Chapter 1: Literature review
1.10 Diversity estimation methods
Several techniques have been used to determine variability in crop species. However all the techniques are based on genetic markers that have been classified into three groups (Semagn et al., 2006a).
- Phenotypical markers based on agro-morphological trait differences between plants,
- Biochemical markers, based on proteins or their activity as detected through gene expression, - Molecular markers based on the detection of DNA polymorphisms between individuals.
1.10.1. Phenotypic markers
Phenotypical traits were the first generation of markers to be used for diversity studies, both on animals and plants. Tateoka, (1963) used spikelet and awns length to differentiate wild forms within the Oryza complex, based on their geographical origin. Later, several agro-morphological traits were used to estimate the genetic diversity in African rice species (Chang et al., 1977;
Second et al., 1977). Similarly, phenotypic markers were used to study the genetic diversity of tomato landraces from Greece (Terzopoulos and Bebeli, 2010). Phenotypic traits allow for visual assessments, based on a direct observation of the trait of interest. Several documents describing traits to be measured and measurement methods have been developed (IRRI, 2002; Bioversity International et al., 2007). However, phenotypic markers are cost effective and reliable only when skilled personnel are available.
1.10.2. Biochemical markers
Biochemical markers have greatly enhanced the improvement of various traits of some crops especially where phenotypical traits or molecular markers are not efficient. Biochemical markers can be grouped into two types, including metabolic-based markers and protein-based markers.
The first type is based on products from the secondary metabolism of plants, such as polyphenols (De Vicente et al., 2004). Thus, biochemical analysis methods are required to accomplish this
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kind of study. Plants like tea, coffee and tobacco have benefited from this technique. Catechins were successfully used to classify Kenyan and Western Himalayan tea cultivars (Magoma et al., 2000; Karthigeyan et al., 2008). Apart from classifying the varieties into groups, the content of catechins could be used as an indicator of the quality of tea. The second type is based the detection of protein activity. Isozymes are the most used and the most beneficial to both Asian (Glaszmann, 1987) and African rice studies (Second, 1985; Bezançon et al., 1989). The technique is based on the detection of enzymes, diverging in nucleotide sequence but catalyzing the same chemical reaction. Although biochemical markers were very useful in assessing genetic diversity of many important crops, they have been supplanted by molecular markers, which are considered to be more reliable and repeatable to as confirmation of visual or phenotypical assessment of variation in plant populations.
1.10.3. Genomic Markers
Genomic markers are identifiable DNA sequences that can be used as landmarks to orientate the genome of individuals. They are associated exclusively with a segment of the genome, and follow a Mendelian mode of inheritance across generations (Semagn et al., 2006a). Genomic markers detect variation between individuals based on changes due to mutations, substitutions or deletions within the DNA sequence. The relationship between the genomic marker and the genomic segment (which can be considered to be the target trait) depend on the distance between them: The more tightly they are linked, the greater their probability to segregate together. The distance between a genomic marker and a target trait is calculated based on frequencies of recombination between them in centimorgan (cM). Three categories of genomic markers were proposed by Semagn et al. (2006a), based on the mode of transmission (biparental nuclear inheritance, maternal nuclear inheritance, maternal organelle inheritance, or paternal organelle inheritance), the mode of gene action (dominant or codominant markers) and finally, the method of analysis (hybridization-based or PCR-based markers).
PCR-based markers
With the automation of the Polymerase Chain Reaction (PCR), and the advances in genome sequencing, PCR-based markers have become the commonly used genomic markers. Moreover, the development of site-targeted PCR techniques from known DNA sequences and several thousand Simple Sequence Repeats (SSRs) or microsatellites were developed for rice (Temnykh
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et al., 2000; Temnykh et al., 2001) and have become the most commonly used genotyping tool for rice. These sequences are usually 1 to 6 base pairs of non-coding DNA, inherited co- dominantly and created by mutation. Microsatellites are often used in population genetic studies, gene mapping and marker-assisted selection. Forward and Reverse sequences of SSRs that will recognize and hybridize the target region of the DNA template could be added to the PCR mix reaction and the final product separated on agarose or acrylamide gel. The high mutations rate of the neutral region of the genome permits the detection of several alleles for a given SSR (Langridge and Chalmers, 2005). Microsatellites were used for the last global study of the two cultivated rice species (Garris et al., 2005; Semon et al., 2005).
Several variants of microsatellites exist. Inter-simple sequence repeat amplification (ISSR), for instance, is a PCR-based amplification of DNA using a single primer developed from the interval sequence between two microsatellite sequences. In this case the target to be amplified is the variable region between the sequences, and is conducted without prior knowledge of the sequence (Wu et al., 2004). Another but dominant and arbitrary PCR-based marker is RAPD (random amplified polymorphic DNA). This method is difficult to reproduce.
The latest generation of PCR-based genomic markers involves Single Nucleotide Polymorphisms (SNPs). This type of marker is based on detecting single base pair changes between species or paired chromosomes. They are widely and densely distributed in Oryza sativa L. genome, with a rate of 0.65% in the O. sativa subspp. indica and O. sativa subspp. japonica subspecies (Liu and Zhang, 2006). The SNP rate is 0.65%, indicating a bright future for this technique in rice.
Hybridization-based Markers
Restriction fragment length polymorphism (RFLP) is the most widely used hybridization-based molecular marker. It uses restriction enzymes to digest the DNA fragment into sequence pieces, which are then separated using a gel electrophoresis system. From the gel, the sequences are transferred to a membrane via the Southern blot procedure, involving probes with RNA and finally revelation using autoradiography (Semagn et al., 2006a). Presently, this technique has been overtaken by the PCR-based techniques. A derived technique is the amplified fragment length polymorphism (AFLP), which combines the enzymatic digestion of RFLP with the
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amplification of PCR-based. This technique generates lots of variation and has been used in diversity studies and gene mapping.