Introduction

Celosia argentea L. is a widely cultivated vegetable crop in Amaranthaceae family as traditional food (Denton, 2004). It is commonly known as Lagos spinach or sokoyokota in Yoruba that is, “make husbands fat and happy” (Grubben and Denton, 2004). Young leaves of C. argentea harvested 5-7 weeks after sowing have the best nutritional value; it contains water, energy, protein, fat, carbohydrate, fibre, Calcium, Phosphorus, Iron, Vitamins A and C with high content of micronutrients comparable with some vegetables. Again, the leaves contain phytic acid and oxalic acid which makes it less suitable for fresh consumption (Ayodele and Olajide, 2011). The composition is strongly influenced by environmental factors which include soil fertility, fertilizer application and age of plant at harvest (Denton, 2004). The genetic variability of this crop had been characterized phenotypically with high significant variation detected in this population (Olawuyi et al., 2016). The need for genetic base and distinctive useful traits which may be eroded in crop adaptation, improvement, and biodiversity conservation necessitated this study. Hence, there is need for molecular evaluation of C. argentea to validate it. Polymerase Chain Reaction (PCR) of many molecular techniques has been developed for plant genomic analysis (Esayas and Bryngelsson, 2006). They are more reliable for genetic studies than morphological characteristics, time efficient and cover the whole organism’s genome. Molecular techniques made it possible to isolate and analyze specific genes, enhance the understanding of organism’s genome, generate genetic maps and advancement of gene therapy technologies. Moreover, they have played crucial roles in phylogeny studies and species evolution using DNA sequence in manipulation of genetic variation within populations. Recently, the use of Amplified Fragment Length Polymorphism (AFLP) marker has become the main tool for disclosing a high number of polymorphic markers by single reaction (Vos et al., 1995). It is a useful technique for breeders to facilitate plant improvement, using molecular genetics maps to undertake marker-assisted selection and positional cloning for some characters. The identification of genetic relationship among the cultivars based on biochemical and molecular analysis is essential in genetic improvement and selection of crossing combinations from bulk parental genotypes. AFLP markers have successfully been used for analyzing genetic diversity in some species of Amaranthaceae such as Brazilian Ginseng (Figueira et al., 2011), Palmer Amaranths (Aman et al., 2013) and Weed Amaranths (Wassom and Tranel, 2005). However, there is limited information on molecular variability of Celosia using AFLP marker. This study therefore aimed at investigating variability of C. argentea using molecular technique.

1 Results, Recommendations and Suggestions

The AFLP molecular marker revealed genetic variability among the C. argentea genotypes based on the number of amplified fragments with polymorphic information content of 89.1% and gene diversity of 90%. The variability of morphological and yield traits in the breeding materials have their roots in the organism’s genome. This will ensure germplasm conservation and development of strategies in improvement of C. argentea. The highest concentrations of genomic DNA of 13.30 μL and volume of 2 217.59 μL for total genomic DNA were recorded for NG/TO/MAY/09/015 and NG/MA/MAY/09/015 respectively. These genotypes could be recommended for crop improvement breeding program. Hybridization breeding of these genotypes is suggested for its crop improvement with desired traits in the parent lines. Also, primer sequence and compatibility are seemly unending challenges in molecular studies, therefore; the primer combinations in its compatibility for the crop studied could be documented and recommended for pilot study in molecular evaluation of other vegetable crops.

2 Discussion

The nano drop DNA quantization for the extracted C. argentea genotypes was found at 260/280 ng/μL (Table 1). The quality of genomic DNA concentration for NG/TO/MAY/09/015 had the highest value of 13.30 μL, while the volume of total genomic DNA was 376.12 μL. Though, genotype NG/MA/MAY/09/015 produced the highest quantity, but lesser quality, while the lowest quality of genomic DNA at 2.08 μL from total volume of 464.70 μL was recorded for NHGB/09/160.

The polymorphic information content and amplification patterns of the genome of C. argentea are shown in Table 2. The percentage gene diversity recorded was 90%, while the polymorphism in the population was diverse at 89.1% for standardized DNA size of 100 base pairs. There are variations in number of amplified fragments, number of polymorphic bands and total number of bands. Primer sequence combination of AAC + CAG produced the highest number of bands, amplified fragments and number of polymorphic bands of 400, 40, and 156.000 0 respectively.

The dendrogram result showed the delineation of ten genotypes of C. argentea into three cluster groups (Figure 1). The cluster group 1 comprises of genotypes NGB/01260, NG/MA/MAY/09/015, NHGB/09/160 and NIHORT/0001, in which NGB/01260 and NG/MA/MAY/09/015 as well as NHGB/09/160 and NIHORT/0001 are more genetically related to each other. Also, the second cluster group comprises of NG/MAY/09/015 and NG/SA/07/213, while NHGB/01260, NG/AO/MAY/09/015, NG/MR/MAY/09/015 and NG/TO/MAY/09/015 genotypes constituted the third cluster group. Genotypes NHGB/01260 and NG/AO/MAY/09/015 as well as NG/MR/MAY/09/015 and NG/TO/MAY/09/015 are also genetically related to each other.

The result from Figure 2 showed the optimization reactions of genomic DNA of C. argentea genotypes carried out in four reactions using six different primer combinations. Reaction 3 produced the best scorable amplified bands followed by reaction 1. In reaction 1, G1, G4, G5 and G10 revealed the most noticeable bands compared to other genotypes.

The separation of DNA amplification fragment by polyacrylamide gel electrophoresis (PAGE) shown in Figure 3 revealed the polymorphism in the population studied. The primer ladder, which is a 100- base pairs marked out the polymorphic bands within the genotypes in each of the three primer combinations. Primer combination E- AAC + M- CAG produced the highest number of fragments amplified and number of polymorphic bands.

Polymorphic information content (PIC) of C. argentea at 89.1% showed that the variability in this population exists in the organism’s genome as similarly reported by Denton (2004). This indicates these genotypes could be useful as breeding material in the improvement of this crop. Cluster analysis and dendrogram indicate that cluster groups consist of genotype from different geographical background and such wide adaptability has been attributed to population genetic architecture, selection history and approach under domestic cultivation and developmental traits (Ganapathy et al., 2011; Olawuyi et al., 2015).

3 Materials and Methods

3.1 Germplasm collection of C. argentea seeds

Ten C. argentea genotypes sourced from National Institute of Horticultural Research (NIHORT) and National Centre for Genetic Resources and Biotechnology (NACGRAB) in Ibadan, Nigeria were; NGB 01260, NG/MA/MAY/09/015, NHGB/09/160, NIHORT/0001, NG/MAY/09/015, NG/SA/07/213 and NHGB/01260.

3.2 Experimental locations and planting procedure

The molecular studies were carried out in Bioscience Laboratory of International Institute of Tropical Agriculture (IITA), Ibadan, while the field experiment was conducted at the research farm of the Department of Botany, University of Ibadan. The cultivars were raised for 2 weeks in nursery bags; thereafter fresh young apical leaves were collected into ice bags before transported to the laboratory for molecular studies.

3.3 DNA extraction

Extraction of DNA in young apical leaves was carried out using AFLP technique to assess genetic variability of ten C. argentea genotypes. Total genomic DNA was extracted from frozen leaf tissue (50 - 100 mg) using the procedure described in D2 Bio Technologies DNA X-Tract.

3.4 DNA quantization and AFLP protocol

DNA quantization kit was used to determine DNA concentration in the final preparation using nano drop DNA quantization. Nano drop DNA quantization showed the extracts’ ratio of absorptions at 260/280 nm. This ratio is used to validate purity of the nucleic acids in terms of the quality and quantity of total genomic DNA extracted from the plant samples. The AFLP protocol initially described by Vos et al. (1995) was performed using components from various commercial AFLP kits. AFLP Primers; EcoRI and MseI were adopted for the study (Table 3).

3.5 Digestion of genomic DNA and ligation of oligonucleotide adapters

Digestion of genomic DNA by the restriction enzymes EcoRI and MseI and ligation of oligonucleotide adapters were accomplished in a single reaction mixture of 11 μL. Prior to each use, the adaptor pairs were pre-heated to 95ºC for 5 min, and then allowed to cool slowly for ten minute at room temperature of 25ºC. The mixture was incubated overnight at room temperature in order to digest template DNA completely.

3.6 Pre-selective PCR amplification of DNA

Pre-selective PCR amplification was performed using the Applied Biosystems AFLP kit. The 20 μL reaction contained 4 μL of the diluted restricted/ligated DNA and 16 μL of a mixture with 1 μL of EcoRI+A and MseI+C AFLP pre-selective primers with 15 μL of AFLP core mix. The PCR program for the pre-selective amplification was: 72ºC for 3 min, followed by 20 repetitive cycles of 94ºC for 20 s, 56ºC for 30 s, and 72ºC for 2 mins, with a final hold at 60ºC for 30 mins.

3.7 Selective PCR amplification of DNA

For selective PCR amplification of restriction fragments, primers were prepared for recognition of EcoRI and MseI adapters. Fragments are visualized by attaching a D4, D3 or D2 WellRED™ dye to the 5´ end of each EcoRI selective amplification primer without modification ofMseI primer.

3.8 Preparation of DNA amplification fragments for separation of PCR product by polyacrylamide gel electrophoresis (PAGE)

Loading solution was prepared with a 100-base-pair (bp) DNA size standard labeled with WellRED™ dye D1 (approximately 100:1; Beckman Coulter 608082 and 608098). This solution was thoroughly mixed by vortexing for a minimum of two minutes. A 30-μL aliquot of this cocktail was added to 1.5 μL of the selective amplification product and loaded into the prepared well.

3.9 Data scoring and analysis of AFLP product

Total bands were scored visually from three primer combinations and polymorphic bands were observed as presence (1) or absence (0). Also, cluster analysis and dendrogram was constructed using Powermarker software V3.25, to reveal the phylogenetic relationship following UPGMA method of Jaccard’s similarity coefficient.

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