258096"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1007\/s00335-015-9565-z","article-title":"CRISPR\u2013Cas9-mediated genome editing and guide RNA design","volume":"26","author":"Wiles","year":"2015","journal-title":"Mamm. Genome"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1038\/s41580-020-00288-9","article-title":"Applications of CRISPR\u2013Cas in agriculture and plant biotechnology","volume":"21","author":"Zhu","year":"2020","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1537","DOI":"10.1093\/jxb\/erp058","article-title":"Wheat","volume":"60","author":"Shewry","year":"2009","journal-title":"J. Exp. Bot."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Garc\u00eda-Molina, M.D., Gim\u00e9nez, M.J., S\u00e1nchez-Le\u00f3n, S., and Barro, F. (2019). Gluten Free Wheat: Are We There?. Nutrients, 11.","DOI":"10.3390\/nu11030487"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1093\/jexbot\/53.370.947","article-title":"Cereal seed storage proteins: Structures, properties and role in grain utilization","volume":"53","author":"Shewry","year":"2002","journal-title":"J. Exp. Bot."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"eaar8602","DOI":"10.1126\/sciadv.aar8602","article-title":"Genome mapping of seed-borne allergens and immunoresponsive proteins in wheat","volume":"4","author":"Belova","year":"2018","journal-title":"Sci. Adv."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1279","DOI":"10.1016\/j.jprot.2011.03.014","article-title":"Assessment of allergenicity of diploid and hexaploid wheat genotypes: Identification of allergens in the albumin\/globulin fraction","volume":"74","author":"Larre","year":"2011","journal-title":"J. Proteom."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"S7","DOI":"10.1097\/01.mpg.0000450393.23156.59","article-title":"The new epidemiology of celiac disease","volume":"59","author":"Catassi","year":"2014","journal-title":"J. Pediatr. Gastroenterol. Nutr."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"587","DOI":"10.3109\/07853890.2010.505931","article-title":"The prevalence of celiac disease in Europe: Results of a centralized, international mass screening project","volume":"42","author":"Mustalahti","year":"2010","journal-title":"Ann. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1210","DOI":"10.1136\/gutjnl-2013-306578","article-title":"Diagnosis and management of adult coeliac disease: Guidelines from the British Society of Gastroenterology","volume":"63","author":"Ludvigsson","year":"2014","journal-title":"Gut"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1007\/s00251-019-01141-w","article-title":"Update 2020: Nomenclature and listing of celiac disease\u2013relevant gluten epitopes recognized by CD4+ T cells","volume":"72","author":"Sollid","year":"2020","journal-title":"Immunogenetics"},{"key":"ref_13","first-page":"41ra51","article-title":"Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease","volume":"2","author":"Stewart","year":"2010","journal-title":"Sci. Transl. Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1456","DOI":"10.1038\/ajg.2016.439","article-title":"Fecal Gluten Peptides Reveal Limitations of Serological Tests and Food Questionnaires for Monitoring Gluten-Free Diet in Celiac Disease Patients","volume":"111","author":"Comino","year":"2016","journal-title":"Am. J. Gastroenterol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"116","DOI":"10.3389\/fnut.2018.00116","article-title":"Gluten-Free Products for Celiac Susceptible People","volume":"5","author":"Rai","year":"2018","journal-title":"Front. Nutr."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"17023","DOI":"10.1073\/pnas.1007773107","article-title":"Effective shutdown in the expression of celiac disease-related wheat gliadin T-cell epitopes by RNA interference","volume":"107","author":"Tollefsen","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"902","DOI":"10.1111\/pbi.12837","article-title":"Low-gluten, nontransgenic wheat engineered with CRISPR\/Cas9","volume":"16","author":"Ozuna","year":"2018","journal-title":"Plant Biotechnol. J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"258","DOI":"10.3389\/fpls.2021.630396","article-title":"Global Regulation of Genetically Modified Crops Amid the Gene Edited Crop Boom\u2014A Review","volume":"12","author":"Turnbull","year":"2021","journal-title":"Front. Plant Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"794","DOI":"10.1111\/tpj.12851","article-title":"Diversification of the celiac disease \u03b1-gliadin complex in wheat: A 33-mer peptide with six overlapping epitopes, evolved following polyploidization","volume":"82","author":"Ozuna","year":"2015","journal-title":"Plant J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5181","DOI":"10.1038\/s41598-018-23570-5","article-title":"Dynamic evolution of \u03b1-gliadin prolamin gene family in homeologous genomes of hexaploid wheat","volume":"8","author":"Huo","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_21","first-page":"742","article-title":"The \u03b1-gliadins in Bread Wheat: Effect of Nitrogen Treatment on the Expression of the Major Ceeliac Disease Immunogenic Complex in Two RNAi Low-Gliadin Lines","volume":"12","author":"Barro","year":"2021","journal-title":"Front. Plant Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e168","DOI":"10.1093\/nar\/gku936","article-title":"Easy quantitative assessment of genome editing by sequence trace decomposition","volume":"42","author":"Brinkman","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1431","DOI":"10.1016\/j.molp.2015.05.009","article-title":"DSDecode: A web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations","volume":"8","author":"Liu","year":"2015","journal-title":"Mol. Plant"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e58","DOI":"10.1093\/nar\/gky164","article-title":"Easy quantification of template-directed CRISPR\/Cas9 editing","volume":"46","author":"Brinkman","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Hsiau, T., Conant, D., Rossi, N., Maures, T., Waite, K., Yang, J., Joshi, S., Kelso, R., Holden, K., and Enzmann, B.L. (2018). Inference of CRISPR Edits from Sanger Trace Data. bioRxiv.","DOI":"10.1101\/251082"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1089\/crispr.2020.0022","article-title":"Deconvolution of Complex DNA Repair (DECODR): Establishing a Novel Deconvolution Algorithm for Comprehensive Analysis of CRISPR-Edited Sanger Sequencing Data","volume":"4","author":"Bloh","year":"2021","journal-title":"Cris. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2053","DOI":"10.1111\/pbi.12938","article-title":"Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes","volume":"16","author":"Liang","year":"2018","journal-title":"Plant Biotechnol. J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2968","DOI":"10.1093\/bioinformatics\/btu427","article-title":"Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA)","volume":"30","author":"Yang","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1038\/nbt.3583","article-title":"Analyzing CRISPR genome-editing experiments with CRISPResso","volume":"34","author":"Pinello","year":"2016","journal-title":"Nat. Biotechnol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1428","DOI":"10.1016\/j.molp.2015.06.001","article-title":"AGEseq: Analysis of genome editing by sequencing","volume":"8","author":"Xue","year":"2015","journal-title":"Mol. Plant"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"30330","DOI":"10.1038\/srep30330","article-title":"BATCH-GE: Batch analysis of Next-Generation Sequencing data for genome editing assessment","volume":"6","author":"Boel","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1093\/bioinformatics\/btw561","article-title":"Cas-analyzer: An online tool for assessing genome editing results using NGS data","volume":"33","author":"Park","year":"2017","journal-title":"Bioinformatics"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s11427-018-9402-9","article-title":"Hi-TOM: A platform for high-throughput tracking of mutations induced by CRISPR\/Cas systems","volume":"62","author":"Liu","year":"2019","journal-title":"Sci. China Life Sci."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3811","DOI":"10.1093\/bioinformatics\/btx518","article-title":"CRISPR-DAV: CRISPR NGS data analysis and visualization pipeline","volume":"33","author":"Wang","year":"2017","journal-title":"Bioinformatics"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4194","DOI":"10.1038\/s41598-019-40896-w","article-title":"CRIS. py: A versatile and high-throughput analysis program for CRISPR-based genome editing","volume":"9","author":"Connelly","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"lqaa012","DOI":"10.1093\/nargab\/lqaa012","article-title":"CRISPRpic: Fast and precise analysis for CRISPR-induced mutations via p refixed i ndex c ounting","volume":"2","author":"Lee","year":"2020","journal-title":"NAR Genom. Bioinform."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1089\/crispr.2020.0068","article-title":"GOANA: A Universal High-Throughput Web Service for Assessing and Comparing the Outcome and Efficiency of Genome Editing Experiments","volume":"4","author":"Reti","year":"2021","journal-title":"Cris. J."},{"key":"ref_38","unstructured":"Edgar, R. (2010). Usearch, Lawrence Berkeley National Lab. (LBNL)."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"eaar7191","DOI":"10.1126\/science.aar7191","article-title":"Shifting the limits in wheat research and breeding using a fully annotated reference genome","volume":"361","author":"Appels","year":"2018","journal-title":"Science"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Edgar, R.C. (2016). UNOISE2: Improved error-correction for Illumina 16S and ITS amplicon sequencing. bioRxiv, 81257.","DOI":"10.1101\/081257"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2639","DOI":"10.1038\/ismej.2017.119","article-title":"Exact sequence variants should replace operational taxonomic units in marker-gene data analysis","volume":"11","author":"Callahan","year":"2017","journal-title":"ISME J."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"R25","DOI":"10.1186\/gb-2010-11-3-r25","article-title":"A scaling normalization method for differential expression analysis of RNA-seq data","volume":"11","author":"Robinson","year":"2010","journal-title":"Genome Biol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Van Herpen, T.W.J.M., Goryunova, S.V., Van Der Schoot, J., Mitreva, M., Salentijn, E., Vorst, O., Schenk, M.F., Van Veelen, P.A., Koning, F., and Van Soest, L.J.M. (2006). Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes. BMC Genom., 7.","DOI":"10.1186\/1471-2164-7-1"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Jouanin, A., Schaart, J.G., Boyd, L.A., Cockram, J., Leigh, F.J., Bates, R., Wallington, E.J., Visser, R.G.F., and Smulders, M.J.M. (2019). Outlook for coeliac disease patients: Towards bread wheat with hypoimmunogenic gluten by gene editing of \u03b1- And \u03b3-gliadin gene families. BMC Plant Biol., 19.","DOI":"10.1186\/s12870-019-1889-5"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"102903","DOI":"10.1016\/j.jcs.2019.102903","article-title":"Optimisation of droplet digital PCR for determining copy number variation of \u03b1-gliadin genes in mutant and gene-edited polyploid bread wheat","volume":"92","author":"Jouanin","year":"2020","journal-title":"J. Cereal Sci."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/j.jcs.2019.04.008","article-title":"Development of the GlutEnSeq capture system for sequencing gluten gene families in hexaploid bread wheat with deletions or mutations induced by \u03b3-irradiation or CRISPR\/Cas9","volume":"88","author":"Jouanin","year":"2019","journal-title":"J. Cereal Sci."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Prodan, A., Tremaroli, V., Brolin, H., Zwinderman, A.H., Nieuwdorp, M., and Levin, E. (2020). Comparing bioinformatic pipelines for microbial 16S rRNA amplicon sequencing. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0227434"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e2584","DOI":"10.7717\/peerj.2584","article-title":"VSEARCH: A versatile open source tool for metagenomics","volume":"4","author":"Rognes","year":"2016","journal-title":"PeerJ"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Pereira, M.B., Wallroth, M., Jonsson, V., and Kristiansson, E. (2018). Comparison of normalization methods for the analysis of metagenomic gene abundance data. BMC Genom., 19.","DOI":"10.1186\/s12864-018-4637-6"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1089\/crispr.2017.0010","article-title":"Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat","volume":"1","author":"Wang","year":"2018","journal-title":"Cris. J."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1007\/s11032-019-0954-y","article-title":"Enhanced rice salinity tolerance via CRISPR\/Cas9-targeted mutagenesis of the OsRR22 gene","volume":"39","author":"Zhang","year":"2019","journal-title":"Mol. Breed."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1038\/nbt.2969","article-title":"Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew","volume":"32","author":"Wang","year":"2014","journal-title":"Nat. Biotechnol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"7489","DOI":"10.1038\/s41598-017-07871-9","article-title":"CRISPR\/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus","volume":"7","author":"Yang","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1425","DOI":"10.1104\/pp.18.00200","article-title":"Editing of an alpha-kafirin gene family increases digestibility and protein quality in sorghum","volume":"177","author":"Li","year":"2018","journal-title":"Plant Physiol."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1016\/j.biochi.2011.09.004","article-title":"Catalogue of alleles of gliadin-coding loci in durum wheat (Triticum durum Desf.)","volume":"94","author":"Melnikova","year":"2012","journal-title":"Biochimie"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"10903","DOI":"10.1093\/nar\/gku806","article-title":"Large chromosomal deletions and heritable small genetic changes induced by CRISPR\/Cas9 in rice","volume":"42","author":"Zhou","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"1533","DOI":"10.3389\/fpls.2018.01533","article-title":"Development and validation of an effective CRISPR\/Cas9 vector for efficiently isolating positive transformants and transgene-free mutants in a wide range of plant species","volume":"871","author":"Tang","year":"2018","journal-title":"Front. Plant Sci."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1038\/s41467-020-15053-x","article-title":"Multiplexed CRISPR technologies for gene editing and transcriptional regulation","volume":"11","author":"McCarty","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"1519","DOI":"10.1007\/s00299-015-1900-z","article-title":"A multiplex CRISPR\/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis","volume":"35","author":"Zhang","year":"2016","journal-title":"Plant Cell Rep."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1093\/bioinformatics\/btp616","article-title":"edgeR: A Bioconductor package for differential expression analysis of digital gene expression data","volume":"26","author":"Robinson","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"lqab019","DOI":"10.1093\/nargab\/lqab019","article-title":"Sequencing error profiles of Illumina sequencing instruments","volume":"3","author":"Stoler","year":"2021","journal-title":"NAR Genom. Bioinforma."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"759","DOI":"10.1111\/j.1755-0998.2011.03024.x","article-title":"Field guide to next-generation DNA sequencers","volume":"11","author":"Glenn","year":"2011","journal-title":"Mol. Ecol. Resour."},{"key":"ref_63","unstructured":"R Core Team (2013). R: A Language and Environment for Statistical Computing, R Core Team."},{"key":"ref_64","first-page":"790","article-title":"Package \u2018pheatmap\u2019","volume":"1","author":"Kolde","year":"2015","journal-title":"R Packag."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1111\/2041-210X.12628","article-title":"ggtree: An R package for visualization and annotation of phylogenetic trees with their covariates and other associated data","volume":"8","author":"Yu","year":"2017","journal-title":"Methods Ecol. Evol."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"1547","DOI":"10.1093\/molbev\/msy096","article-title":"MEGA X: Molecular evolutionary genetics analysis across computing platforms","volume":"35","author":"Kumar","year":"2018","journal-title":"Mol. Biol. Evol."},{"key":"ref_67","doi-asserted-by":"crossref","unstructured":"Edgar, R.C. (2004). MUSCLE: A multiple sequence alignment method with reduced time and space complexity. BMC Bioinform., 5.","DOI":"10.1186\/1471-2105-5-113"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"3059","DOI":"10.1093\/nar\/gkf436","article-title":"MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform","volume":"30","author":"Katoh","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"4673","DOI":"10.1093\/nar\/22.22.4673","article-title":"CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice","volume":"22","author":"Thompson","year":"1994","journal-title":"Nucleic Acids Res."},{"key":"ref_70","unstructured":"Van Rossum, G., and Drake, F.L. (1995). Python Reference Manual, Centrum voor Wiskunde en Informatica Amsterdam."},{"key":"ref_71","unstructured":"Stacey, J., and Isaac, P.G. (1994). Isolation of DNA from plants. Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Springer."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1007\/s00425-010-1290-y","article-title":"Identification of suitable reference genes for normalization of qPCR data in comparative transcriptomics analyses in the Triticeae","volume":"233","author":"Atienza","year":"2011","journal-title":"Planta"},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1038\/s41588-019-0381-3","article-title":"Durum wheat genome highlights past domestication signatures and future improvement targets","volume":"51","author":"Maccaferri","year":"2019","journal-title":"Nat. Genet."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"e45","DOI":"10.1093\/nar\/29.9.e45","article-title":"A new mathematical model for relative quantification in real-time RT\u2013PCR","volume":"29","author":"Pfaffl","year":"2001","journal-title":"Nucleic Acids Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/23\/13076\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:39:11Z","timestamp":1760168351000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/23\/13076"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,12,3]]},"references-count":74,"journal-issue":{"issue":"23","published-online":{"date-parts":[[2021,12]]}},"alternative-id":["ijms222313076"],"URL":"https:\/\/doi.org\/10.3390\/ijms222313076","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,12,3]]}}}