The segregation of different submicroscopic imbalances underlying the clinical variability associated with a familial karyotypically balanced translocation

Abstract\ud \ud Background\ud About 7 % of karyotypically balanced chromosomal rearrangements (BCRs) are associated with congenital anomalies due to gene or regulatory element disruption, and cryptic imbalances on rearranged chromosomes. Rare familial BCRs segregating with clinical features are a powerful source for the identifying of causative genes due to the presence of several affected carriers.\ud \ud \ud Case presentation\ud We report on a karyotypically balanced translocation t(2;22)(p13;q12.2) associated with variable learning disabilities, and craniofacial and hand dysmorphisms, detected in six individuals in a three-generation family. Combined a-CGH, FISH and mate-pair sequencing revealed a ten-break complex rearrangement, also involving chromosome 5. As the consequence of the segregation of the derivative chromosomes der(2), der(5) and der(22), different imbalances were present in affected and clinically normal family members, thus contributing to the clinical variability. A 6.64 Mb duplication of a 5q23.2-23.3 segment was the imbalance common to all affected individuals. Although LMNB1, implicated in adult-onset autosomal dominant leukodystrophy (ADLD) when overexpressed, was among the 18 duplicated genes, none of the adult carriers manifested ADLD, and LMNB1 overexpression was not detected in the two tested individuals, after qRT-PCR. The ectopic location of the extra copy of the LMBN1 gene on chromosome 22 might have negatively impacted its expression. In addition, two individuals presenting with more severe learning disabilities carried a 1.42 Mb 2p14 microdeletion, with three genes (CEP68, RAB1A and ACTR2),which are candidates for the intellectual impairment observed in the previously described 2p14p15 microdeletion syndrome, mapping to the minimal overlapping deleted segment. A 5p15.1 deletion, encompassing 1.47 Mb, also detected in the family, did not segregate with the clinical phenotype.\ud \ud \ud Conclusion\ud The disclosing of the complexity of an apparently simple two-break familial rearrangement illustrates the importance of reconstructing the precise structure of derivative chromosomes for establishing genotype-phenotype correlations.This work was funded by FAPESP - Fundação de Amparo à Pesquisa do Estado\ud de São Paulo (Grants: CEPID-Human Genome and Stem Cell Research Center\ud 2013/08028-1; student fellowships 2011/14293-4 and 2013/01146-9); the Lundbeck\ud Foundation (2013–14290), the UCPH Programme for Interdisciplinary Research\ud (Global Genes, Local Concerns) and The Danish Council for Independent Research\ud - Medical Sciences (4183-00482B). The authors thank Mrs. Maria Raimunda L. S.\ud Pinheiro for technical support

Balanced chromosomal rearrangements offer insights into coding and noncoding genomic features associated with developmental disorders

Balanced chromosomal rearrangements (BCRs), including inversions, translocations, and insertions, reorganize large sections of the genome and contribute substantial risk for developmental disorders (DDs). However, the rarity and lack of systematic screening for BCRs in the population has precluded unbiased analyses of the genomic features and mechanisms associated with risk for DDs versus normal developmental outcomes. Here, we sequenced and analyzed 1,420 BCR breakpoints across 710 individuals, including 406 DD cases and the first large-scale collection of 304 control BCR carriers. We found that BCRs were not more likely to disrupt genes in DD cases than controls, but were seven-fold more likely to disrupt genes associated with dominant DDs (21.3% of cases vs. 3.4% of controls; P = 1.60×10$^{−12}$). Moreover, BCRs that did not disrupt a known DD gene were significantly enriched for breakpoints that altered topologically associated domains (TADs) containing dominant DD genes in cases compared to controls (odds ratio [OR] = 1.43, P = 0.036). We discovered six TADs enriched for noncoding BCRs (false discovery rate < 0.1) that contained known DD genes (MEF2C, FOXG1, SOX9, BCL11A, BCL11B, and SATB2) and represent candidate pathogenic long-range positional effect (LRPE) loci. These six TADs were collectively disrupted in 7.4% of the DD cohort. Phased Hi-C analyses of five cases with noncoding BCR breakpoints localized to one of these putative LRPEs, the 5q14.3 TAD encompassing MEF2C, confirmed extensive disruption to local 3D chromatin structures and reduced frequency of contact between the MEF2C promoter and annotated enhancers. We further identified six genomic features enriched in TADs preferentially disrupted by noncoding BCRs in DD cases versus controls and used these features to build a model to predict TADs at risk for LRPEs across the genome. These results emphasize the potential impact of noncoding structural variants to cause LRPEs in unsolved DD cases, as well as the complex interaction of features associated with predicting three-dimensional chromatin structures intolerant to disruption

Heterologous expression of FtsH- m1 protease in Nicotiana tabacum L.

As proteases FtsHs, encontradas em eubactérias e eucariotos, pertencem à família das proteínas AAA (ATPases associadas a diferentes atividades celulares) e são classificadas no grupo das metaloproteases. A proteína FtsH foi primeiro identificada em Escherichia coli em mutantes ftsH (filamentation temperature sensitive) como uma protease que age facilitando a degradação de proteínas intermembrânicas e citosólicas. Ortólogas a FtsH já foram encontradas em leveduras, metazoários e plantas. Nestas últimas, a FtsH pode estar localizada nos plastídeos, como uma proteína integral da membrana dos tilacóides ou na membrana interna das mitocôndrias, como em leveduras. Estudos sobre as isoformas plastidiais são abundantes e indicam a participação dessas proteases na renovação da proteína D1, componente do fotossistema II, na resposta hipersensível e na biogênese das membranas do tilacóide. Os estudos sobre as isoformas mitocôndriais são escassos e resumem-se a informações sobre o direcionamento subcelular dessas proteases e evidências de seu envolvimento no acúmulo da subunidade 9 da ATP-sintase na membrana interna. Com o objetivo de caracterizar a função da FtsH-m1 em plantas, foi realizada a transformação genética de Nicotiana tabacum via Agrobacterium tumefaciens contendo um plasmídeo em que o cDNA da FtsHm1 de cana-de-açúcar encontra-se sob o controle de um promotor transcricional constitutivo. Esse plasmídeo apresenta o gene seletivo nptII, que confere resistência à canamicina. As plantas regeneradas in vitro, 17 no total, foram aclimatadas e transferidas para a casa de vegetação até a produção de sementes. As plantas regeneradas foram testadas quanto à presença do cDNA da FtsH-m1 de cana-de-açúcar por meio da técnica de PCR. A seleção de plantas da geração T1 que possuíam o transgene de interesse foi conduzida em ensaios in vitro e em casa de vegetação baseados na resistência dessas plantas à canamicina. A presença do transgene foi confirmada por meio da técnica de PCR. A análise da expressão heteróloga foi realizada via RT-PCR e revelou níveis elevados de RNA mensageiro nas plantas transformadas. Ao todo, foram obtidas dez plantas de fumo em que a presença do transgene de interesse foi confirmada. Não foram observadas alterações fenotípicas marcantes nas plantas das gerações T0 e T1 que pudessem ser relacionadas à expressão heteróloga da FtsH-m1 de cana-de-açúcar. Devem ser realizadas análises mais finas, que podem incluir ensaios para a investigação de possíveis alterações na função mitocondrial, no metabolismo respiratório ou na ultraestrutura das mitocôndrias em plantas a partir da geração T2.FtsH proteases of eubacteria and eucaryotes belong to the AAA protein family (ATPases associated with different celular activities) and are included among the metaloproteases. The FtsH protein was first described in ftsh (filamentation temperature sensitive) mutant of Escherichia coli as a protease involved in the degradation of transmembrane and cytosolic proteins. FtsH-ortologous proteins have been described in yeast, metazoa and plants. In the latter, FtsHs are localized to plastids, as an integral membrane protein of thylakoid or, as in yeast, to the inner mitochondrial membrane. The various studies on the plastid isoforms point to their involvement in the turnover of D1 protein, a member of the photosystem II, in hypersensitive response, and in thylakoid membrane biogenesis. On the other hand, data on the mitochondrial isoforms are scarce and restricted to their subcellular localization, and involvement in the accumulation of ATP-synthase subunit 9 in the inner membrane. With the aim of disclosing the function of FtsH-m1 in plants, Nicotiana tabacum plants were transformed by Agrobacterium tumefaciens carrying a designed plasmid containing the sugar-cane FtsH-m1 cDNA under the control of a constitutive-transcription promoter. This plasmid also contained the selectable kanamycin-resistance gene nptII. The seventeen plants obtained in vitro were acclimatized and kept in a greenhouse until seeding. These plants were then tested by PCR for the presence of the sugar-cane FtsH-m1 cDNA. Selection of T1 plants for the presence of the transgene of interest was carried out both in vitro and in the greenhouse, based on kanamycin resistance. The transgene carrier status of the selected plants was confirmed by PCR. Higher levels of FtsH-m1 messenger RNA were detected by RT-PCR in transformed T1 plants. A total of ten tobacco plants carrying the sugar-cane FtsH-m1 cDNA were obtained. T0 and T1 plants did not show conspicuous phenotype alterations which could be related to the heterologous gene expression. Refined analyses are needed in plants from T2 generation on, including the evaluation of mitochondrial structural abnormalities and disfunction, as well as respiratory alterations

Studies on the dual targeting of plant proteins

Na célula eucariota, os processos metabólicos estão compartimentalizados em organelas e proteínas sintetizadas no citosol são endereçadas para elas por meio de sistema celular específico. Devido a sobreposições funcionais entre organelas, uma dada proteína pode ser requerida em mais de um compartimento. É o caso de proteínas com duplo direcionamento, em que um gene nuclear é capaz de gerar produtos protéicos direcionados para mais de uma organela. Cerca de 60 proteínas de plantas já tiveram seu duplo direcionamento demonstrado, a maioria para mitocôndrias e cloroplastos, portanto um fenômeno não tão raro como imaginado. Estudos recentes procuram esclarecer os mecanismos que permitem esse duplo direcionamento, mesmo existindo fatores celulares que garantem a especificidade do transporte para cada organela. O presente trabalho está dividido em três partes. Na primeira, foram investigados aspectos evolutivos do duplo direcionamento. Na análise comparativa de famílias gênicas com membros cujos produtos protéicos apresentam duplo direcionamento, em Arabidopsis thaliana e Oryza sativa, foi demonstrada a conservação do duplo direcionamento de monodeidroascorbato redutase, metionina aminopeptidase e, provavelmente, de THI1 (enzima da biossíntese de tiazol), entre as duas espécies. Os dados sugeriram um mesmo padrão de evolução em famílias incluindo membros com duplo direcionamento. Na segunda parte, o foco foi a seqüência de duplo direcionamento. Documentado o duplo direcionamento, para mitocôndrias e cloroplastos, de proteínas de ligação ao RNA, RBP1a, RBP1b e RPS19, mutações sítiodirigidas foram introduzidas na seqüência de direcionamento de RBP1b. A importância de aminoácidos positivos para o direcionamento de proteínas para mitocôndrias foi confirmada. Demonstrou-se que a mutação da alanina na posição 2, conservada em seqüências de direcionamento ambíguas para mitocôdrias e cloroplastos, não afeta o duplo direcionamento de RBP1b. A informação para o duplo direcionamento foi localizada entre os 17 primeiros aminoácidos da região amino-terminal. Os sinais de direcionamento para cloroplastos se distribuíram ao longo da seqüência. Enquanto a metade amino-terminal da seqüência foi suficiente para determinar o duplo direcionamento, a seqüência compreendendo os 13 aminoácidos seguintes afetaram a eficiência do transporte. Nessas análises, mostrou-se a adequação de método quantitativo na medida dos sinais de fluorescência da GFP, para ser aplicado em estudos quantitativos do direcionamento de proteínas para mitocôndrias e cloroplastos, in vivo. Na terceira parte, o foco foi o mecanismo traducional do duplo direcionamento de THI1, em A. thaliana. Entretanto, não foi possível testar a hipótese da presença de um sítio interno de entrada do ribossomo (IRES) no mRNA de thi1, pois não se encontrou um sistema de expressão transiente capaz de reproduzir dados da literatura que mostraram o duplo direcionamento de THI1. Nos sistemas testados, a proteína foi encontrada somente em cloroplastos, o que inviabilizou o prosseguimento da investigação.Compartimentalization of the metabolic processes in organelles, each one having a characteristic protein pool and distinct functions, is a property of eukaryote cells. A highly specific cellular system directs proteins, which are synthesized in the cytosol, to the proper organelles. However, due to functional overlaps between organelles, a given protein may be needed in different compartments. This is the case of dual-targeted proteins, which are the product of single nuclear genes, but are somehow directed to different organelles. About 60 plant proteins have had their dual targeting demonstrated, most of them to mitochondria and cloroplasts, the phenomenon being not so rare as previously supposed. Investigations have focused on the mechanisms, which enable protein dual targeting, even in the presence of other cell mechanisms that guarantee the specific protein transport to each organelle. The present work on the subject can be divided in three parts. In the first part, evolutionary aspects of dual targeting were investigated. A comparative analysis of gene families that included members encoding dual-targeted proteins in Arabidopsis thaliana and Oryza sativa demonstrated that the dual targeting of monodehidro-ascorbate reductase, methyonine aminopeptidase and, problably, of the thiazole biosynthetic enzyme THI1 was evolutionary conserved between the two species. In addition, the data suggested the same pattern of evolution for families with members presenting dual targeting. The focus of the second part was the ambiguous sequence for dual targeting. After showing that the RNA-binding proteins RBP1a, RBP1b and RPS19 were dual-targeted to mitochondria and cloroplasts, sitedirected mutations were introduced in the targeting sequence of RBP1b. The importance of positive-charged amino acids for directing the protein to mitochondria was confirmed. Mutation of alanine at position 2, which is conserved in ambiguous sequences, was shown not to affect RBP1b dual targeting. Information for dual targeting was localized among the 17 first amino acids in the amino-terminal region. The signals for directing the protein to cloroplasts appeared distributed along the targeting sequence. While the amino-terminal half of the sequence was sufficient for RBP1b dual targeting, the sequence comprising the next 13 amino acids appeared to affect the efficiency of the transport. In these analyses, a quantitative method to measure the intensity of fluorescent signals of GFP had its efficacy demonstrated to be adopted for in vivo quantitative analysis of dual targeting to mitochondria and cloroplasts. In the third part, the focus was the translational mechanism enabling THI1 dual targeting to mitochondria and cloroplasts, in A. thaliana. It was hypothesized that an internal ribosomal entry site (IRES) was present in thi1 mRNA. However, a transient expression system could not be found that reproduced the literature data demonstrating THI1 dual targeting. In the tested systems the protein was addressed solely to cloroplasts, thus preventing the objective to be pursued

Heterologous expression of FtsH- m1 protease in Nicotiana tabacum L.

As proteases FtsHs, encontradas em eubactérias e eucariotos, pertencem à família das proteínas AAA (ATPases associadas a diferentes atividades celulares) e são classificadas no grupo das metaloproteases. A proteína FtsH foi primeiro identificada em Escherichia coli em mutantes ftsH (filamentation temperature sensitive) como uma protease que age facilitando a degradação de proteínas intermembrânicas e citosólicas. Ortólogas a FtsH já foram encontradas em leveduras, metazoários e plantas. Nestas últimas, a FtsH pode estar localizada nos plastídeos, como uma proteína integral da membrana dos tilacóides ou na membrana interna das mitocôndrias, como em leveduras. Estudos sobre as isoformas plastidiais são abundantes e indicam a participação dessas proteases na renovação da proteína D1, componente do fotossistema II, na resposta hipersensível e na biogênese das membranas do tilacóide. Os estudos sobre as isoformas mitocôndriais são escassos e resumem-se a informações sobre o direcionamento subcelular dessas proteases e evidências de seu envolvimento no acúmulo da subunidade 9 da ATP-sintase na membrana interna. Com o objetivo de caracterizar a função da FtsH-m1 em plantas, foi realizada a transformação genética de Nicotiana tabacum via Agrobacterium tumefaciens contendo um plasmídeo em que o cDNA da FtsHm1 de cana-de-açúcar encontra-se sob o controle de um promotor transcricional constitutivo. Esse plasmídeo apresenta o gene seletivo nptII, que confere resistência à canamicina. As plantas regeneradas in vitro, 17 no total, foram aclimatadas e transferidas para a casa de vegetação até a produção de sementes. As plantas regeneradas foram testadas quanto à presença do cDNA da FtsH-m1 de cana-de-açúcar por meio da técnica de PCR. A seleção de plantas da geração T1 que possuíam o transgene de interesse foi conduzida em ensaios in vitro e em casa de vegetação baseados na resistência dessas plantas à canamicina. A presença do transgene foi confirmada por meio da técnica de PCR. A análise da expressão heteróloga foi realizada via RT-PCR e revelou níveis elevados de RNA mensageiro nas plantas transformadas. Ao todo, foram obtidas dez plantas de fumo em que a presença do transgene de interesse foi confirmada. Não foram observadas alterações fenotípicas marcantes nas plantas das gerações T0 e T1 que pudessem ser relacionadas à expressão heteróloga da FtsH-m1 de cana-de-açúcar. Devem ser realizadas análises mais finas, que podem incluir ensaios para a investigação de possíveis alterações na função mitocondrial, no metabolismo respiratório ou na ultraestrutura das mitocôndrias em plantas a partir da geração T2.FtsH proteases of eubacteria and eucaryotes belong to the AAA protein family (ATPases associated with different celular activities) and are included among the metaloproteases. The FtsH protein was first described in ftsh (filamentation temperature sensitive) mutant of Escherichia coli as a protease involved in the degradation of transmembrane and cytosolic proteins. FtsH-ortologous proteins have been described in yeast, metazoa and plants. In the latter, FtsHs are localized to plastids, as an integral membrane protein of thylakoid or, as in yeast, to the inner mitochondrial membrane. The various studies on the plastid isoforms point to their involvement in the turnover of D1 protein, a member of the photosystem II, in hypersensitive response, and in thylakoid membrane biogenesis. On the other hand, data on the mitochondrial isoforms are scarce and restricted to their subcellular localization, and involvement in the accumulation of ATP-synthase subunit 9 in the inner membrane. With the aim of disclosing the function of FtsH-m1 in plants, Nicotiana tabacum plants were transformed by Agrobacterium tumefaciens carrying a designed plasmid containing the sugar-cane FtsH-m1 cDNA under the control of a constitutive-transcription promoter. This plasmid also contained the selectable kanamycin-resistance gene nptII. The seventeen plants obtained in vitro were acclimatized and kept in a greenhouse until seeding. These plants were then tested by PCR for the presence of the sugar-cane FtsH-m1 cDNA. Selection of T1 plants for the presence of the transgene of interest was carried out both in vitro and in the greenhouse, based on kanamycin resistance. The transgene carrier status of the selected plants was confirmed by PCR. Higher levels of FtsH-m1 messenger RNA were detected by RT-PCR in transformed T1 plants. A total of ten tobacco plants carrying the sugar-cane FtsH-m1 cDNA were obtained. T0 and T1 plants did not show conspicuous phenotype alterations which could be related to the heterologous gene expression. Refined analyses are needed in plants from T2 generation on, including the evaluation of mitochondrial structural abnormalities and disfunction, as well as respiratory alterations

The clinical impact of chromosomal rearrangements with breakpoints upstream of the SOX9 gene: two novel de novo balanced translocations associated with acampomelic campomelic dysplasia

Abstract Background The association of balanced rearrangements with breakpoints near SOX9 [SRY (sex determining region Y)-box 9] with skeletal abnormalities has been ascribed to the presumptive altering of SOX9 expression by the direct disruption of regulatory elements, their separation from SOX9 or the effect of juxtaposed sequences. Case presentation We report on two sporadic apparently balanced translocations, t(7;17)(p13;q24) and t(17;20)(q24.3;q11.2), whose carriers have skeletal abnormalities that led to the diagnosis of acampomelic campomelic dysplasia (ACD; MIM 114290). No pathogenic chromosomal imbalances were detected by a-CGH. The chromosome 17 breakpoints were mapped, respectively, 917–855 kb and 601–585 kb upstream of the SOX9 gene. A distal cluster of balanced rearrangements breakpoints on chromosome 17 associated with SOX9-related skeletal disorders has been mapped to a segment 932–789 kb upstream of SOX9. In this cluster, the breakpoint of the herein described t(17;20) is the most telomeric to SOX9, thus allowing the redefining of the telomeric boundary of the distal breakpoint cluster region related to skeletal disorders to 601–585 kb upstream of SOX9. Although both patients have skeletal abnormalities, the t(7;17) carrier presents with relatively mild clinical features, whereas the t(17;20) was detected in a boy with severe broncheomalacia, depending on mechanical ventilation. Balanced and unbalanced rearrangements associated with disorders of sex determination led to the mapping of a regulatory region of SOX9 function on testicular differentiation to a 517–595 kb interval upstream of SOX9, in addition to TESCO (Testis-specific enhancer of SOX9 core). As the carrier of t(17;20) has an XY sex-chromosome constitution and normal male development for his age, the segment of chromosome 17 distal to the translocation breakpoint should contain the regulatory elements for normal testis development. Conclusions These two novel translocations illustrate the clinical variability in carriers of balanced translocations with breakpoints near SOX9. The translocation t(17;20) breakpoint provides further evidence for an additional testis-specific SOX9 enhancer 517 to 595 kb upstream of the SOX9 gene

Identification of duplicates in cassava germplasm banks based on single-nucleotide polymorphisms (SNPs)

Genetic redundancy in cassava (Manihot esculenta Crantz) presents a challenge to efficient management of genetic resources. This study aimed to identify and define the genetic structure of duplicates in cassava germplasm from various Embrapa research units, using single-nucleotide polymorphism (SNP) markers. We evaluated 2,371 accessions with 20,712 SNPs. The identification of duplicates was performed based on multilocus genotypes (MLG), adopting a maximum genetic distance threshold of 0.05. The population structure was defined based on discriminant analysis of principal components (DAPC). A total of 1,757 unique and 614 duplicate accessions were identified. The redundancy of the collections ranged from 17 % (Belém, PA – Brazil) to 39 % (Petrolina, PE – Brazil), with an average of 21 %. This redundancy between different research units is probably due to the historical sharing of accessions, as well as collections carried out in the same region, or even to the intense germplasm exchange between farmers with different genotype names. In terms of genetic structure, the 250 principal components explained 88 % of the genetic variation of the SNP markers and defined the hierarchical structure of the duplicate cassava germplasm in 12 groups. Since heterotic groups have not yet been identified for cassava, crosses between accessions of the 12 DAPC groups may be promising. All MLGs were allocated within the same DAPC group, corroborating duplicate analyses yet still revealing high variability between groups that were quite distinct based on the first two discriminant functions. Our results contribute to optimizing the conservation of genetic resources, together with understanding diversity and its use in crop improvement

A novel soybean hairy root system for gene functional validation.

Agrobacterium rhizogenes-mediated transformation has long been explored as a versatile and reliable method for gene function validation in many plant species, including soybean (Glycine max). Likewise, detached-leaf assays have been widely used for rapid and mass screening of soybean genotypes for disease resistance. The present study combines these two methods to establish an efficient and practical system to generate transgenic soybean hairy roots from detached leaves and their subsequent culture under ex vitro conditions. We demonstrated that hairy roots derived from leaves of two (tropical and temperate) soybean cultivars could be successfully infected by economically important species of root-knot nematodes (Meloidogyne incognita and M. javanica). The established detached-leaf method was further explored for functional validation of two candidate genes encoding for cell wall modifying proteins (CWMPs) to promote resistance against M. incognita through distinct biotechnological strategies: the overexpression of a wild Arachis α-expansin transgene (AdEXPA24) and the dsRNA-mediated silencing of an endogenous soybean polygalacturonase gene (GmPG). AdEXPA24 overexpression in hairy roots of RKN-susceptible soybean cultivar significantly reduced nematode infection by approximately 47%, whereas GmPG downregulation caused an average decrease of 37%. This novel system of hairy root induction from detached leaves showed to be an efficient, practical, fast, and low-cost method suitable for high throughput in root analysis of candidate genes in soybean

Identification of duplicates in cassava germplasm banks based on single-nucleotide polymorphisms (SNPs)

ABSTRACT: Genetic redundancy in cassava (Manihot esculenta Crantz) presents a challenge to efficient management of genetic resources. This study aimed to identify and define the genetic structure of duplicates in cassava germplasm from various Embrapa research units, using single-nucleotide polymorphism (SNP) markers. We evaluated 2,371 accessions with 20,712 SNPs. The identification of duplicates was performed based on multilocus genotypes (MLG), adopting a maximum genetic distance threshold of 0.05. The population structure was defined based on discriminant analysis of principal components (DAPC). A total of 1,757 unique and 614 duplicate accessions were identified. The redundancy of the collections ranged from 17 % (Belém, PA – Brazil) to 39 % (Petrolina, PE – Brazil), with an average of 21 %. This redundancy between different research units is probably due to the historical sharing of accessions, as well as collections carried out in the same region, or even to the intense germplasm exchange between farmers with different genotype names. In terms of genetic structure, the 250 principal components explained 88 % of the genetic variation of the SNP markers and defined the hierarchical structure of the duplicate cassava germplasm in 12 groups. Since heterotic groups have not yet been identified for cassava, crosses between accessions of the 12 DAPC groups may be promising. All MLGs were allocated within the same DAPC group, corroborating duplicate analyses yet still revealing high variability between groups that were quite distinct based on the first two discriminant functions. Our results contribute to optimizing the conservation of genetic resources, together with understanding diversity and its use in crop improvement