MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF NINTEEN ACCESSIONS OF CAPSICUM ANNUUM L.

ABSTRACT

Genetic variation constitutes the core component in the breeding progress of plants. In order to explore the structure of the genetic diversity and similarity in pepper (Capsicum annuum), morphological and molecular characterization were of great necessity. This study assessed the morphologic traits of different varieties of pepper and determined their DNA (Deoxyribonucleic acid) sequences. Nineteen accessions of pepper were collected from various locations in Enugu State and planted in Botanical Garden of the University of Nigeria Nsukka. The experiment was laid out in a completely randomized design (CRD) with three replications, where each replication contained seven poly bags of each accession. Morphological data collected were subjected to analysis of variance (ANOVA) and significant means were separated using Least Significant Difference (LSD) Test. The second phase of this work involved the molecular evaluation of the accessions.   The DNA of each accession was extracted using tender leaves. The extracted DNA samples were amplified using polymerase chain reaction (PCR) and then visualized with agarose gel electrophoresis. The DNA sequencing was carried out in collaboration with Genewiz, LLC Company, South Plainfield; United States of America. It was used to determine specific nucleotides among accessions. The phylogeny was inferred using Neighbor-Joining method and phylogeny tree was computed using Maximum Composite Likelihood method. The results of the performance of the various accessions after planting showed that there were considerable variations among the accessions with respect to the morphologic traits considered. Plant height at 30 days after transplanting (DAT), ranged from 18.62 ± 0.14 cm to 58.02 ± 21.44 cm. T16 and T13 had the tallest and shortest plants, respectively. At 44 DAT, the plant height ranged from 24.67 ± 0.88 cm to 91.64 ± 0.37 cm, with T12 having the tallest plants.  Number of leaves and number of nodes showed significant differences across the accessions. At 30 DAT, T10 had the highest number of leaves with a mean value of 104.3 ± 35.02 which was significantly different from other accessions evaluated.  At 44 DAT, T14 had the highest number of leaves (84.67 ± 30.60). Other morphological characters like number of nodes, length of internodes, mature leaf length, petiole length, fruits length, fruits width, fruits weight, number of seeds and pericarp thickness were evaluated and they showed considerable variation among the accessions. The results of molecular evaluation of the accessions  showed  high  genetic  similarities  among  accessions  with  a  point  variation  which occurred at the 337th base pairs where the nucleotide base ‘A’ (Adenine) was substituted with ‘G’ (Guanine). The phylogenetic tree showed that the accessions analysed were all C. annuum with a narrow polymorphism which grouped the nineteen accessions into two groups, with group 1 having four  accessions  and  group  2  having  fifteen.  The  result  of  this  research  provides  adequate information  on  the  characterization  of  the  various  accessions  which  can  be  used  in  crop improvement and plants breeding programs in future.

CHAPTER ONE

INTRODUCTION

1.1       Background of the study:

The importance of peppers (Capsicum annuum) in the tropics cannot be over emphasized as they are highly rated among cultivated vegetables (Abu et al., 2011).  C. annuum is a good source of vitamins A and C and it is used as spice in daily diet due to the presence of capsaicin (Materska and Perucka, 2005).

Peppers are naturally self pollinating plants, but some species exhibit tremendous variability and genetic divergence (Grubben and El-Tahir, 2004; Abu and Uguru, 2006; Adetula and Olakojo, 2006; Fonseca et al., 2008 and Aktas et al., 2009). Within C. annuum complex, there is phenotypic diversity in plant habit and especially in plant height, number of leaves, mature leaf length, shapes and other morphological characters (Greenleaf, 1986; Andrews, 1995, 1999; DeWitt and Bosland, 1996).  Paran  et al. (2004) using Random Amplified  Polymorphic Deoxyribonucleic acid  (RAPD) also found variations in morphological collections such as plant height at first branching, stem diameter, total fruit weight, fruit weight per plant, seed weight and leaf colour of C. annuum. The observed variation can be considered important for genetic improvement of pepper, since according to Sahagun (1992), the morphological and genetic variations are fundamental condition for the start of a genetic improvement program. A comparison of the plant phenotype is the simplest approach for the evaluation of genotypes and the assessment of genetic variations; however, phenotypic evaluation is influenced by enviro nment and might not distinguish between closely related genotypes (Rodriguez et al., 1999). Molecular DNA marker analyses which are not affected by environment have been suggested for the determination of genetic similarity and diversity among genotypes (Gilbert et al., 1999).

The use of molecular markers is a powerful approach to the characterization of various organisms because it can show the differences at the DNA level. Molecular markers have been reported as useful tools in the characterization of C.   annuum (Conicella et al., 1990; Lefebvre et al., 1993; Zewdie and Zeven, 1997; Geleta et al., 2004). These markers act as ‘sign posts’ to specific genes and such markers when very tightly linked to genes of interest, can be used  in indirect selection of desirable alleles and can also show genetic variation among various genotypes (Jonah  et al., 2011). DNA barcoding is emerging as a preferred global standard in taxonomy, which promises to bring a significant  level  in  characterization  and  identification  of  plants.  DNA  barcoding  involves sequencing a short stretch of DNA (rbcL gene) which is universally present in all plant species with a  high  degree  of  nucleotides  sequence  variation.  In  plant,  DNA  fragments  of  the  chloroplast genome have been widely used for phylogenetic reconstruction and species-level identification. The common universal DNA fragment that serves as a primer in plant is rbcL (Ribulose 1,5- bisphosphate carboxylase)  gene; which is a gene that encodes for the plant universal enzyme called RuBisCO (Ribulose 1,5- bisphosphate carboxylase oxygenase) (Group et al., 2009).

Several works have been done on the morphological and molecular characterization of Capsicum spp in general but little has been done in sequencing the DNA which is pertinent in evaluating the variability among varieties of pepper.

1.2       Justification of the study

The widely cultivated pepper is an important vegetable and also an integral spice in many countries due to its appealing flavour, taste and pungency. There is need for seed producers and end-users to know the identity of their plant species and cultivars. Peppers are one of the most diverse crops, yet the diversity have not been maximized and utilized due to the limited information in the molecular component   of   the   plant.   To   enhance   breeding   programs,   detailed   characterizations   of

Capsicum diversity including morphological and molecular data are required. Currently, molecular data characterizing Capsicum diversity is limited especially among the landraces in Nigeria. To date, limited information is also available on the aspect of DNA sequencing of landraces of pepper in Nigeria which is also an important consideration required for the development of breeding lines. This work was intended to contribute to the knowledge of diversity in Capsicum by providing the essential information via morphological and molecular evaluation.

1.3       Aim and Objectives

This work is aimed at evaluating the morphological and molecular characteristics of Capsicum annuum in order to:

1.   Assess the morphological diversity in the expression of traits in nineteen   accessions of pepper

2.   Determine DNA sequencing of nineteen accessions of pepper and to construct the phylogenetic tree of the plant.

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