m m
2 1
2 −
Backcrossing results in effective recombination only in the gametes of the hybrid progeny and not in the gametes coming from recurrent parent. The lack of
effective recombination in the recurrent parent is due to the homozygous nature of that parent in a self-fertilized species. Backcrossing could be used to incorporate
traits from donor parent with good enough recovery of the recurrent parent feature Stoskopf et al. 1993.
Backcross population has been used as a mapping population. Xiao et al. 1998 reported that 300 families of BC
2
population V20AO. rufipogonV20BV20BCe64 had been used to map 68 QTLs for 12
agronomically important traits. A set of 122 RFLP and microsatellite markers was used to identify the QTLs. Mei et al. 2005 reported the utilization of 254
RILs of LemontTeqing and two backcross hybrid BC F
1 1
of that RIL to study the gene action controlling several traits, such as heading date, plant height, flag leaf
length, flag leaf width, panicle length, and spikelet fertility. Backcrossing has also been used to introgress traits assisted by molecular marker termed as
Molecular Assisted Backcrossing Hospital 2005.
2.4. Molecular Markers and QTL Mapping
There are three kinds of markers, morphological plant traits, biochemical proteins and isozymes, and molecular DNARNA. These have been used
to construct genetic maps and marker assisted selection. Morphological markers are limited in number, influenced by the environment, developmental
stage and sometimes have pleiotropic effects. Isozymes markers offer more polymorphism but their number is limited. Scoring of both of these marker types
depends on gene expression, which may be sensitive to environmental influence, genetic background, stage of plant development, and tissue type. Both types of
markers have limited abundance Van Den Berg et al. 1997. The third kind of markers are called molecular markers. Molecular
markers are numerous in number and their discovery represents a milestone in genetics as they provide the capacity for complete coverage of crop genome.
These markers show Mendelian inheritance, are stably inherited, have no
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pleiotropic effects and are unaffected by the environment and express at all developmental stages. Majority of DNA polymorphisms are selectively neutral.
The genetic variation can result from of a simple point mutation, DNA insertiondeletion event or change in repeat copy number at some
microsatellites or termed as SSR Brar 2002. The most commonly used molecular markers are: SSR, AFLP, and RAPD Sasaki 2002.
The first generation of molecular markers-RFLP Restriction Fragment Length Polymorphisms were discovered in 1974. RFLP markers are
developed by extracting nuclear DNA and digesting it with a restriction enzyme. The resulting restriction fragments are separated according to size by
electrophoresis, denatured, and transferred onto a solid support such as a nitrocellulose or nylon filter. The DNA fragments are then hybridized with
radioactive- or hapten-labelled probe DNAs. The results are visualized by X- Ray film or fluorescence. Different DNA gives specific product, and it is
genetically inherited Van Den Berg et al. 1997. However, this technique requires large amount of DNA, and is relatively tedious and expensive.
The potential of using markers for the construction of complete genetic linkage maps was recognized soon after. The power of RFLPs resides in their
ability to directly reveal differences in the nucleic acid base sequences of homologous chromosomes. Unlike scoring of conventional morphological and
biochemical markers, scoring of RFLPs does not depend on gene expression. RFLPs are numerous, insensitive to the influence of environment and genetic
background, developmentally stable, often inherited in a simple Mendelian fashion, and alleles are usually co-dominant Van Den Berg et al. 1997.
The invention of PCR technique ushered in the availability of markers for practical applications. PCR is the technique involving denaturization,
annealing and amplifcation of DNA in the presence of dNPTs, Taq polymerase primers and buffer. The process is generally repeated thirty
times typically in a thermocycler and produces more than 1 x 10
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copies of amplified DNA fragments Innis et al. 1990.
Random Amplified Polymorphic DNA RAPD technique uses a single primer that bind to specific sites of DNA and amplifies sequences flanked
9
between primer binding sites. Primers are typically decamers and don’t require sequence information and several sets of primers are readily available
from biotechnological companies such as Operon. The amplified products are separated according to the length of fragment by electrophoresis. The DNA
fragments are stained by ethidium bromide, and visualized by exposure to ultra violet light Waugh 1997, Karp et al. 1997.
Another PCR based approach is called AFLP and is in fact a combination of RFLP and RAPD. Firstly DNA is restricted by two-restriction
enzymes follwed by ligation with adapters. Using appropriate primers, the restricted DNA is amplified with PCR. Amplicons are stained and visualized
similar to RAPD technique Karp et al. 1997. Simple sequence repeats SSR markers generate variation based on the
variable number of simple-sequence DNA repeats. Primers are designed to get the simple sequence repeats as PCR amplification product. SSR markers need
prior sequencing of the DNA to design primer pairs in conserved regions. SSR markers are co-dominant, contrary to RAPD which are dominant in nature. SSR
markers can distinguish different alleles of a locus that make it more powerful. Therefore, SSRs have become the markers of choice for a wide spectrum of
genetic, population, and evolutionary studies Powell et al. 1996. Simple Sequence Repeats markers are simple, tandem repeated of di- to
tetra-nucleotide sequence motifs flanked by unique sequences. They are valuable as genetic markers because they are co-dominant, detect high levels of allelic
diversity, and easily and economically assayed by PCR techniques. SSR is a co- dominant marker like RFLP, but is much easier to detect by the PCR and allows
detection of more alleles than RFLP Sasaki 2002. Initial results from screening of a rice genomic library suggested that there
are an estimated 5700-10 000 microsatellite markers in rice, with the relative frequency of different repeats decreasing with increasing size of repeat motifs. A
map consisting of 120 microsatellite markers demonstrated that they are well distributed throughout the 12 chromosomes of rice. Study of allelic diversity have
documented up to 25 alleles at a single locus in cultivated rice germplasm and
10
provide evidence that amplification in wild relatives of Oryza sativa is generally reliable McCouch et al. 1997.
Simple Sequence Repeats markers have been established by screening a genomic library with a synthetic DNA containing a target repeat, and then by
designing PCR primers flanking repeat sequences from the positive clones. As the genomic sequence of rice is gradually generated, more SSR markers should be
easily found and widely used. Currently, a total of 2240 unique experimentally validated SSR markers are available in rice, or approximately one SSR every 157
kb McCouch et al. 2002. The total number of co-dominant markers such as RFLP, SSR or CAPS
Cleaved Amplified Polymorphic Sequence so far published is about 5000. This means that the average marker density within the rice genome is one marker in
every 80 kb Sasaki 2002. Recently, 18,828 SSR markers have been identified and annotated on rice genome International Rice Genome Sequencing Project
2005. Genetic maps are constructed using mapping populations such as F
2
, Backcross, Doubled Haploid, and Advanced Backcross population or
Recombinant Inbred Lines RIL. Mapping populations are derived from parents those are genetically distinct, such as japonica and indica varieties. This
combination is generally chosen to generate more polymorphism. Sasaki 2002 reported that the frequency of polymorphism observed within the cross of
japonica varieties decreased to 10–30 compared to that obtained between crossing japonica and indica rice varieties.
Molecular markers are of great value in applying genetic technologies to crop improvement such as determining genetic diversity, marker assisted
selection, gene-pyramiding, QTL mapping, map-based cloning of important genes, monitoring introgression from exotic and wild species germplasm,
DNA fingerprinting of crop germplasm and pathogen populations Brar 2002. One of the most important applications of DNA markers and
molecular linkage maps is to dissect the genetic variation of quantitative traits into individual Mendelian factors through QTL mapping analyses Li 2001.
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Quantitative Trait Loci mapping can be defined as the marker-facilitated genetic dissection of variation of complex phenotypes through appropriate
experimental design and statistical analyses of segregating materials. In QTL mapping, genes controlling genetic variation of quantitative traits in segregating
populations are resolved into individual Mendelian factors by detecting marker-trait associations. The primary objective of a QTL mapping experiment is to
understand the genetic basis of specific quantitative traits by determining the number, locations, gene effects, and actions of loci involved and their interactions
with other loci epistatic and with environments QTL x environment, or QE, interactions. Another major purpose of QTL mapping is to identify DNA markers
diagnostic for particular phenotypes of interest so that marker-aided selection MAS can be used to efficiently manipulate progenies carrying alleles
for target traits grown under non target environments Li 2001. Most important traits dealt with by plant breeders are quantitative in nature.
The classical multiple-factor hypothesis considered the continuous variation of quantitative traits as the collective effects of many genes, each with a small effect
Mather Jinks 1982. Most of the yield traits are polygenically inherited and are strongly influenced by the environment. Therefore, determination of
genotypic values from phenotypic expression is not precise and selection strategies must take into account low heritability. Breeders generally select
for yield when uniform breeding lines are obtained. Up to now, it has not been possible to select for individual QTL having positive effect on yield
in segregating populations. Recently individual QTL for yield component traits have been tagged with molecular markers in rice. Quantitative Trait
Loci for several traits in rice such as blast resistance, salinity tolerance, submergence tolerance and root traits length, thickness, dry weight and
root shoot ratio have also been identified and tagged using linked molecular markers Khush Virk 2002.
Trait improving QTLs can also be transferred from exotic germplasm to elite-lines via MAS. In rice, two yield enhancing QTLs yld1,yld2 linked to
molecular markers have been identified in Oryza rufipogon Xiao et al., 1996. These results demonstrate that discovery and transfer of QTLs for complex
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traits such as yield can be facilitated via molecular marker technology Khush Virk 2002.
Although each marker system is associated with some advantages and disadvantages, the choice of marker system is dictated to a large measure by the
intended application, convenience and the cost involved. High throughput approaches have also been developed, thus making it possible to scale-up the
use of some of these markers.
2.5. The Physiology of Iron and Zinc in Rice Plants
