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Capsicum annuum is a herbaceous plant of the Solanaceae family. Capsicum is historically associated with the voyage of Columbus. Columbus is given credit for introducing chilli to Europe and subsequently to Africa and asia (Heiser, 1976). Capsicum annuum is widely cultivated throughout the world, more specifically in the tropical and sub-tropical regions. The fruits are rich in vitamin, especially in vitamins A and C. It is a very important and indispensable item in every kitchen for its pungency, spicy taste, besides the appealing colour which it adds to the food. Due to its several medicinal values, the demand of chilli in the pharmaceutical industries is increasing day to day (Bose et al., 2003). Scientists are carrying out studies on chillies and their compounds as a pain management tool and in the treatment of cancer. Capsicum species are diploids, with most having 24 chromosomes (n=x=12), but some wild species have 26 chromosomes (n=x=13) (Tong and Bosland, 2003).
The chief constituent of chilli pericarp is a crystalline colourless pungent principle known as capsaicin (C18H27NO3), a condensation product of 3-hydroxy-4-methoxy benzylamine and decylenic acid which produces a highly irritating vapour on heating (Anon, 1952). It is secreted by the outer walls of the fruit. The content of capsaicin differs in cultivars of chillies (Kamalan and Rajamani, 1963). Pungency can be considered as one of the most important desirable traits of chilli. Many innovative uses of pungency are being studied. Besides new medicinal applications, it has been tried as anti-mugger aerosols with chilli pungency as the active ingredient. The aerosols have replaced tear gas in more than a thousand police departments in the United States. The spray will cause attackers to grasp and twitch helplessly for 20 minutes (Bosland, 1996).
1.3 Botanical description
The cultivated Capsicum annuum shows much variability, particularly in regard to the fruits. It is a herb or subshrub, erect and much branched, 45-100 cm tall; it is usually early maturing and is grown as an annual. The main shoot is radial, but later branches are cincinnal, one of the branches at each node remaining undeveloped and the subtending bract or bracts are adnate and are carried up a lateral shoot to the node above. The simple leaves are very variable in size with the petiole 0.5-2.5 cm long. The lamina is broadly lanceolate to ovate, entire and thin, 1.5-2.0 cm long and 0.5-7.5 cm wide; the tip is acuminate and the base is cuneate or acute. The flowers are usually borne singly and are terminal, but because of the form of branching they appear to be axillary. The fruit is an indehiscent many-seeded berry, pendulous or erect, and is usually borne singly at the nodes. It is extremely variable in size, shape and colour, and in the degree of pungency. It is generally over 8 mm wide and 0.8-30.0 cm long and they may be linear, conical, or globose. The unripe fruit may be green, yellowish or purplish, ripening to red, orange, yellow, brown or purplish. The flattened seeds are pale yellow and are 3-5 mm at their largest diameter. Some fruits, like the sweet peppers, are lacking in pungency and the degree of pungency varies from this to very pungent (Purseglove et al., 1987).
1.4 Chilli in Mauritius
Chilli is an indispensable ingredient in the Mauritian cuisine. Chillies are used as a condiment and as spices. They are appreciated mostly for their pungency, spicy taste, and also for the colour it adds to food. Chilli is both a self-pollinated and a cross pollinated crop. Bees, ants and thrips are the possible agents of pollination. The acreage under cultivation of long chillies is 170 ha and about 28 ha for small chillies in Mauritius. A yearly average of about 1300 tonnes of chilli is produced in Mauritius (AREU, 2008).
1.4.1 Chilli varieties
In Mauritius, the long chilli type (the 'Cipaye' variety) is the only type of long chilli being cultivated on a commercial scale by planters. However, different types of small chillies can be found. Most of the small chilli fruits except piment blanc are green in colour and turn red when they have ripen. Small chilli plants of piment blanc produces white fruits which turn reddish pink when ripen. All the small chilli fruits differ in shape. Some have elongated shape and are pointed at the end like the piment petard. The fruits of piment blanc are roundish in shape with a rounded end.
The varieties cultivated in Mauritius are as follows:
Long chilli type: 'Cipaye' variety
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Figure ?? : Long chilli plant Figure ?? : Fruits of long chilli
Small chilli type: local variety
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Figure ?? : Small chilli plant Figure ?? : Fruits of small chill
'Piment carri' type: local variety
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Figure ?? : Piment carri plant Figure ?? : Fruits of piment carri
'Piment blanc' and 'piment petard' are also cultivated in Mauritius but these varieties are becoming rarer and are not grown on a commercial scale.
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Figure ?? : Piment blanc plant Figure ?? : Fruits of piment blanc
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Figure ?? : Piment petard plant Figure ?? : Fruits of piment petard
1.4.2 Crop cycle
Small chillies have a crop cycle of 12 months. Long chillies and 'Piment carri' have a crop cycle of 6 months respectively (AREU, 2008).
1.4.3 Composition of chilli
Chilli is rich in vitamin C (140 mg per 100 g). Its composition per 100g of fresh chilli is shown in the table as follows:
Table ?? : Composition of chilli
Source: AREU, 2008
1.4.4 Climatic requirements
Chilli is grown in both tropical and subtropical areas where its climatic requirements are mainly affected by three main factors:
Most cultivars are adapted to temperatures in the range of 20-25 Â°C but excessively hot weather produce infertile pollen and reduce fruit set.
Short day light conditions from 9-10 hours have seen to stimulate growth and increase productivity and quality of fruits.
Water and humidity
Rainfall levels from 600-1200mm are generally considered adequate, but excessive rainfall affects flowering and fruit set and may also fruit rot and decay. Warm humid climate favours growth while dry weather enhances fruit maturity.
1.4.5 Cultivation period
In region of low humidity, chilli is grown at the beginning of winter whereas in moderate and high humid region, the crop is cultivated at the beginning of summer. Chilli can be cultivated all year round if supplied with good irrigation. Chilli is cultivated mainly in the northern and eastern region of the island (Mr. Padaruth, 2010, pers.comm.,).
1.4.6 Harvesting of chilli
Harvesting is normally done in early morning and late afternoon. During these hours the fruits are not exposed to sun and heat which can affect the quality and shelf-life. The harvesting of chilli starts about 2 months after plantation and can extend up to 5 to 6 months depending on the variety. The fruits must be collected at maturity index. The fruit should be smooth and firm to the touch. Sight, touch, smell, taste, and experience are the factors which help in identifying the harvest maturity. Chillies required for fresh consumption are harvested green while those needed to make dehydrated ones are picked red.
1.5 DNA extraction
The isolation of plant nucleic acids is a fundamental requirement for most genome characterization and mapping procedures involving the use of genetic markers, and for the identification and isolation of plant genes for genetic engineering. In general, a good extraction procedure for the isolation of DNA should fulfil three major criteria:
It should yield DNA of reasonable purity for the subsequent manipulations to which it is to be subjected.
The DNA should be sufficiently intact to give accurate and reproducible migration patterns following gel electrophoresis.
The yield of DNA must be adequate.
The procedure should be quick, simple and cheap and, if possible, avoid the use of dangerous chemicals (Clark, 1997).
The extraction process involves, first of all, breaking or digesting away cell walls in order to release the cellular constituents. This is followed by disruption of the cell membranes to release the DNA into the extraction buffer. This is normally achieved by using detergents such as cetyl methyl ammonium bromide (CTAB). EDTA is often included in the extraction buffer to chelate magnesium ions, a necessary co-factor for nucleases, for this purpose. The initial DNA extracts often contain a large amount of RNA, proteins, polysaccharides, tannins, and pigments which may interfere with the extracted DNA and difficult to separate. Most proteins are removed by denaturation and precipitation from the extract using chloroform and/or phenol. RNAs on the other hand are normally removed by treatment of the extract with heat treated RNAse. Polysaccharide-like contaminants are, however, more difficult to remove (Puchooa, 2004). They can inhibit the activity of certain DNA-modifying enzymes and may also interfere in the quantification of nucleic acids by spectrophotometric methods (Wilkie et al., 1933). NaCl at concentrations of more than 0.5M, together with CTAB is known to remove polysaccharides (Murray and Thompson, 1980; Paterson et al., 1993).
1.5.1 The importance of chemicals used in extraction
CTAB is one of the most widely used detergents for plant DNA extraction buffers. CTAB dissolves DNA by carrying a net positive charge that interacts with the negatively charged DNA, resulting in a soluble complex. CTAB also binds to polysaccharides which are then removed during chloroform extraction (Murray and Thomson, 1980).
EDTA inactivates cellular nucleases that breakdown nucleic acids. EDTA also weakens the cell membrane by binding to divalent cations (magnesium and calcium ions) which are important for membrane stability.
NaCl increases the solubility of polysaccharides in alcohol (isopropanol or ethanol) thus preventing it to be co-precipitated with DNA.
Chloroform is efficient in removing proteins. Chloroform also remove complexes formed when CTAB binds to polysaccharides (Lodhi et al., 1994).
ß-mercaptoethanol prevents quick formation of oxidized phenolic complexes that usually occur after disruption of plant cells after the homogenization process.
PVP binds to polyphenols by hydrogen bonding, forming complexes. Polyphenols are thus separated from the DNA.
RNAse treatment degrades the RNA into small ribonucleosides that are not detectable by gel electrophoresis. Remaining ribonucleosides would be in very small amounts to compete with large quantities of added primers in the RAPD reaction, making interference by ribonucleoside primer highly unlikely (Porebski et al., 1997).
1.6 Estimation of DNA concentration and purity (using spectrophotometric method)
Quantification of reasonably pure DNA solutions may be achieved by UV absorbance spectrophotometry over the wavelength range 200-300 nm. The DNA should show a clear absorbance peak at 260 nm. The A 260 / A 280 ratio provides an estimation of purity of the DNA. Pure DNA has a ratio of 1.8 +/- 0.1. Where DNA extract is less pure, data obtained may be misleading because of interference by RNA or non-nucleic acid contaminants (Clark, 1997). The amount of UV absorbed by a DNA sample is directly proportional to the amount of DNA in the sample (Brown, 1992).
1.7 Agarose gel electrophoresis
Agarose gel electrophoresis is a method used to separate DNA, or RNA molecules by size. Smaller molecules migrate faster and further than larger ones. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field. Agarose is a purified powder which is isolated from agar, a material from sea weeds. When prepared, the gel forms small pores which act as molecular sieves, allowing larger molecules to move more slowly than the smaller molecules (Dubey, 2001). The electrophoresis box contains a positive electrode and a negative electrode which are connected to a power supply. The DNA is mixed with a dye and loaded onto the agarose gel. The gel is then stained with Ethidium Bromide and viewed under a UV transilluminator.
1.8 POLYMERASE CHAIN REACTION (PCR)
Polymerase chain reaction (PCR) is an in vitro method of nucleic acid synthesis by which a particular segment of DNA can be specifically replicated. Since the unveiling of the method by Kary Mullis and colleagues (Saiki et al., 1985, 1988), numerous modifications, improvements and novel applications of PCR have been devised (Clark, 1997).
1.8.1 Principle of the method
PCR involves several cycles and each cycle consists of three separate steps:
Denaturation of the DNA strands by heating to high temperatures usually at 94Â°C.
Annealing (hybridization) of primers to templates at an appropriate annealing temperature.
Extension of primer by Taq at 72 Â°C.
The number of cycle is repeated several times to obtain a satisfactory amount of amplified product (Brown, 2002). The product is then run onto a gel by electrophoresis.
1.8.2 PCR parameters
Successful amplification depends on DNA template quantity and purity. The DNA must be pure and free from contaminants to ensure Taq DNA polymerase activity.
This enzyme, isolated from Thermus aquaticus requires the presence of magnesium ions to catalyse the primer- dependent incorporation of nucleotides into duplex DNA in the 5' to 3' direction.
RAPD primers are oligonucleotides, typically 10 bases long, hybridizing to opposite strands flanking the region of interest in the target DNA.
An optimal dNTP concentration usually lies between 0.2mM to 1.5mM. A lower concentration leads to a reduction in sensitivity whereas a high concentration inhibits Taq polymerase.
The function of the buffer is to regulate the pH of the reaction which affects the DNA polymerase activity.
Taq requires free magnesium ions for its activity. Template DNA, chelating agents present in the sample (e.g. EDTA), dNTPs concentration and the presence of proteins can all affect the amount of free magnesium ion in the reaction.
1.9 MOLECULAR MARKERS
Genetic markers fall into one of the three broad classes: those based on visually assessable traits (morphological and agronomic traits), those based on gene product (biochemical markers), and those relying on a DNA assay (molecular markers). Molecular markers should not be considered as normal genes, as they usually do not have any biological effect, and instead can be thought of as constant landmarks in the genome. They are identifiable DNA sequences, found at specific locations of the genome and transmitted by the standard laws of inheritance from one generation to the next (Semagn et al., 2006).
1.9.1 Advantages of molecular markers
The advantages of molecular markers over morphological and biochemical markers are:
They are not affected by the variable environment
Sample to be analysed is required in very small amount
They are consistent
1.9.2 Types of molecular markers
The main types of molecular markers are:
Restriction Fragment Length Polymorphism (RFLP)
Amplified Fragment Length Polymorphism (AFLP)
Single Nucleotide Polymorphism (SNP)
Random Amplified Polymorphic DNA (RAPD)
Microsatellites or simple sequence repeats (SSRs)
126.96.36.199 Restriction Fragment Length Polymorphism (RFLP)
RFLP markers were first used in 1975 to identify DNA sequence polymorphisms for genetic mapping of a temperature-sensitive mutation of adeno-virus serotypes (Grodzicker et al., 1975). It was then used for human genome mapping (Botstein et al., 1980), and later adopted for palnt genomes (Helentjaris et al., 1986). RFLP is based on restriction enzymes that reveal a pattern difference between DNA fragment sizes in individual organisms. Two individuals will always differ at a few nucleotides. Some of the differences in DNA sequences at the restriction sites can result in the gain, loss or relocation of a restriction site. Hence, fragments resulting from digestion with restriction enzymes may vary in number and size among individuals, populations, and species (Semagn et al., 2006).
188.8.131.52 Amplified Fragment Length Polymorphism (AFLP)
AFLP technique combines the power of RFLP with the flexibility of PCR-based technology by ligating primer-recognition sequences to the restricted DNA (Lynch and Walsh, 1998). The key feature of AFLP is its capacity for "genome representation": the simultaneous screening of representative DNA regions distributed randomly throughout the genome (Semagn et al., 2006). The amplification reaction is versatile and robust, and appears to be quantitative. While AFLP is capable of producing very complex fingerprints, it is a technique that requires DNA of reasonable quality and is more experimentally demanding (Karp et al., 1996; Caetano- Anolles, 1998).
Microsatellites (Litt and Luty, 1989) also known as simple sequence repeats (SSRs), short tandem repeats(STRs) or simple sequence length polymorphisms (SSLPs) are the smallest class of simple repetitive DNA sequences. Microsatellites can be used to determine genetic diversity within a species, as well as being able to distinguish varieties and even individuals, as well as parentage (Kap et al., 1996). These markers appear to be hypervariable, in addition to which their co-dominance and reproducibility make them ideal for genome mapping as well as for population genetic studies (Dayanandan et al., 1998).
184.108.40.206 Random Amplified Polymorphic DNA (RAPD)
The simplest of all PCR-based markers is RAPD. The key innovation of RAPD is the use of a single arbitrary oligonucleotide primer to amplify template DNA without prior knowledge of the target sequence. The amplification of nucleic acids with arbitrary primers is mainly driven by the interaction between primer, template annealing sites and enzymes, and determined by complex kinetic and thermodynamic processes (Caetano-Anolles, 1997). When the single primer binds to sites on opposite strands of the genomic DNA that are within an amplifiable distance (generally less than 3000 base pairs) at an appropriate annealing temperature, a discrete PCR product is produced. In all RAPD, polymorphisms (band presence or absence) result from changes in DNA sequence that inhibit primer binding or interfere with amplification of a particular marker in some individuals, therefore, they can be simply detected as DNA fragments that are amplified from one individual but not from another.
1.9.3 Major advantages of PCR-based markers (AFLP, RAPD, SSR) over hybridization-based methods (RFLP)
The advantages include:
Detection is not radioactive
A small amount of DNA is required
The ability to amplify DNA sequences from preserved tissues
Accessibility of methodology for small laboratories in terms of equipment, facilities, and cost
No prior sequence knowledge is required for many applications
The ability to screen many genes simultaneously
High polymorphism that enables to generate many genetic markers within a short time
1.9.4 Use of molecular markers in crops and fruits
There are several reports on the use of molecular markers in crops and in fruit trees. Yang and Quiros (1993) applied RAPD markers to the identification and classification of celery cultivars. Hu and Quiros (1993) were able to distinguish 14 broccoli and 12 cauliflower cultivars. RAPD was successful in detecting genetic polymorphism between coffee species (Orozco- Castillo et al., 1994). RAPD has been used to assist the genetic enhancement of banana (Jarret et al.,1995) and to assess genetic diversity in lychee cultivars (Taylor, 1994).
1.9.5 Use of molecular markers in chilli
Several molecular studies were done on the large diversity of chilli varieties in other countries. Lefebvre et al. (1993) studied nuclear RLFP between pepper cultivars of Capsicum annum. Forty-one nuclear probes, encompassing the different linkage groups of a previously published map, were used to examine RFLPs of cultivated peppers. Wang et al. (1996) surveyed fourteen diverse pepper (Capsicum spp) accessions by RAPD analysis. A high degree of polymorphism was obtained from 4 random primers which produced 11 reproducible and effective amplification fragments useful for identification between species. The results indicated that RAPD markers could be effectively and reliably used for the classification of Capsicum spp.