Research Report
Identification and Molecular Analysis of Pro-vitamin A Carotenoid Genes in Cassava (Manihot esculenta Crantz) 
,
Adenubi Adesoye1 
,
Melaku Gedil2 2 Bioscience Center, International Institute for Tropical Agriculture (IITA), PMB 5320 Ibadan, Oyo, Nigeria
Author Correspondence author
Molecular Plant Breeding, 2017, Vol. 8, No. 4 doi: 10.5376/mpb.2017.08.0004
Received: 01 Mar., 2017 Accepted: 01 Jun., 2017 Published: 30 Jun., 2017
Udoh L.I., Adesoye A., and Gedil M., 2017, Identification and molecular analysis of pro-vitamin A carotenoid genes in cassava (Manihot esculenta Crantz), Molecular Plant Breeding, 8(4): 38-44 (doi: 10.5376/mpb.2017.08.0004)
Cassava is one of the most important sources of calories in the tropics and consumed as a staple food. Several cassava varieties are deficient in vitamin A. The present work is envisaged towards the genetic improvement of carotenoid content in cassava, by identifying single nucleotide polymorphism (SNP) attributed to variation in carotenoid concentration among some cassava genotypes. Phytoene synthase (PSY), β-carotene hydroxylase (HYD), lycopene β and ε cyclase (LYCB and LYCE), have been found to play a role in increasing levels of β-carotene in plants. A total of 40 lines with varying total carotene content were drawn from a cassava recurrent selection breeding program. Primers for HYD, LYCB, LYCE, and PSY genes designed from cassava ESTs were used to genotype the cassava lines. Amplified PCR products were purified and sequenced. A total of 169 polymorphisms were detected among the sequences under study both in the coding and non-coding regions of the genes. Analysis of SNPs in relation to carotene content of each accession revealed a variation G/T at 1295th position of the LYCE reference gene that caused an amino acid exchange from tryptophan (Trp) to leucine (leu) in a high carotene line 07/0593. High total carotene lines recorded the highest number of polymorphism with 42%of entire polymorphisms observed. This indicates that if the whole gene is sequenced unique polymorphisms associated with high carotene cassava can be retrieved for marker development to aid selection for high carotenoid containing cassava germplasm.
Introduction
Cassava (Manihot esculenta Crantz) is a drought tolerant crop grown in tropical and sub-tropical areas where many people are afflicted with undernutrition, making it a potential valuable food security source (Montagnac et al., 2009). Its storage roots form the basic carbohydrate element of the diet and the leaves are eaten as a preferred green vegetable in many parts of Africa. Tuber flesh colour and good culinary quality are important traits for consumption of cassava as staple food. In most of the cultivated cassava the tuber flesh is white or cream which contain negligible amount of carotenoids (Bradbury and Holloway, 1988). Nutritionally, cassava tubers are rich in carbohydrates, but deficient in many proteins and essential micronutrients (Montagnac et al., 2009; Salvador et al., 2014).
The yellow flesh colour of cassava varieties is associated with presence of carotenoids and the nutritive importance of carotenoids is attributed to its conversion to vitamin A when consumed. Carotenoids are important as they help prevent heart attacks or cancer, lower cataract risks and macular disorders, and enhance the immune system (Pfeiffer and McClafferty, 2007; Saltzman et al., 2013). The consumption of tuberous roots of β-carotene-rich cultivars may contribute significantly to addressing vitamin A deficiency in sub-Saharan Africa. Vitamin A deficiency is a serious public health problem in many parts of sub-Saharan Africa, affecting mostly preschool children, pregnant and lactating mothers. It is the cause of childhood blindness, reduced immune function and mortality resulting from gastrointestinal diseases (West and Mehra, 2010; Saltzman et al., 2013). The continued prevalence of micronutrient deficiency in many developing regions of the world necessitates the development of new varieties of staple food crops that are enriched in limiting nutrients. The consumption of carotene rich foods is the most effective intervention for vitamin A deficiency and increasing the carotenoid content of staple food crops such as cassava can be achieved through marker assisted breeding. Conventional breeding is currently underway to release varieties rich in vitamin A (IITA, 2014). Because of the long breeding cycle of cassava (six to ten years), there is a need to apply molecular markers at some evaluation stages were about one hundred thousand seedlings are being evaluated for preselection.
DNA marker tools could be utilized for the development of molecular markers at important target genes involved in carotenoid biosynthetic pathway which can aid selection for high carotene cultivers. Single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels) represent the most frequent form of naturally occurring genetic variation in populations (Chen and Sullivan, 2003; Zhou et al., 2016). In some cases, SNPs have correlation with non-conservative amino acid changes which may result to phenotypic variation. This permits the association of genotypic and phenotypic variations (Gray et al., 2000; Rafalski, 2002; Neuhaus and Horn, 2004; Zhou et al., 2016). Genes such as phytoene synthase (PSY), β-carotene Hydroxylase (HYD), Lycopene β and ε cyclases (LYCB and LYCE), have been reported to be critical in increasing levels of β-carotene in the carotenoid pathway of plants (Harjes et al., 2008; Yan et al., 2010; Welsch et al., 2010).
The identification of genes and alleles that modify β-carotene trait is the most direct approach using genomics for plant breeding. In this study, direct sequencing approach (Rafalski, 2002) of amplified PCR products of the key carotenoid genes (HYD, PSY, LYCE and LYCB) was carried out and carotenoid sequences obtained from cassava accessions with diverse total carotene content was accessed for SNPs polymorphism. The informativeness of each SNP as regards its association with the phenotype of the cassava tuber color was carefully examined. The polymorphisms, generated as a result of effects of transition, transversion, and InDels were assessed. This approach will contribute to provide an insight into the genetic and molecular basis of carotenoid biosynthesis to enhance genetic improvement of nutritional quality of cassava storage roots through marker assisted selection.
1 Results
1.1 Primer design, PCR amplification and sequence analysis
A total of 20 cassava specific primers were designed at different points on the mRNA transcript gotten from Phytozome Manihot esculenta (v5.0) database. Selected primers are those that showed single band amplification on agarose gel (Figure 1) for all accessions studied.
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Figure 1 PCR Amplification of HYD primers, run on 1.5% agarose gel against a 50bp DNA ladder |
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1.2 Sequencing and sequence analysis
Bands of the appropriate size were purified and sequenced in forward and reverse directions, and analyzed on Codon Code Aligner (v2.0.6) software for identification of nucleotide variations among the sequences. After careful editing of Sequences, a total of 305 sequences were generated across all genes (HYD1, 73; LYCB, 78; PSY, 76; LYCE, 78). Afterwards, polymorphic positions identified were (2 for LYCB, 6 for LYCE, 4for HYD1 and 4 for PSY1), which were then called for the presence of usable SNPs. The gene sequences for LYCE, LYCB, PSY, and HYD from Phytozome Manihot esculenta database are 5 320 bp, 2 224 bp, 1 895 bp, 2 292 bp respectively. A total of 169 polymorphisms were detected among the sequences under study. Upon analysis, 97 (57.4%) of the polymorphism detected were transition (C/T or A/G), 60 (35.5%) were transversion (C/G, A/T, A/C, or G/T), while InDels (insertion-deletions) were 12 and constituted 7.1% of the polymorphisms (Figure 2). Transitions recorded the highest number of SNPs in the LYCB genes while InDels were lowest. Among the accessions studied, high total carotene lines had 77 (42%), low lines 40 (23.7%) and white 58 (34.3%) of the entire polymorphisms observed (Figure 3).
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Figure 2 Transitions, transversions and InDels observed among the provitamin A sequences |
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Figure 3 Number of SNP polymorphisms observed in the studied genes in relation to high (H) and low (L) total carotene lines and white (W) cassava accessions |
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1.2.1 Analysis of SNPs at Exon and Intron region in relation to total carotene content
SNP polymorphisms were analyzed across both the exon and intron region across all sequences. When nucleotide sequences were converted to amino acids using the codon Code aligner software, most of the SNPs did not cause an amino acid exchange. A variation was found in the amino acid sequences of HYD, but it occurred in both high and low carotene lines. Those found in PSY occurred in white, low and high carotene lines, but one occurred in only white line 30572; also, those found in LYCB occurred in white, low and high carotene lines. In LYCE lines, a variation (G/T) was found G (guanine) in the reference sequence and T (thymine) in high carotene accession 07/0539) that occurred only in high carotene accession and in the exon region at position 1295th nucleotide of the reference gene (cassava43823.valid.ml). Upon conversion of nucleotides to amino acid sequence in the cassava accession this resulted in an amino acid change from Trp (tryptophan) to Leu (leucine) (Figure 4).
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Figure 4 SNP G/T in 07/0593 caused an amino acid exchange in a high carotenoid cassava accession |
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1.3 Searches with sequences in public databases
Searches of sequences with homologs in other species on NCBI Basic Local Alignment Search Tool (BLAST) database (http://www.ncbi.nlm.nih.gov/blast/) using Blastn algorithm revealed matches with 82%, 88%, 73%, and 92% similarity to publicly available sequences of HYD, PSY, LYCE and LYCB respectively (Table 1). A summary of the first two closest putative ortholog and their percentage identity is given in Table 2.
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Table 1 Percentage of NCBI accessions matching our putative carotenoid gene sequences using BLASTn algorithm in NCBI |
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Table 2 BLASTn results from NCBI database showing accessions of closest putative ortholog producing significant alignment with putative carotenoid sequences of cassava |
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2 Discussion and Conclusion
Sequences obtained in this study were considered to be putative cassava carotenoid genes because of the high percentage similarity of the matches with the genes in other crops. This indicates that these genes actually code for the enzymes in the carotenoid pathway responsible for accumulation of β-carotene and are also found in cassava. Analysis of SNPs detected was carried out in both exon and intron region of the genes under study, with most of the SNPs located in the intronic region. Those found in the exon region of the genes were of particular interest because they can be expressed in the phenotype, thus affecting a trait (Zhou et al., 2016). Polymorphisms found in the intron are useful as it can also be used for characterization of accessions. Careful analysis of SNPs in association with carotenoid content of the various cassava accessions revealed nucleotide variation G/T in the exon region of the LYCE gene at 1295th nucleotide position of the reference gene. Guanine (G) was the wild type while Thymine (T) was the mutant nucleotide located in a high carotene accession 07/0593, although five accessions comprising of high and low carotene content were heterozygote for these nucleotides. The polymorphism can be for exploited to develop molecular marker for marker assisted selection. Although (Welsch et al., 2010) discovered SNP A/C at 572bp in PSY2 that associated with yellow root color of cassava, no SNP was found to associate with high carotene lines in PSY and perhaps we have sequenced a different PSY gene in cassava. Also, the sequences obtained in this study were partial genomic sequences. The LYCE have been reported to be of critical value in the carotenoid pathway for β-carotene accumulation (Harjes et al., 2008). A HPLC analysis showed lycopene to be the major carotenoid in yellow rooted cassava although ά-carotene and cis-lycopene were also found (Nassar et al., 2009).
In the past, polymorphisms capable of increasing the proportion of provitamin A carotenoids have been identified in LYCE and HYD genes for maize genotypes (Harjes et al., 2008; Yan et al., 2010; Azmach et al., 2013; Babu et al., 2013). A single SNP leading to an amino acid replacement in a conserved position of LYCB was found to define red and yellow fleshed watermelon (Citrullis vulgaris) (Bang et al., 2014). High total carotene lines recorded the highest number of polymorphism with 42% of entire polymorphisms observed. The SNPs obtained in this study can further be validated for the development of functional markers which are important for accurate discrimination of contrasting alleles in marker assisted selection (Rafalski, 2002; Zhou et al., 2016). This increases the efficiency of selection in a plant breeding program and also shortens the long cycles involved in recurrent selection and recombination.
3 Materials and Methods
3.1 Plant materials and evaluation of carotene content
A total of 40 cassava accessions with varying total carotene content were selected from a recurrent selection breeding program to increase β-carotene in cassava to levels better than those currently found in the International Institute of Tropical Agriculture (IITA) cassava germplasm were collection. Plant materials were derived from a cross between yellow root and white root cassava. The total carotene content for these clones at the clonal evaluation stage, were evaluated by the IITA cassava breeders using the standard Harvest Plus procedure. All cassava accessions were collected based on their total carotene content to constitute a panel of high carotene (6 to 15 µg), low carotene (0 to 5 µg) and white checks. Genomic DNA was extracted from 0.2 g (200 mg) of fresh leaf of the selected plant materials as described in (Rabbi et al., 2014).
3.2 PCR primer design and target genes
Genes involved in the study and their abbreviation are: β-carotene hydroxylase (HYD1), Phytoene synthase (PSY1), Lycopene epsilon cyclase (LYCE), and Lycopene beta cyclase (LYCB). In the initial experiment, Primers were designed using Primer3 (v.0.4.0) (Rozen and Skaletsky, 2000) from cassava ESTs (accessions; LYCE DB947186, PSY1 BH794911.1, LYCB DV445903, HYD1 DB955222), using the process of comparative genomics by performing a BLAST algorithm (Altschul et al., 1997) with putative carotenoid genes from Castor Genome Database, given the homology of castor bean and cassava.
The second set of primers were designed from carotenoid genes (accessions: cassava7257.valid.m1, cassava992.valid.m1, cassava32745.m1, and cassava43823.valid.m1) of the cassava genome database (Phytozome v5.0 Manihot esculenta) for HYD1, LYCB, PSY1, and LYCE respectively, using BLASTn algorithm performed with putative carotenoid sequences of cassava obtained with initial primers (Supplementary Table 1). Primers were used to obtain partial DNA sequences of the carotenoid genes of interest from the cassava genome database. Selected primers were submitted to NCBI (National Center for Biotechnology Information) BLASTn algorithm (Altschul et al., 1997) and blast results analyzed to avoid primer pairs that can cause amplification of targets other than the input template.
3.2.1 PCR amplification and agarose gel electrophoresis
The PCR was performed in volumes of 25 µl reaction with 200 ng/µl DNA template, 10 x buffer, 50 mM MgCl2, and 2.5 µM dNTPs, 1 unit of Taq polymerase, 4-5% DMSO, and 5% pmole of forward and reverse primers. Amplification was carried out with a touchdown program performed on a BIORAD peltier Thermal cycler with initial DNA denaturation at 94°C for 2 min, followed by 9 cycles of 93°C for 15 s, at -1°C per cycle, and 72°C for 30 s followed by 24 cycles of 93°C for 15 s, 55-65°C for 20 s, and 72°C for 30 s. A final extension step was performed at 72°C for 5min. PCR reaction products were electrophoresed on a 1.5% agarose gel containing 0.5mg/ml ethidium bromide in 1xTBE buffer.
3.3 Sequencing and sequence analysis
Amplified PCR products that showed only single bands were purified using QIAgen kit, and eluted with 24 μl of GIBCO water (Invitrogen Corporation). PCR products obtained after a purification stage were sequenced in both forward and reverse directions with the same PCR primers; by the ABI Prism 3130X1 Genetic automated sequencer (Applied Biosystems) and BigDye terminator√3.1 kit (Applied Biosystems). Sequencing was performed on ABI sequence analyzer (Applied Biosystems) and transferred to CodonCode Aligner (v2.0.6) for sequence editing and SNP analysis; DNA and deduced amino acid sequences were aligned by Clustal W (Thompson et al., 1994). Sequences were examined for SNPs polymorphism as indicated by superimposed peaks in a chromatogram or nucleotide replacement, indels (insertion deletions) were indicated by a missing nucleotide or gap in the sequence chromatogram. Sequences were compared with other homologs in NCBI database (Altschul et al., 1997) using Blastn options (http://www.ncbi.nlm.nih.gov/blast). Sequences that had significant matches were considered as putative genes. Sequences were accessed for transitions, transversions and InDel polymorphic sites.
Authors' contributions
LIU carried out the laboratory analysis and wrote the manuscript. AA supervised and reviewed the manuscript. MG conceived the study and supervised the laboratory analysis.
Acknowledgements
We will like to thank the harvestplus breeding program that provided the funds for the study and Bioscience center of International Institute of tropical Agriculture where the study was carried out.
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