Journal of Life Sciences Volume 6 Number (4)
J LS
Journal of Life Sciences
Volume 6, Number 6, June 2012 (Serial Number 50)
Contents
Biochemical and Microbiology
587 Antigenicity of Synthetic Peptides Derived from Plasmodium Apoptosis-Linked Pathogenicity
Factors
Ulrick Bisvigou, Estelle Sonya Zang-Edou, Florian Noulin, Rafika Zatra, Ludovic Mevono, Jean-Bernard Lékana-Douki, Dominique Mazier, Frédérick Gay and Fousseyni S. Touré Ndouo
Genotype × Environment Interaction of Carotene Content of Yellow-Fleshed Cassava Genotypes in Nigeria
Norbert G. Maroya, Peter Kulakow, Alfred G.O. Dixon, Busie Maziya-Dixon and Moshood A. Bakare 602
Assessment of HRP2-Based RDT in Diagnosing Malaria Cases
Yémou Dieng, Doudou Sow, Thérèse Dieng, Abdourahmane Sow, Babacar Faye, Jean Louis Ndiaye, Daouda Ndiaye, Roger Clément Tine, Khadime Sylla, Oumar Faye, Oumar Ndir and Oumar Gaye
Selection of Relevant Variables to the Enzyme Production on Red Grape Pomace by Solid-State Fermentation
Martha Dina Vallejo, María L. Martín, Pablo Monetta and Silvia C. Gouiric
615 The in Vitro Effect of a Symbiotic Combination Between Cellobiose and Two Probiotic Strains
Toward Two Pathogenic Bacteria
Amar Yacine, Tirtouil Meddah Aïcha, Meddah Boumédiene, El Keurti Khadidja, Mokhtari Bochra and Kridech Souhila
Some Antimicrobial Activity Plants Growing in Rize Region Used in Alternative Medicine
Gülsüm Yaldiz
Botany and Zoology
632 Distribution Boswellia sacra in Dhofar Mountains, Sultanate of Oman: Economic Value and
Environmental Role
Mohsin Musalim Hassan Alaamri
637 Antihymolytic and Antioxidant Effects of Medicinal Plant Capparis spinosa L.
Sabah Boumerfeg, Djamila Ameni, Moufida Adjadj, Meriem Djarmouni, Seddik Khennouf, Lekhmici Arrar and Abderrahmane Baghiani
Late Season Sugarcane as Affected by Deficit Irrigation at the Yield Formation Stage in Northern Ivory Coast
Crépin B. Péné, Marco H. Ouattara and Sylvain G. Koulibaly 652 Antihyperglycemic Effect of Zygophyllum Geslini Aqueous Extract in Streptozotocin-Induced
Diabetic Wistar Rats
Houria Medjdoub, Boufeldja Tabti, Malika Baatouche, Leila Baou, Souhila Zehhaf and Karima Azzeddine
The Relationship Between Behavior with Some Productive Traits of Rabbits
Dorota Kowalska, Pawe ł Bielański and Mariusz Pietras 663
Spatiotemporal Dynamics of Forest and Mountain Formations in Aurès Area, Algeria
Yassine Beghami, Mahdi Kalla, Michel Thinon and Hassen Benmessaoud
Interdisciplinary Researches
670 Determination of Creatinine in PETs and Laboratory Calculations of the Peritoneal Dialysis
Adequacy
Gorana Predovan and Dragan Klari ć 679
Marine and Inland Water Resources in Eneolithic Communities. New Data from Sardinia (Italy)
Maria Grazia Melis, Marco Zedda and Laura Manca 694 Efficient School Health Services and Sport Participation Among Nigerian Universities
Undergraduates
Patience Edoho Samson-Akpan and Lucas Olusegun Ogunjimi 701
Forecasting World Market Structure of Iran’s Pistachio Exports
Hossein Mehrabi Boshrabadi and Ebrahim Javdan 708
Lead Dynamics in the Body of Waterfowl That Swallowed Lead Pellets
Eskov Evgenii Konstantinovich, Eskova Maiya Dmitrievna and Kiriyakulov Viacheslav Michailovich
Journal of Life Sciences 6 (2012) 587-594
Antigenicity of Synthetic Peptides Derived from Plasmodium Apoptosis-Linked Pathogenicity Factors
1 1 Ulrick Bisvigou 3 , Estelle Sonya Zang-Edou , Florian Noulin , Rafika Zatra , Ludovic Mevono , Jean-Bernard Lékana-Douki , Dominique Mazier 1, 4 , Frédérick Gay 5, 6 5, 6 and Fousseyni S. Touré Ndouo 1*
1. Unit of Medical Parasitology, International Centre for Medical Research of Franceville (CIRMF), Franceville B.P. 769, Gabon 2. Département de Santé Publique de Médecine Légale et du Travail, Université des Sciences de la Santé, Libreville B.P. 4009, Gabon 3. Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon 4. Département de Parasitologie-Mycologie Médecine Tropicale, Université des Sciences de la Santé, Libreville B.P. 4009, Gabon 5. Inserm/UPMC UMR S 945, Université Pierre et Marie Curie, Paris, France 6. AP-HP Groupe Hospitalier Pitié-Salpêtrière, Service Parasitologie-Mycologie, Paris, France
Received: October 25, 2011 / Accepted: January 12, 2012 / Published: June 30, 2012.
Abstract: Background: Plasmodium falciparum malaria remains a major life-threatening disease. Recently, the Plasmodium apoptosis-linked pathogenicity factors (PALPF) have been identified. These antigens PALPF are expressed only by P. falciparum -infected erythrocytes triggering endothelial cell apoptosis (apoptogenic). Methods: We designed ten synthetic peptides (P1 to P10) from PALPF: PF07_0032, PF10_0226, PFI0130c, PFD0875c and MAL13P1.206, and analyzed their antigenicity with an ELISA method using plasma samples from subjects living in Dienga, Gabon. Results: Four peptides showed good reactivity with human antibodies. The prevalence rate of specific IgG was 61%, 51%, 44% and 34% for P5, P6, P4 and P2, respectively. The median optical density of total IgG anti-P2 was higher than that directed against P4 and P6 (P = 0.009; P = 0.012 respectively). The prevalence rate of IgG subclasses determined with plasma samples recognizing peptide 5 for IgG1, 2, 3 and 4 isotypes was 69%, 45%, 76% and 62%, respectively. All the subjects had at least one immunoglobulin subclass, while 13 (44%) had both IgG1 and IgG3 antibodies. There was no significant difference in the prevalence rate of anti-P5 IgG1, IgG3 and IgG4. Conclusion: These results warrant further immunogenicity studies of peptides 2, 4, 5 and 6 with a view of a tentative to antimalarial vaccine development.
Key words: Malaria, vaccine candidate, antigenicity, immunoglobulin, IgG, PALPF.
1. Introduction uncomplicated malaria or remain asymptomatic [2]. The pathophysiological mechanisms underlying
Plasmodium falciparum malaria remains a major severe malaria are poorly understood. Several studies public health issue in intertropical countries, have shown the involvement of parasitized red blood especially in Sub-Saharan Africa. There are an cell (PRBC) sequestration in the brain and lungs [3-7], estimated 300 million cases annually, and about due to PRBC adherence, agglutination and rosetting [5, 781,000 people (mainly children) died of the disease
8, 9]. PRBC adhesion to endothelial cells (EC) in in 2009 [1]. For still unknown reasons, some non brain and lung capillaries can induce mechanical immune persons infected by P. falciparum develop blockade, over-expression of proinflammatory severe malaria and die, while others have cytokines [10, 11] and EC apoptosis [12, 13]. Despite
pharmacologic parasite clearance, between 15% and * Equal contributors.
20% of patients with severe malaria die and others Corresponding author: Fousseyni S. Touré Ndouo, Ph.D.,
professor, research fields: immunology, cell biology. E-mail:
have permanent sequelae [14].
fousseyni@yahoo.fr.
Antigenicity of Synthetic Peptides Derived from Plasmodium
Apoptosis-Linked Pathogenicity Factors
According to some authors, the antimalarial PF10255, PFI0130c, PFD0875c, and MAL13P1.206). vaccination could be feasible [15]. WHO has listed
Cytoadherence was inhibited by PFD0875c and about 80 candidate vaccines in preclinical MAL13P1.206 knock down. These five genes are development and another 30 are being tested in
known as Plasmodium apoptosis-linked pathogenicity clinical trials. Only 2 vaccines have reached phase IIb
factors (PALPF).
of clinical development, namely MSP1 [16] and RTS, Here we used PALPF-derived peptides synthesized S/AS02A [17]. The principal candidate is RTS, © by GenScript (NJ, USA) to measure naturally
S/AS02A, a pre-erythrocytic vaccine based on P. occurring specific antibodies present in plasma falciparum circumsporozoite (CS) protein. A samples collected from people living in a highly randomized phase IIb trial showed that RTS, S/AS02A
endemic area.
protected children against severe malaria with 57.7%
2. Materials and Methods
efficacy [17], while the latest phase IIb trial in Kenya and Tanzania gave an efficacy of 53%. These results
2.1 Study Site and Design
were presented at the 5th Conference of Multilateral The plasma samples used in this study were Initiative Malaria (MIM), which was held in collected from individuals living in Dienga, a village
November 1, 2009 in Nairobi, Kenya. RTS, S/AS02A located in Ogooué-Lolo province in south-eastern has now entered phase III trials, the results of which
Gabon, during an epidemiological investigation should be known in about three years. According to
carried out in 2004. All the subjects were Christian Loucq, Director of PATH MVI, an effective
asymptomatic at the time of sampling. vaccine will be marketable in about five years. A
“second-generation” vaccine (RTS, S/AS02A +) 2.2 Peptide Expression combining RTS, S and other parasite antigens is
The PALPF-derived peptides are annotated as already in development, with the aim of reaching 80%
follows in PlasmoDB (http://plasmodb.org/plasmo/): protection in 2020-2025.
PF07_0032, PF10_0226, PFI0130c, PFD0875c, and In 2004, we showed that approximately 20% of P.
MAL13P1.206. Expasy © software was used to design falciparum field isolates in Franceville, Gabon,
peptides with T and B cell epitopes from the induced human lung endothelial cell (HLEC) nucleotide sequences. After confirming the amino acid apoptosis, by cytoadherence [18, 19]. In addition,
sequences and the presence of the two epitopes, apoptogenic isolates were more frequent in children
GenScript (New Jersey, the USA) synthesized the with neurological signs (prostration or coma) [18].
peptides.
Whole-transcriptome analysis showed that 59 genes
2.3 Serology
were more intensely transcribed in apoptogenic isolates than in non apoptogenic ones [20]. The
The synthetic peptides are designated in Table 1. complete P. falciparum genome has been sequenced
Plasma was collected from 50 adults and 30 children. (PlasmoDB) [21]. The transcriptome includes 10
Antibody responses were determined with an surface antigens and several enzymes involved in
enzyme-linked immunosorbent assay (ELISA) using essential functions such as parasite replication.
5 µg/mL each peptide, 100-fold diluted plasma, and Functional analysis using double-strand RNA 2,000-fold diluted goat anti-human IgG (Fc-specific) interference to knock down the expression of 8 of
conjugated to alkaline phosphatase (Sigma, St. Louis, these genes enabled us to significantly reduce the
MO, USA). The amount of bound enzyme was apoptogenic response in 5 genes (PF07_0032, derived from absorbance at 405 nm after adding
Antigenicity of Synthetic Peptides Derived from Plasmodium
Apoptosis-Linked Pathogenicity Factors
Table 1 The amino acid sequence of the 10 peptides and
asymptomatic at the time of sampling. Five adults
corresponding gene in PlasmoDB nomenclature.
were smear-positive for P. falciparum (8 to 160 Gene nomenclature in Synthetic peptide and amino-acid
PlasmoDB® parasites/µL), as were six children (5 to 800
PF07_0032 P1: CNTNPITRYLDRRRK parasites/µL). Plasma from 10 European adults was
228 aa P2: CEEAAEKEAESEEQE used to determine ELISA cut-off values. PF10_0226
P3: CRQKMKEKATQISEE 79 aa
P4: CGESEKKLEGDVTEL
3.2 Choice of Peptides
PFI0130c P5: CNKNEKDKYDKLGKD* 434 aa
P6: SEKTYKRYNLNGDGC* Two 15-amino-acid peptides were selected, 2 for
PFD075c P7: RRRERGRERGREKGC each PALPF (GenScript , New Jersey, the USA). 1215 aa
P8: CDEGDKNYDDNKKND MAL13P1.206
P9: CNGNSNEGNGKGGDP
3.3 Total IgG Assay
687 aa P10: CLPGDKNVKSQQDDS
3.3.1 PF07_0032
p-nitrophenylphosphate. Peptide 1: CNTNPITRYLDRRRK IgG subclass analysis used 50-fold diluted plasma,
The prevalence rate of anti-P1 total IgG was 6% 1000-fold diluted mouse anti-human IgG1, IgG2,
(3/50). The median optical density (OD) in the whole IgG3 and IgG4 antibodies (Sigma, St. Louis, MO,
adult population was 0.122 (Fig. 1). USA), and 2,000-fold diluted goat anti-mouse IgG
Peptide 2: CEEAAEKEAESEEQE (Sigma, St. Louis, MO, USA). Bound enzyme was
The prevalence of anti-P2 total IgG antibodies was detected as described above. Positive and negative
34% (17/50) in adults and 20% (6/30) in children. The control plasma samples were included in each plate.
median OD was 0.176 in the whole adult population The positive cut-off was the mean optical density (OD)
and 0.117 for children. The median OD of positive plus 2 SD of plasma samples from 10 non-immune
adults was 1.532 (Fig. 1).
subjects.
3.3.2 PF10_0226
Peptide 3: CRQKMKEKATQISEE
2.4 Statistical Analysis The prevalence rate of anti-P3 total IgG was 4%
(2/50). The median OD in the whole adult population Data were analyzed with Statview and EPI INFO
was 0.148 (Fig. 1).
software. The characteristics included in the statistical Peptide 4: CGESEKKLEGDVTEL analysis were the sample size, age, sex, parasitemia, The prevalence rate of anti-P4 total IgG was 44% and optical density (OD). (22/50). The median OD in the whole adult population
We analyzed demographic characteristics, the was 0.199 and 0.455 for positive subjects (Fig. 1).
prevalence rate of IgG antibodies and isotypes, and
3.3.3 PFI0130c
the prevalence rate of multiple subclass positivity.
2 Peptide 5: CNKNEKDKYDKLGKD Proportions were analyzed with the -test, Student The prevalence rate of anti-P5 total IgG was 61%
Mann-Withney and Wilcoxon tests. Significance was (30/49). The median OD in the whole adult population
assumed at P < 0.05. was 0.391 and 0.718 for positive subjects (Fig. 1).
3. Results and Discussion
Peptide 6: SEKTYKRYNLNGDGC The prevalence rate of anti-P6 total IgG was 51%
3.1 Study Population (25/49). The median OD in the whole adult population The study population consisted of 50 adults aged
was 0.288 and 0.512 in positive individuals (Fig. 1). from 17 to 67 years (median 36) and 30 children aged
3.3.4 FD075c and MAL13P1.206 from 1 to 5 years (median 2). All the subjects were
The prevalence rate of total IgG specific for peptides
Antigenicity of Synthetic Peptides Derived from Plasmodium
Apoptosis-Linked Pathogenicity Factors
P7, P8, P9 and P10 was 0%, 2%, 2% and 4% IgG2: Anti-P5 IgG2 was detected in 45% subjects respectively (Fig. 2).
(13/29). The median OD was 0.217 for the whole population and 0.522 for positive subjects (Fig. 3).
3.4 IgG Isotypes IgG3: Anti-P5 IgG3 was detected in 76% of
IgG isotypes were analyzed only for 29 of 50 subjects (22/29). The median OD was 0.493 for the plasmas, which gave positive reaction with peptide 5.
whole population and 0.526 for positive subjects (Fig. 3). IgG1: Anti-P5 IgG1 was detected in 20 of the 29
IgG4: Anti-P5 IgG4 was detected in 62% of subjects tested (69%). The median OD was 0.342 for
subjects (18/29). The median OD was 0.429 for the the whole population and 0.845 for positive subjects
whole population and 0.643 for positive subjects (Fig. 3).
E A E Peptide P1 0,0 A C
Fig. 1 Total IgG antibodies against peptide 1 to 6, in adults from Dienga. Europeans (E) as non-immune control, Adults (A) from Dienga and Children (C), the cut-off by a red dotted line.
Fig. 2 Prevalence rate of Total IgG.
Antigenicity of Synthetic Peptides Derived from Plasmodium
Apoptosis-Linked Pathogenicity Factors
E IgG1 0,0 A E
Fig. 3 Anti-P5 subclass Immunoglobulin in Dienga adult patients.
3.5 Antibody Responses to Multiple Peptides
3.5.1 Total IgG Overall, 10.2% (5/49) of the tested plasma samples recognized 4 peptides (P2, 4, 5 and 6), and 20.4% (10/49) recognized 3 peptides (P4, P5 and P6). Peptides P5 and P6 were recognized by 38.8% (19/49) of samples, P2 and P5 by 24.5% (12/49), P2 and P6 by 22.4% (11/49); P4 and P5 by 28.6% (14/49) and P4 and P6 by 26.5% (13/49). Four samples (8%)
Fig. 4 Prevalence rate of IgG isotypes against peptide 5.
recognized none of the peptides. falciparum antigens. Four peptides (P2, P4, P5 and P6)
3.5.2 IgG Isotype Distribution were recognized by plasma samples from a large All 29 adults tested had at least one Ig subclass
proportion of the subjects (34%, 44%, 61% and 51%, directed against P5, while 13 adults (44%) had both
respectively). Peptides P5 and P6 were derived from P5-specific IgG1 and IgG3. There was no significant
antigen PFI0130c, while P2 and P4 were derived from difference in the prevalence of IgG1, IgG3 and IgG4,
antigens PF07_0032 and PF10_0226, respectively, while IgG2 was significantly less prevalent than IgG3
showing the potential interest of antigen PFI0130c. (P = 0.015) (Fig. 4).
Nevertheless, a minority of plasma samples did not recognize either P5 or P6, and plasma from 8% of the
4. Discussion
subjects (4/50) reacted with none of the peptides. We analyzed the prevalence rate of natural IgG
These subjects could be restricted by their major antibody reactivity to 10 peptides derived from P.
histocompatibility complex (MHC). Similar results
Antigenicity of Synthetic Peptides Derived from Plasmodium
Apoptosis-Linked Pathogenicity Factors
have been obtained with other peptides, such as SPf66 [22], LSA-1 [23, 24], GLURP [25], EBA 175 [26] and MSP1 19 [27].
A large proportion of the subjects had total IgG antibodies reacting simultaneously with at least 2 peptides. These antibodies could be implicated in protection. In zones with perennial transmission, antibodies directed against monomorphic or variable epitopes participate in protection [28-30]. The genes encoding antigens PF07_0032 (P2) and PF10_0226 (P4) are conserved but their function is unknown. These two antigens are expressed at the schizont stage. It is not known whether the gene encoding PFI0130c (corresponding to peptides P5 and P6) is conserved. However, the protein, expressed at both trophozoite and schizont stages, is known to be exported (http://plasmodb.org/plasmo/).
Among reactive subjects, the median optical density of total IgG directed against P2 was higher than that directed against P4 and P6 (P = 0.009; P = 0.012 respectively), while the median OD in P5 responders was significantly higher than that in P4 (P = 0.04). Five of the remaining 6 peptides (P1, P3, P8, P9 and P10) were recognized by 2% to 6% subjects, while P7 was not recognized by any of the samples. It is possible that the antigens from which these peptides are derived are expressed by developmental parasite stages, making them less accessible to the immune system. This could also explain the results obtained with antigens expressed by mature parasites, and particularly the schizont stage, such as PF07_0032 (P1) and PF10_0226 (P3), contrary to antigens expressed at all developmental stages. It is noteworthy that the whole-transcriptome analysis used to identify these new P. falciparum antigens was carried out with messenger RNA from field isolates at the schizont stage.
PFD087c (P7 and P8) and MAL13P1.206 (P9 and P10) are described as adhesins, as they are strongly associated with cytoadherence [20]. These antigens may be more accessible to the immune system because they are expressed at all developmental stages,
with maximal expression at the ring and schizont stages (http://plasmodb.org/plasmo/). Overall, these results show that the presence of B and T cell epitopes is not sufficient to ensure good antigenicity.
Approximately 20% of the children had specific total IgG directed against peptide 2. Only total IgG against peptide 2 was measured in children. Some children had antibodies directed against PALPF antigens, and particularly PF07_0032. The protective nature of these antibodies remains to be studied.
Most of the adults had IgG1, IgG2, IgG3 and IgG4 antibodies recognizing antigen PFI0130c, and particularly peptide P5. All the subjects had at least one reactive subclass, while 13 subjects (44%) had both IgG1 and IgG3. There was no significant difference in the prevalence of IgG1, IgG3 and IgG4, but IgG2 was significantly less prevalent than IgG3 (P = 0.015). This clearly shows competition between the different isotypes. A similar isotype distribution was found in a cohort study using recombinant EBA-175 [31], EBA-175 peptide 4 [26], MSP1 [32] and MSP2 [33, 34]. Anti-P5 IgG1 and IgG3 were most prevalent in our study. These anti-P5 antibodies could cooperate with cells to destroy the parasite and could thus be involved in protection. The prevalence of IgG4 was also high (62%). This isotype can recognize the same epitopes as IgG1 and IgG3 and could thus compete with them, limiting their role in protection.
It would be interesting to investigate whether age/exposure-related acquisition of anti-PALPF antibodies may contribute to the development of clinically protective immunity. Rodent studies of the most prevalent peptides are warranted. A synthetic “tree” peptide presenting multiple antigenic epitopes might also be of interest. Alternatively, a synthetic nucleotide sequence of these arborescent peptides could be used to produce a recombinant antigen.
5. Conclusion
In conclusion, three P. falciparum apoptogenic antigens (PF07_0032, PF10_0226, and PFI0130c)
Antigenicity of Synthetic Peptides Derived from Plasmodium
Apoptosis-Linked Pathogenicity Factors
were recognized by plasma samples from a large [10] H. Brown, S. Rogerson, T. Taylor, M. Tembo, J. proportion of subjects living in an endemic zone, Mwenechanya, M. Molyneux, et al., Blood-brain barrier function in cerebral malaria in Malawian children, The
warranting further analysis as potential vaccine American Journal of Tropical Medecine and Hygiene 64 candidates.
(2001) 207-213. [11] S.C. Wassmer, V. Combes, G.E. Grau, Pathophysiology
Acknowledgments
of cerebral malaria: Role of host cells in the modulation of cytoadhesion, Annals of the New York Acadademy of
The authors thank the study participants and the
Sciences 992 (2003) 30-38.
staff of the Medical Parasitology Unit of Centre [12] P. Pino, I. Vouldoukis, J.P. Kolb, N. Mahmoudi, I. International de Recherches Médicales de Franceville
Desportes-Livage, F. Bricaire, et al., Plasmodium falciparum -infected erythrocyte adhesion induces caspase
(CIRMF). activation and apoptosis in human endothelial cells, The
CIRMF is funded by Total Gabon, the Gabonese Journal of Infectious Diseases 187 (2003) 1283-1290. government and the French Ministry of Foreign and
[13] V. Combes, N. Coltel, D. Faille, S.C. Wassmer, G.E. European affairs (MAEE).
Grau, Cerebral malaria: Role of microparticles and platelets in alterations of the blood-brain barrier, The
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[33] R.R. Taylor, S.J. Allen, B.M. Greenwood, E.M. Riley, genotypes related to clinical status in children from
IgG3 antibodies to Plasmodium falciparum merozoite Bakoumba, Gabon, Clinical Medecine and Research 4
surface protein 2 (MSP2): Increasing prevalence with age (2006) 7-11.
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American Jornal of Tropical Medecine and Hygiene 58 between treatment outcome and both anti-MSP119
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Journal of Life Sciences 6 (2012) 595-601
Genotype × Environment Interaction of Carotene Content of Yellow-Fleshed Cassava Genotypes in Nigeria
1 1 2 1 Norbert G. Maroya 1 , Peter Kulakow , Alfred G.O. Dixon , Busie Maziya-Dixon and Moshood A. Bakare
1. International Institute of Tropical Agriculture, PMB5320, Ibadan, Oyo State, Nigeria
2. Sierra Leone Agricultural Research Institute, PMB1313, Freetown, Sierra Leone
Received: November 20, 2011 / Accepted: January 16, 2012 / Published: June 30, 2012.
Abstract: Cassava is an important starchy root crop and a major staple for more than 70 million people in Nigeria. New yellow-fleshed genotypes are being developed to combat vitamin A deficiency. Trials of 18 yellow-fleshed genotypes and two officially released white-fleshed clones, used as checks for 2008/2009 and 2009/2010 seasons in five major cassava growing agroecological zones of Nigeria. The trial locations were Ikenne (humid forest), Ibadan (forest-savanna transition), Ubiaja (subhumid forest), Mokwa (southern Guinea savanna) and Zaria (northern Guinea savanna). At each location, the trial was established in a randomized complete block design with four replications. The objective of the study was to assess genotype performance and genotype × environment interaction for total carotene concentration (TCC), total carotene content per root (TC-R), and total carotene content per plant (TC-P). Significant differences (P < 0.001) among genotypes, environments and genotype × environment interaction for all the traits evaluated were observed. For TCC, TC-R and TC-P, the best genotypes across the 10 environments were TMS I051601, TMS I050311, and TMS I050998. Variation among genotypes accounted for most of the Total Sum of Squares for TCC (67.9%), TC-R (39.0%) and TC-P (35.9%). These characteristics of total carotene were also highly correlated. This study revealed that cassava with total carotene concentration can be assessed using either the TCC, or the TC-R, or the TC-P.
Key words: Yellow-fleshed cassava, carotene, genotype × environment interaction.
1. Introduction thereby influencing the expression of traits [1]. Genotype × environment (G × E) interaction occurs in
Cultivars of cassava (Manihot esculenta Crantz) both short-term and long-term crop performance trials often demonstrate specific adaptation because of their [2]. It is the change in cultivars’ relative performance high sensitivity to the genotype × environment when grown in different environments, resulting from interaction. Genotype (G) refers to the set of genes the differential response of the genotypes to various possessed by individuals that are important for the edaphic, climatic and biotic factors [3]. G × E expression of the traits under investigation. The interaction is a major concern in plant breeding Environment (E) is usually defined as all non-genetic because it reduces progress from selection and makes factors that influence the expression of traits and cultivar recommendation difficult because the choice includes all biophysical factors such as water, of superior cultivars changes with locations [4]. nutrition, temperature, and the diseases or pests that Sub-Saharan Africa (SSA) is the world’s largest influence the growth and development of individuals, producer of cassava, with 121.3 million t of fresh
Corresponding author: Norbert G. Maroya, Ph.D., research storage roots produced in 2008 grown in 12.02 million fields: breeding cassava for high carotene content, high dry matter content and resistance to cassava mosaic diseases.
ha [5]. Nigeria was the leading cassava producing E-mail: n.maroya@cgiar.org.
Genotype × Environment Interaction of Carotene Content of
Yellow-Fleshed Cassava Genotypes in Nigeria
country in SSA, producing 44.58 million ton 3.78 two white-fleshed check genotypes were evaluated at million ha. In Nigeria, more than 70% of the
five locations representing the major cassava growing production is processed into gari at the village level.
agroecological zones in Nigeria. The 20 genotypes Gari is the principal source of calories for 70-80
were cultivated for two 12-month cropping seasons in million Nigerians. In some locations in the South East
2008/2009 and 2009/2010 at Ibadan (forest-savanna of the country, most farmers grow varieties with white
transition); Ikenne (humid forest); Ubiaja (sub-humid roots but processors during gari preparation, add palm
forest); Mokwa (southern Guinea savanna); and Zaria oil. The resultant yellow gari commands a higher
(northern Guinea savanna) (Table 1). The five price, as it is preferred by a larger proportion of the
locations combined with the two cropping seasons populace. Therefore, it is important to breed and
constituted the 10 environments in which the promote yellow-fleshed cassava varieties which are
genotypes were evaluated (Table 2).
A randomized complete block design with four deficiency.
very useful in combating the widespread vitamin A
replications was used at each location with plants
A nationwide food consumption and nutrition established under rain-fed conditions at 1 m × 1 m survey revealed that 29.5% of children under 5 years
spacing on ridges about 0.3 m high. Each experimental of age were vitamin A deficient (serum retinol < 0.70
unit was a 6 m × 6 m plot of 36 plants. No fertilizer was µmol/L) [6]. The proportion differed among applied. Weed control was accomplished manually as agroecological zones with an incidence of 31.3% in
necessary. Harvesting was done in all the locations at the dry savanna, 24.0% in the moist savanna, and
approximately 12 months after planting. A border of 1 29.9% in the humid forest (P < 0.001). β-carotene, the
m was left and only the inner plants (maximum of 16 most potent and widespread form of pro-vitamin A [7],
plants per plot) were harvested.
is the predominant carotenoid in cassava, occurring as
Table 2 List and composition of the 10 environments.
a mixture of trans- and cis-forms [8]. Structurally,
Cropping seasons vitamin A (retinol) is essentially one-half of the
Abbreviation for GGE biplot Locations
E 1 Ibadan 2008-2009 β-carotene molecules. Typical white-fleshed E 2 Ibadan 2009-2010
genotypes contain only small amounts of β-carotene E 3 Ikenne 2008-2009
E 4 Ikenne [9], however, yellow-fleshed cassava contains up to 2009-2010 E 5 Mokwa 2008-2009
about 100 times more [10]. E 6 Mokwa 2009-2010
2. Materials and Methods E 7 Ubiaja 2008-2009 E 8 Ubiaja 2009-2010
A set of 18 yellow-fleshed cassava genotypes at a E 9 Zaria 2008-2009 E 10 Zaria 2009-2010
breeding stage known as Uniform Yield Trial with
Table 1 Characteristics of five locations representing the agroecological zones where the trials were conducted.
Length of
Annual
Location Latitude Longitude Max. T
(m·asl) growing period rainfall FAO dominant (days) (mm) soil type Agroecological zone Ibadan
Min. T
1,312 Ferric Luvisols Forest Savanna transition Ikenne 6.867 3.717 28.2-33.4 22.3-24.8
27.3-33.8 20.7-23.1 227
1,515 Distric Nitosols Humid forest
Mokwa 9.283 5.05
1,149 Distric Nitosols Southern Guinea savanna Ubiaja
29.2-37.3 18.1-24.6 132
27.1-32.6 19.9-22.5 285
1,186 Distric Nitosols Sub-humid forest
Zaria 11.167 7.633 27.4-35.5 13.9-24.3 687
1,076 Ferric Luvisols Northern Guinea savanna T: temperature.
Genotype × Environment Interaction of Carotene Content of
Yellow-Fleshed Cassava Genotypes in Nigeria
For laboratory determination of total carotene Similarly, the total carotene content in storage roots concentration (TCC), five medium sized roots were
per plant (TC-P) was obtained by multiplying the collected from the harvested roots in two replications.
TCC by 85%, the average weight of the flesh in the Three of the five roots were washed, dried, peeled,
storage roots per plant, as indicated in the formula and again washed and dried with tissue paper. Each
below:
TC-P (µg) = TCC (µg·g peeled root was cut longitudinally in half and the two -1 ) * 85% (average root halves were again cut longitudinally into quarters.
weight per plant (g)) (2) Two opposite quarters of the three roots were pooled
Data collected were statistically analyzed using the for total carotene quantification. The six quarters were
GGE biplot. This is a tool for G × E data analysis to
achieve understanding about test environments and After many subdivisions, a sample of approximately
cut in small pieces of about 1 cm 3 and mixed together.
genotypes. It is a set of interpretation methods and 10-15 g of small pieces of root was taken as uniform
refers to a biplot that is based on the principal and representative. The sample was ground in
component analysis of environment-centered or refrigerated acetone solvent using a mortar and pestle.
standardized G × E data that displays both genotype All analyses of the total carotene were completed
main effects (G) and the genotype × environment within 24 hours after each harvest, using the
interaction. These are the two sources of variation that spectrophotometer method described in the are relevant to variety evaluation [13]. HarvestPlus Handbook for Carotenoid Analysis [11].
3. Results and Discussion
During sample preparation, special care was taken to avoid directly exposing the storage roots to sunlight
3.1 Total Carotene Concentration in Fresh Storage and the lights in the laboratory were protected with Roots
UV filters. Samples were covered with aluminium foil The statistical analyses of data recorded for TCC when not under processing.
has shown significant differences among genotypes, For cassava, it was known that the peel weight
environment, and for genotype × environment represents an average of 15% (ranging from 10 to 20%)
interaction (Table 3). The contribution to the total Sum of the weight of the storage root and the edible fleshy
of Squares was 67.9% for genotypes; 6.7% for portion makes up an average of 85% [12]. Based on the
environment, and 14.8% for the genotype × environment fact that the TCC was determined per gram using the
interaction. These three sources of variation together peeled root, the average carotene content per storage
have a contribution of 87.4% to the Sum of Squares. root (TC-R) was calculated as follows:
The highest TCC (5.97 µg·g -1 fresh weight) was TC-R (µg) = TCC (µg·g -1 ) * 85% (average weight
obtained for genotype TMS I0150311 and the lowest of individual fresh root (g)) (1)
average was recorded by the white check TMS I30572
Table 3 Combined analysis of variance table and the contribution of genotype, environment and genotype × environment interaction to the Sum of Squares of total carotene concentration.
Source DF SS MS F P
%SS Total 398 1,086.6
14.8 BLK(E)
10 4.9 0.5 0.8 Error
Genotype × Environment Interaction of Carotene Content of
Yellow-Fleshed Cassava Genotypes in Nigeria
(0.87 µg·g 1 fresh weight). The carotene concentration
the winning niche occupied by genotype G 12 and also
reported in this work ranged from a minimum 0.87 to
contained E 1 ,E 2 ,E 3 ,E 9 , and E 10 . The second fell in
a maximum 5.92 µg·g -1 (Table 4). These values were the sector of genotype G 5 and comprised lower than the range from 1.45 to 7.69 µg·g -1 with an
environments E 4 ,E 5 ,E 6 ,E 7 , and E 9 .
average of 4.26 µg·g -1 obtained in a similar study
3.2 Average Total Carotene Content per Fresh Storage
conducted with seven genotypes in two years at three
Root
locations in Ghana [14]. The average TCC of this study (3.6 µg·g -1) was also lower than the 5.04 µg·g -1 reported
The TC-R per genotype ranged from 218.7 to
for 25 yellow-fleshed genotypes in Nigeria [15].
1,925.4 µg with the mean of 969.7 µg (Table 4). The
In the GGE biplot analysis, the first and second
highest value of TC-R was recorded for TMS
principal components together explained 89.7% of the
I051601 (G 12 ) and TMS I050311 (G 5 ). The lowest
total variation. Genotypes G 12 ,G 5 , and G 6 had the
value was registered for TMS I30572 (G 15 ) and TME
highest value of TCC. Genotypes G 15 and G 10 (the
B1 (G 10 ). These genotypes were those identified
white-fleshed checks) had the lowest TCC. A polygon
using TCC. The variation of TC-R among genotypes
view of the GGE biplot (Fig. 1) showed which
was similar to that observed for TCC. The table of
genotypes performed best in which environments. The
Analysis of Variance of the combined data of the ten
vertex genotypes for TCC were G 12 ,G 5 ,G 15 , and G 10 .
environments (Table 5) also showed significant
Two mega-environments were defined. The first was
differences among genotypes, environments, and
Table 4 Average TCC (µg·g -1 ); TC-R (µg) and TC-P (µg) for 18 yellow-fleshed and two white-fleshed cassava genotypes in 10 environments.
Genotype Abbreviation of genotype TCC (µg·g -1 (fresh weight)) TC-R (µg)
TC-P (µg)
TMS I050024
G 1 2.55 ghi 878.25 cde
4,319.54 cde
TMS I050286
G 3.14 efghi 855.20 cde 4,745.34 2 bcde
TMS I050303
G 3 3.91 cde 1,141.77 cd 4,487.90 bcde
TMS I050099
G 4 4.86 bc 1,058.44 cd 5,134.19 bcd
TMS I050311
G 5.92 a 1,594.15 ab 8,456.50 5 a
TMS I050998
G 6 5.20 ab 1,240.53 bc 8,175.82 a
TMS I050741
G 7 3.46 defgh 945.86 cd 3,853.14 de
TMS I051274
G 8 3.02 efghi 731.36 de 3,848.67 de
TMS I050231
G 3.77 de 888.73 cde 5,290.41 9 bcd
TME B1
TMS I051570
G 11 2.46 hi 533.72 ef 2,591.93 efg
TMS I051601
G 12 5.97 a 1,925.38 a 8,405.69 a
TMS I050127 G
13 4.40 bcd 1,162.89 cd 5,412.54
bcd
TMS I050327
TMS I30572
G 15 0.87 j 218.71 f 1,170.50 fg
TMS I050128
G 16 3.65 def 1,118.17 cd 5,246.74 bcd
TMS I051740
G fg de 17 de 2.71 794.31 3,899.88
TMS I051553
G 18 3.51 defg 832.83 de 4,619.09 bcde
TMS I051814
G 19 2.18 i 811.97 de 3,394.69 def
TMS I050125
Mean 3.6 969.7 4,851.8 SE 0.8 296.6 1,683.7
CV% 21 31 35 LSD (5%)
The same letter in a column are not significantly different at 95% level.
Genotype × Environment Interaction of Carotene Content of
Yellow-Fleshed Cassava Genotypes in Nigeria
genotype × environment interaction. results for TC-P in ranking were very similar to those The contribution of genotype to the total Sum of
of TCC and TC-R. The contribution of the genotype, Squares of TC-R was 39.1% (lower than the 67.9% of
environment, and genotype × environment interaction TCC). The respective contributions of the to the variation in the total Sum of Squares of TC-P environment and the genotype × environment accounted respectively for 35.9%; 27.6%; and 23.6%. interaction were 24.9% and 23.1% higher than those
These three contributions in the Total Sum of of TCC. When the contributions of the three sources
Squares of TC-P were equal to 87.1%, the same as of variation are combined, they accounted for 87.1%
for TC-R and very close to those of TCC (89.4%). of the total Sum of Squares.
The two principal components explained 85.1% The GGE biplot of TC-R (Fig. 2) showed plots of
of the total variation in a polygon view of the GGE two principal components that explained 83.7% of the
biplot (Fig. 3) that showed which genotypes won in
which environments for TC-P. The vertex for G 17 ) and G 13 , anchored sectors that covered all 10 genotypes for the TC-P were G 13 ,G 17 , and G 4 . The environments grouped into two mega-environments
total variation. The vertex genotypes, G 18 (same sector
two mega- environments were defined in the sectors with G 18 linking E 4 ,E 6 ,E 9 , and E 10 and with G 13 of G 13 (or G 12 ) and G 17 , each having five
grouped with environments E 1 ,E 2 ,E 3 ,E 5 ,E 7 , and E 8 .
environments. The first mega-environment was the winning niche of genotypes G 12 and G 13 made up of
3.3 Average Total Carotene Content in Fresh Storage
E 1 ,E 2 ,E 3 ,E 7 , and E 9 . The second fell in the sector Roots per Plant of genotype G 17 and was made up of environments
The genotype means for TC-P ranged from
E 4 ,E 5 ,E 6 ,E 8 , and E 10 .
1,049.37 to 8,456.50 µg (Table 6). The lowest TC-P
3.4 Relationships Among Total Carotene Content was recorded for the white-fleshed check varieties
Characteristics TME B1 (G 10 ) and TMS I30572 (G 15 ). The highest
values were registered for G 5 (TMSI050311); G 12 The correlation coefficients (Table 7) revealed that (TMS I051601), and G 6 (TMS I050998). G 5 and G 12 TCC was significantly correlated with TC-R (0.94) were identified in the first category with the highest
and TC-P (0.95). The TC-R was also significantly content for all the three total carotene traits. The
correlated with TC-P (0.93). These results mean that
Table 5 Combined analysis of variance table and genotype, environment and genotype × environment contributions to the Sum of Squares of TC-R.
Source DF
SS
MS
%SS TOTAL 398 1.5E + 08
G 19 5.9E + 07
3.1E + 06
24.9 G*E
E 9 3.7E + 07
4.2E + 06
23.1 BLK(E)
Table 6 Combined contributions of analysis of variance table, genotype, environment, and genotype × environment interaction to the Sum of Squares of TC-P.
Source DF SS MS F P
%SS TOTAL 398 4.5E + 09
G 19 1.6E + 09
8.6E + 07
27.6 G*E
E 9 1.2E + 09
1.4E + 08
23.6 BLK(E)
1.1E + 09
6.2E + 06
1.7 Error
10 4.9E + 07
4.9E + 06
5.4E + 08
2.8E + 06
Genotype × Environment Interaction of Carotene Content of
Yellow-Fleshed Cassava Genotypes in Nigeria
Fig. 1 Mega-environment defined by different winning cassava genotypes tested in 10 environments for TCC (µg·g -1 fresh weight).
Fig. 2 Mega-environment defined by different winning cassava genotypes tested in 10 environments for TC-R.
Fig. 3 Mega-environment defined by different winning cassava genotypes tested in 10 environments for TC-P.
Genotype × Environment Interaction of Carotene Content of
Yellow-Fleshed Cassava Genotypes in Nigeria
Table 7 Pearson correlation coefficients among total carotene
and adaptability: Analytical methods and implications for
traits based on the average values over the genotypes and the
cassava breeding for low-input agriculture, in: F. Ofori,
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S.K. Hahn (Eds.), Tropical Root Crops in a Developing TCC TC-R TC-P
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International Society for Tropical Root Crops, Accra, TC-R
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pp. 51-84.
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B. Maziya-Dixon, I.O. Akinyele, R.A. Sanusi, T.E.
4. Conclusion
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D.B. Rodriguez-Amaya, Nature and distribution of significant variation among genotypes,
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pp. 547-589.
I051601 and TMS I050311, followed by TMS
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