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Genetic fingerprinting is one of the DNA-based techniques that have permeated a wide gamut of biological research, beginning with forensic biology and medicine and now extending to agriculture. The advent of polymerase chain reaction (PCR) ushered a revolutionary approach in producing genetic fingerprints, supplanting hybridization-based techniques. PCR-based methods can be accomplished using either arbitrary markers of unknown location in the genome or those markers that target specific genome sites. Among agricultural crops, rice and maize are the most intensively characterized for DNA markers. At present, genetic fingerprinting has also been applied in many aspects of crop biology, such as taxonomy and phylogeny, diversity analysis, hybridity testing, gene mapping, molecular breeding, and somaclonal variation. This paper describes genetic fingerprinting technology and discusses its applications in the major crops of the Philippines, highlighting the progress made by Filipino scientists.
Discovery and principle of genetic
fingerprinting
The past century marked groundbreaking discoveries in the biological sciences
particularly in the area of molecular biology. Since the discovery of the
DNA structure in 1953, researchers have been increasingly empowered to analyze
and manipulate genetics using the principles of nucleic acid biochemistry.
The beginnings of DNA profiling or genetic fingerprinting can be traced in
the work of geneticist Alec J. Jeffreys of the University of Leicester in
Great Britain, on the gene for myoglobin, a protein that stores oxygen in
muscle cells (Jeffreys et al., 1985). He found that the myoglobin gene contains
many segments that vary in size and composition from individual to individual
and that have no apparent function. Jeffreys called these segments minisatellites
because they are small and are located near the gene that actually serves
as a genetic blueprint. Minisatellites are tandemly arranged, repetitive DNA
elements scattered throughout eukaryote and prokaryote genomes, the short
basic units of which are mostly imperfectly reiterated at each locus (Epplen
et al., 2005). Minisatellites comprise 10 – 100 nucleotide-long repeat
units.
DNA variation is the substrate of genetic fingerprinting. In simple organisms
with small genomes, DNA variation can be detected simply by using restriction
enzymes or restriction endonucleases that cut DNA strands at specific DNA
sequences. Variation in the cutting sites on the DNA is manifested as a difference
in the size of the digestion products. This difference is called Restriction
Fragment Length Polymorphism (RFLP), which can be detected as follows: digesting
DNA with restriction enzymes and separating the DNA fragments by gel electrophoresis.
In higher forms with large genomes, additional steps are necessary to display
the variation, namely: blotting the digested fragments to a filter, and hybridizing
a probe to the fragments (Southern, 1975). DNA fingerprinting has opened the
possibility to identify genomic variation between individuals, and to follow
DNA exchange in the progeny, making it a powerful tool for genotypic selection
such as in animal and plant breeding.
The discovery of PCR afforded a very convenient way of assaying DNA variation
without the blotting and probing steps in RFLP. With creative designs of primers
and various thermal cycling strategies, PCR has brought about a new class
of DNA profiling markers for fingerprinting and later has also become a major
tool in biotechnology research and product development worldwide.
A wide array of molecular markers is now available for detecting DNA variation.
They fall into three broad categories: (a) Hybridization-based approaches
such as RFLP, (b) PCR arbitrary or multi-locus profiling techniques such as
Random Amplified Polymorphic DNA (RAPD) and Amplified Length Polymorphism
(AFLP), and (c) Site-Targeted-PCR techniques such as microsatellite or Simple
Sequence Repeat (SSR), and Cleaved Amplified Polymorphic DNA (CAPs). RAPD
makes use of short (10-mer) primers and low annealing temperature to amplify
several regions of unknown map location in the genome (Welsh and McClelland,
1990; Williams et al., 1990). AFLP is based on the amplification of a subset
of digested DNA using primers designed for selective extension at digestion
sites fitted with adapters (Vos et al., 1995) (Figure 1).
Figure 1. Principle of the
AFLP strategy. Genomic DNA is cut with two restriction enzymes (here
EcoRI and MseI) and specific adapters are linked to both
ends of all the resulting fragments. Two successive PCRs are then performed
using specific primers complementary to the adapters and the restriction site
with the 3’-ends extending by one or a few bases to effect selective
amplification. Amplification products obtained by the second, selective PCR
are separated on gels. Band detection can be through silver staining or labeling
of one primer by a radioisotope or a fluorochrome (indicated by a star). (Weising
et al., 2005).
SSRs are tandemly repeated simple sequences of about 2 - 6 nucleotides that vary in repeat number to as many as 100 times (Weber and May, 1989). CAPs are PCR products that contain sequence variation that can be detected by cleavage or digestion with restriction enzymes (Konieczny and Ausubel, 1993). Table 1 summarizes the properties of these commonly used markers in crop breeding and genetics (Edwards and McCouch, 2007). DNA markers are continually being modified to boost their utility, becoming more amenable to high-throughput, automated process of genome analysis.
Click
the table for the larger version
The latest marker type is the Simple or Single Nucleotide Polymorphism (SNPs), the ultimate marker representing single base changes or short insertion/deletions in DNA. SNPs are revealed by sequencing, microarray-chip hybridization, or digestion of heteroduplex DNA from pairs of polymorphic lines. The very high densities of SNPs in a genome have made them a preferred molecular marker for fine-mapping studies (Rafalski, 2002).
Marker
abundance in crop genomes
Table 2 provides the present abundance of markers discovered and developed
in the major crop species. Model plant species and major crops are the most
intensively characterized for DNA markers. Rice, being both a model species
and the staple of most people, is the most advanced in terms of genome characterization.
Its small genome and huge economic importance stimulated vigorous international
efforts by the International Rice Genome Sequencing Project/ IRGSP, which
was established in 1997, leading to the completion of rice genome sequencing
in 2005 (Matsumoto et al., 2005). The genome sequence information has served
as an excellent platform to uncover SNPs of rice that is now more than 5 million.
Rice SNP resources are publicly accessible in www.oryzasnp.org
to aid gene localization and expression studies (McNally, 2006). SSR is the
next most abundant marker in rice numbering to 13,000.
Click
the table for the larger version
Corn is the second most important crop worldwide as well as in the Philippines.
Its genome, which is 6 times the size that of rice, is being sequenced by
an international consortium led by the US Department of Agriculture and National
Science Foundation Plant Genome Research Program (http://www.nsf.gov/bio/dbi/dbi_pgr.htm).
The SNPs in corn are now more than 2,000,000, followed by 9,000 RFLPs. Except
for oats, the rest of the major cereals have more than 200,000 SNPs. The recent
completion of sorghum genome sequencing (Paterson et al., 2009) will certainly
lead to the discovery of many more SNPs. In the non-cereal crops, SSR is the
most abundant in pineapple, while it is AFLP in banana and coconut. Table
3 lists the websites hosting genomics information for the major crops.
Applications of genetic fingerprinting
Genetic fingerprinting has given new impetus to the biological sciences. Because
of its versatility, it was rapidly adopted as a research tool in medicine
(Nakamura et al., 1987), forensic science (Lewin, 1989), and animal behavior
(Burke, 1989). At present, genetic fingerprinting has also been applied in
many aspects of crop biology, from analysis of genetic diversity within breeding
populations in plants, to differentiation between cultivars, as well as to
identification of plants containing a gene of interest, to name a few. Table
4 shows the current crop research activities involving DNA markers, which
are being lead by Filipino scientists.
Click
the table for the larger version
Taxa
identification and phylogeny
The use of molecular markers is a major advance in evaluating genetic variation
and in elucidating the genetic relationships within and among species. The
first application of microsatellites in agriculture has been in cultivar identification,
wherein breeders/scientists are able to determine cultivated varieties from
traditional varieties and vice versa thru the use of microsatellites. This
paved the way for diverse materials like rice, wheat, grapevine (Vitis
unifera), and soybean to be genotyped, using unique genetic information
instead of phenotypic expression or physical appearance to discern between
closely similar or identical materials or varieties.
In addition, the microsatellite markers are useful in sex identification of
dioeceous plants. Normally, sex-specific morphological differences of these
types of plants only show during the flowering stage, however, a microsatellite
probe (GATA)4, used as a diagnostic marker, reveals sex-specific DNA variation
at any stage of plant development, which allows sex-identification possible
beyond the limitation of morphologically expressed differences of the plant.
Likewise, a RAPD marker detected pseudo-autosomal plant sex chromosome in
Silene dioica (L.) (Joshi et al., 1999).
Umali of Del Monte Fresh Produce Division in Davao developed CAP markers for
banana identification during his graduate work in Japan. The markers, which
were based on a maternally inherited chloroplast DNA mutation, helped establish
the lineage of banana cultivars as well as the contribution of wild progenitors
to cultivated types (Umali and Nakamura, 2003).
Diversity
analysis
Diversity analysis measures the level of genetic similarity or differences
among materials, which is vital information in crop conservation and varietal
development. DNA markers are extensively used in assessing the genetic diversity
in most crop species due to its high efficiency, easy to use, co-dominance
nature, reproducibility, and high degree of polymorphism. This information
can serve as basis for rational use and conservation of genetic resources,
since the collection, storage and maintenance of germplasm generally requires
expensive equipment, highly controlled environments and infrastructure. In
conjunction with pedigree records, DNA fingerprinting provides an effective
method of assessing the diversity of varieties as well as breeding lines.
Rivera et al. (1999) of the Philippine Coconut Authority in Zamboanga City
used microsatellites in assessing the diversity of their coconut collection.
The microsatellites revealed high polymorphism in the coconut germplasm, indicating
broad genetic diversity, and thus attesting to the agency’s effective
collecting and gene-banking strategies. A highly polymorphic set of coconut
microsatellites has been assembled for routine evaluation of both in situ
and ex situ genetic resources of coconut.
Hybridity
testing
In hybrid technology, hybridity or the 50:50 parental gene contributions in
the progeny should be ascertained to draw maximum performance of the hybrid
variety in farmers’ fields. Identification and selection is necessary:
a) for proper identification and varietal protection, b) for genetic identity
stability, c) for complete characterization and measurement of crop genetic
diversity, and d) for uniformity of appearance and agronomic performance of
produced variety that will meet the demand of the farmers and consumers (Smith
and Register, 1998). Detection of hybridity is, however, hampered by the paucity
of markers. Seed characteristics are unreliable as they are largely controlled
by the maternal parent. Biochemical markers are of limited use as they can
be affected by developmental stage and environment, and can only be assayed
with considerable tissue material. Genetic fingerprinting is therefore an
ideal approach to hybridity testing.
At the Philippine Sugar Research Institute (PhilSURIN), Manigbas and Villegas
screened fifty SSR markers and found one SSR that amplified highly polymorphic
bands (Manigbas and Villegas, 2004). The SSR correctly identified the true
hybrids among 918 progenies derived from four sugarcane crosses. With this
molecular hybridity testing system, the conventional 7-year breeding cycle
for sugarcane can be reduced by 1-2 years.
Gene
mapping
With the growing density of genetic markers in crop genomes and the increasing
power of associated statistical methods, it is now possible to determine specific
genomic regions that are responsible for the expression of important physiological
and agronomic traits, many of which are quantitative traits. DNA markers have
been efficiently employed in tagging numerous individual traits that are extremely
vital for a breeding program like yield, disease resistance, stress tolerance,
seed quality, etc. Moreover, a causal link between coding structural genes
and quantitative variation can be established by analyzing quantitative trait
loci (QTL). QTL can lead to the identification of candidate genes for the
trait of interest.
Molecular marker technology has been useful in detecting QTL in rice by Filipino
graduate students and researchers. Redoña, while in University of California,
Davis, identified the QTL for rice seedling vigor through RAPD markers (Redoña
and Mackill, 1996). Tabien used RFLP markers to determine the QTL for blast
resistance as a student in Texas A&M (2000). At Cornell, Sebastian localized
the QTL for tungro resistance using both RFLP and RAPD markers (Sebastian
et al., 1996). At PhilRice, Romero et al., (2008) used SSR and CAPS to move
closer to the tungro resistance QTL. Gregorio’s team at the International
Rice Research Institute (IRRI) determined rice genotypes with salt tolerance
using microsatellite markers associated with the saltol QTL (Mohammadi-Nejad
et al., 2008).
At the Institute of Plant Breeding (IPB) at the University of the Philippines
in Los Banos (UPLB), QTL analysis with DNA markers was used in investigating
disease resistance. Balatero and Hautea (2002) discovered that seven AFLP
markers and one Resistance Gene Analogs (RGA) marker were putatively associated
with resistance to bacterial wilt strains Tm-22 and Tm-151.
Marker-aided
introgression
Introgression or transfer of desirable traits into target varieties or organisms
can be facilitated or accomplished thru the aid of DNA markers. For traits
that are recessive, expressed late in development and/or require progeny testing,
the use of markers can significantly hasten and improve selection efficiency.
DNA markers allow plant breeders to monitor genetic variation and identify
genotype in the absence of a phenotype; thus, accelerating breeding work to
produce and select crops with desirable traits.
PhilRice has developed several products of marker-aided breeding. Tabien et
al. (2003) introduced bacterial leaf blight resistance genes into three popular
varieties namely IR64, PSB Rc14, and BPI RI-10, producing elite breeding lines
for anther culture and hybridization. Recently, two irrigated lowland rice
varieties namely NSIC Rc142 (Tubigan 7) and NSIC Rc154 (Tubigan 11) were released
with resistance genes Xa4 and Xa21 against BLB (Babb et al., 2007; Padolina
et al., 2008). These are the first two rice varieties produced under molecular
marker technology in the Philippines.
Somaclonal
variation
Somaclonal variation refers to variation seen in plants being produced via
plant tissue culture. It requires multiple genetic and/or epigenetic events
which affect patterns of expression, or result in mutational alteration of
genes. Several molecular mechanisms such as DNA damage and mutation, alteration
of cell ability to repair damaged and mutated DNA, alteration of genes for
cell-cycle control mechanisms, and DNA methylation (Merlo et al., 1995) are
responsible somaclonal variation. The physical bases include deletions, amplifications,
translocations, insertions, recombination, and chemical alteration (Stoler
et al., 1999).
Their widespread distribution in the genome makes microsatellites an effective
detector of random plant genomic instability. Its variation, Inter-Simple
Sequence Repeats (ISSR), can reveal genetic instability at early stages in
in vitro culture. This method has been applied in in vitro cultures of cauliflower
leaves and calli (Leroy et al., 2000).
Plant patenting
DNA fingerprinting occupies a unique place in the area of plant variety protection.
Conventionally, differences in morphology, cultural characteristics, and pedigree
serve as the initial basis to differentiate the proposed variety from existing
ones (Anderson and Wu, 2007). When these are deemed inadequate, DNA fingerprinting
becomes the final solution to document the distinctness of the new variety
in the patenting process. Although not yet acceptable as proof of unique identity
by The International Union for Protection of New Varieties of Plants (UPOV),
the DNA fingerprint may strengthen claim for protection as supplementary information.
In India, three chilli varieties sold under the brand name of an elite variety
were found by fingerprinting to be spurious (Bhat, 2005). DNA analysis confirmed
the adulteration in retail Basmati rice in the UK (Food Standards Agency,
2004) and established the origin of the different Basmati types in the US
(Woolfe et al., 2001). Efforts are now underway at the Rice Technical Working
group of the Philippine National Seed Industry Council to establish DNA fingerprinting
using SSR to screen applications for registration and accreditation of new
rice hybrids.
Conclusion
DNA fingerprinting is advancing the frontiers of crop biology research in
broad strides, ushering a new age for genomic research and biotechnology.
What began as a human profiling tool is now a prominent fixture in almost
all areas of basic and applied biology including agriculture. DNA fingerprinting
techniques which were tedious and time-consuming have now evolved into simpler,
rapid and straightforward endeavors owing largely to the flexibility of PCR.
DNA fingerprinting has greatly expanded the arsenal of scientists in the study
and improvement of crop genetics. Many of its potential applications in agriculture
are beginning to be realized and steady progress is also being made in the
Philippines.
