Agronomic and biochemical evaluation of cassava clones with roots that have pink pulp / Avaliação agronômica e bioquímica de clones de mandioca com polpa rosada

ABSTRACT: Sweet cassava breeding programs are focused on the development of bio fortified cultivars that combine significant amounts of carotenoids in their reserve roots with desirable agronomic. The objective of this research was to evaluate agronomic and biochemical traits in sweet cassava clones with roots that have pink pulp. The nine genotypes were evaluated in two seasons in a randomized block design with three replications. Among the evaluated clones, the following stood out: i) for the height of the first branch (390/08, 345/08 and the control IAC 576-70); ii) for plant height (390/08, 345/08 e 378/08); iii) for shoot weight without original steam cutting (390/08, 406/08, 378/08 e 341/08); iv) for the percentage of starch in roots (378/08, 413/08, 390/08 and the control IAC 576-70); and v) for the root yield (the control IAC 576-70 and 341/08, 390/08, 406/08 e 387/08). In the 2011/2012 season, all clones cooked within 30 minutes, indicating that they all have good culinary qualities. Regarding the total carotenoid content in the roots, the clones that stood out were 406/08 and 341/08. All clones evaluated had HCN content below 100 mg kg-1. Clones 341/08 and 406/08 have agronomic and biochemical potential for direct cultivation by producers in the Cerrado region of Central Brazil and / or for use as stock in sweet cassava breeding programs.

RESUMO: Os programas de melhoramento genético de mandioca de mesa estão focados no desenvolvimento de variedades biofortificadas que aliem aos caracteres agronômicos a presença de carotenoides nas raízes de reserva. Neste trabalho, objetivou-se avaliar características agronômicas e bioquímicas em clones de mandioca de mesa com polpa rosada. Os nove genótipos foram avaliados por duas safras em delineamento experimental de blocos casualizados com três repetições. Dentre os clones avaliados se destacaram: i) para altura da primeira ramificação (390/08, 345/08 e a testemunha IAC 576-70); ii) para altura da planta (390/08, 345/08 e 378/08); iii) para peso da parte aérea sem a cepa (390/08, 406/08, 378/08 e 341/08); iv) para porcentagem de amido nas raízes (378/08, 413/08, 390/08 e a testemunha IAC 576-70); v) para produtividade de raízes (IAC 576-70 e 341/08, 390/08, 406/08 e 387/08). Na safra 2011/2012, todos os clones cozinharam em até 30 minutos, indicando que todos apresentam boas qualidades culinárias. Com relação ao teor de carotenoides totais nas raízes, os clones que se destacaram foram 406/08 e 341/08. Todos os clones avaliados apresentaram teores de HCN nas raízes de reserva de mandioca, inferiores a 100 mg kg-1. Os clones 341/08 e 406/08 apresentam potencial agronômico e bioquímico para cultivo direto pelos produtores na região do Cerrado do Brasil Central e/ou para a utilização como genitores em programas de melhoramento genético de mandioca de mesa.

Characterization of Carotenoid-protein Complexes and Gene Expression Analysis Associated with Carotenoid Sequestration in Pigmented Cassava (Manihot Esculenta Crantz) Storage Root.

Carotenoid-protein complex (CPC) was isolated from chromoplast-enriched suspensions of cassava storage root (CSR) using size exclusion chromatography and characterized. Peptide sequences (LC_MS/MS spectrum) obtained from CPC and their corresponding proteins were obtained using publically available databases. Small Heat Shock Proteins (sHSPs) were the most abundant proteins identified in the CPC. Western blot analysis showed that Fribrillin and Or-protein were present in chromoplast-enriched suspensions of yellow root but not in the complex or white root. Results from qRT-PCR helped identify an isoform of HSP21 possessing four single point mutations in the intense yellow CSR that may be responsible for increased sequestration of b-carotene.

Caracterização molecular de acessos de mandioca açucarados e não açucarados / Molecular characterization of sugary and non-sugary cassava accessions

Acessos de mandioca que armazenam açúcares livres em suas raízes de reserva e não somente amido, como os acessos de mandioca cultivados comercialmente, vêm recebendo destaque atualmente. Esses acessos são popularmente conhecidos como mandiocas açucaradas ou mandiocabas, e evidenciam potencial de uso na produção de álcool combustível. Entretanto, esse germoplasma ainda não foi suficientemente estudado quanto àsua variabilidade genotípica, o que é fundamental para sua conservação e utilização no melhoramento genético. Neste tabalho, objetivou-se caracterizar por meio de marcadores RAPD 14 acessos de mandioca açucarados e não açucarados. Em laboratório, os acessos foram avaliados por meio de 12 iniciadores RAPD. Posteriormente, foi estimada a matriz de dissimilaridade genética entre os acessos, por meio da utilização do complemento do índice de Jaccard. Os iniciadores geraram 131 marcadores RAPD, dos quais 73 por cento foram polimórficos, evidenciando a existência de variabilidade e a eficiência da técnica de RAPD. Os acessos mais similares foram BGMC 1207 e BGMC 1209, ambos açucarados e os menos similares foram o açucarado BGMC 1219 e a cultivar BGMC 436. Os acessos foram divididos em quatro grupos, sendo o primeiro grupo formado pelos 10 acessos açucarados, o segundo grupo formado por dois acessos locais não açucarados, o terceiro e o quarto foram formados pelos acessos melhorados BGMC 753 e BGMC 436. Os resultados obtidos revelaram a existência de elevada variabilidade genética entre os acessos avaliados e diferenciaram os acessos açucarados das cultivares locais não açucaradas e das cultivares comerciais não açucaradas.

Emphasis has been given lately to cassava accessions that accumulate free sugars in their storage roots, besides starch, such as the commercial cultivars. These accessions are popularly known as sugary cassavas or "mandiocabas", and present a high potential for the use in ethanol biofuel production. Nevertheless, this germplasm was not yet sufficiently studied regarding its genotypic variability, which is of fundamental importance for its conservation and use in genetic improvement. The objective of this work was to characterize, via RAPD marker, 14 sugary and non-sugary cassava accessions. Within Laboratory conditions, the accessions were evaluated by using 12 RAPD primers. Afterwards, the matrix of genetic dissimilarity among the acessions was estimated by using the complement of Jaccard index. The primer generated 131 RAPD markers, among which 73 percent were polymorphic, highlightening the existence of genetic variability and efficiency of the RAPD technique. The most similar accessions were BGMC 1207 and BGMC 1209, both sugary ones, and the least similar, the sugary accession BGMC 1219 and the cultivar BGMC 436. The accessions were divided into four groups, the first group was composed by the 10 sugary accessions, the second group consistecs of two local non-sugary accessions, the third and fourth groups were composed by the improved accessions BGMC 753 and BGMC 436. The results obtained reveal the existence of high genetic variability among the evaluated accessions and differed the sugary accessions from local non-sugary and the non-sugary commercial cultivars.

Caracterização molecular e variabilidade genética de acessos elite de mandioca para fins industriais / Molecular characterization and genetic variability of elite cassava accessions for industrial purpose

Marcadores moleculares são ferramentas úteis na caracterização molecular de acessos de mandioca, em razão de apresentarem elevada capacidade de detecção das informações contidas no genoma. O objetivo deste trabalho foi caracterizar, por meio de marcadores RAPD, 20 acessos de mandioca para fins industriais conservados no Banco Regional de Germoplasma de Mandioca do Cerrado (BGMC). Em laboratório, os acessos foram avaliados por meio de marcadores RAPD, sendo posteriormente estimada a matriz de similaridade genética entre os acessos, por meio do índice de Jaccard. A análise, feita através de 11 iniciadores, gerou um total de 120 marcadores RAPD, dos quais 74 (62 por cento) foram polimórficos, revelando a presença de elevada variabilidade genética no grupo de acessos avaliados. A análise de agrupamento revelou a formação de apenas um agrupamento forte, formado pelos acessos BGMC 1130, BGMC 788, BGMC 1270 e BGMC 1107, o que indica que, no melhoramento genético de mandioca, não devem ser priorizadas hibridações entre esses acessos, sob pena de efeitos de endogamia. Por sua vez, o acesso BGMC 436 foi o mais divergente em relação aos demais e, como expressa elevado potencial produtivo na região do Cerrado do Brasil Central, representa boa opção como genitor para o melhoramento de mandioca para essa região. O estudo comprovou que os marcadores RAPD são eficientes na determinação da variabilidade genética dos acessos avaliados e que neste grupo existe elevada variabilidade genética passível de ser utilizada no melhoramento genético.

Molecular markers are useful tools for the molecular characterization of cassava accessions since they present high capacity to detect information within the genome. The aim of this research was to characterize through RAPD molecular markers, 20 industrial cassava accessions conserved in the Cerrado Cassava Regional Germoplasm Bank ("Banco Regional de Germoplasma de Mandioca do Cerrado"-BGMC). Upon laboratory conditions, the accessions were evaluated though RAPD markers, being afterwards estimated by the matrix of genetic similarity among the accessions, through the Jaccard index. The analyses through 11 primers generated a total of 120 RAPD markers, among which 74 (62 percent) were polymorphic, revealing the presence of high genetic variability within the group of evaluated accessions. The clustering analyses revealed the formation of a single strong group, comprised of the accessions BGMC 1130, BGMC 788, BGMC 1270 and BGMC 1107, which indicates that the cassava genetic breeding hybridizations among these accessions should not be prioritized, upon risk of endogamy effects. The accession BGMC 436 was the most divergent as compared to the others and, as it expresses high productive potential within the Central Brazil Cerrado, it represents a good option as parent for the cassava breeding in this region. The study confirmed that the RAPD markers are efficient for the determination of genetic variability among evaluated accessions and that within the group there is high genetic variability useful for the plant breeding.

A cDNA sequence coding for a glutamic acid-rich protein is differentially expressed in cassava storage roots.

We report the isolation and characterization of a cDNA sequence (Mec1) coding for a glutamic acid-rich protein (Pt2L4) from cassava storage roots. Comparative sequence analysis showed a high identity of Pt2L4 with cassava protein C54, which is expressed in vascular tissues of storage roots. Northern blot analysis showed that the Mec1 transcript expression pattern might be related to the maturation of the storage parenchyma cells.

Comparative gene expression study to identify genes possibly related to storage root formation in cassava.

Cassava storage roots result from swelling of adventitious roots by secondary growth. In the present study we aimed to gain insight into the molecular processes occurring during cassava storage root formation. We report a comparative gene expression study in adventitious and storage roots in order to identify genes possibly related to storage organ formation. Our results revealed five genes with higher expression levels in secondary xylem of storage roots than adventitious roots. Among them, the Mec1 gene coding for Pt2L4 glutamic acid-rich protein and a putative RING Zinc Finger and LEA protein genes were strongly induced in secondary xylem tissue.

Identification and characterization of a novel cassava (Manihot esculenta Crantz) clone with high free sugar content and novel starch.

This study reports the identification of a new class of cassava (Manihot esculenta Crantz) with a storage root showing unusual free sugar accumulation and novel starch. Twenty-seven clones high in free sugar were identified under cultivation in primitive rural community areas in the Amazon. Iodine test and glucose oxidase-peroxidase reagent strips were used, in the field, for identification of starch and glucose, respectively. Five out of these 27 clones of cassava were cultivated at EMBRAPA Genetic Resources and Biotechnology and used for biochemical characterization, starch synthesis enzyme activities and gene expression analysis. Carbohydrates were fractioned into free sugar, polymerized water-soluble and -insoluble alpha-polyglucan. Clones of series CAS36 accumulate over 100 times more free sugar (mainly glucose) than commercial varieties. Monosaccharide composition analysis revealed one clone with distinct water-soluble sugars not present in the commercial cultivar. Structure analysis of the water-soluble and -insoluble alpha-polyglucan revealed the presence of a glycogen-like starch in clone CAS36.1. This clone indicated disruption in the starch synthesis pathway for enzyme activities and protein blot analyses in ADPG-pyrophosphorylase and branching enzyme, and their corresponding protein. Gene expression analysis indicated the lack of transcript for the gene coding for branching enzyme, but not for the gene coding for the ADPG-pyrophosphorylase small subunit. In addition, the pattern of distribution of sugar and starch content showed to be related to tissue age in the storage root.