A) TECHNICAL FIELD
[0001] The present invention generally relates to a technology for characterization and identification of biological specimens such as plant, animal, insect and microbial specimens using genotyping technology or genetic polymorphism. The present invention particularly relates a method for the identification of plants, insects and microbial specimens using DNA bar codes. The present invention more particularly relates to a method and system using the fluorescent assisted bar coding (FAB) technology for characterization, authentication and bar coding of individual plant, animal, insect and microbial specimen.

B) BACKGROUND OF THE INVENTION

[0002] "Genotyping" is the method of determining differences in the genetic makeup or genotype of an individual by examining the DNA sequences in individuals using biological assays and comparing the DNA sequences in one individual with that of the other or a references sequence to identify whether the alleles are inherited from the parental generation to the next gene'ration or not. Traditionally, genotyping is the use of DNA sequences to define a biological population by the use of molecular tools. The genotyping methods include Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Polymerase Chain Reaction (PCR), Single Nucleotide Polymorphism (SNP) based technique, microsatellite based technique, DNA sequencing, DNA microarrays etc. The currently available genotyping method also involves the use of bio-informatic tools for phylogenetic analysis, such as cladogram, CLUSTALW software etc.

[0003] The "taxonomy" is an academic discipline and a branch of biology which deals with defining groups of biological organisms on the basis of shared characteristics and giving name to those groups. The methods employed for taxonomic classification are genotyping, phylogenetics and numerical taxonomy. The researchers arrive at the taxa based on available data, resources, qualitative or quantitative comparisons and computer based analysis of DNA sequences.

[0004] The most common method used for genotyping is the restriction fragment length polymorphism (RFLP). In this method, the genomic DNA is subjected to a restriction digestion process. After the restriction digestion process, the digested fragments are subjected to agarose gel electrophoresis. The digestion profiles are compared for finding polymorphism. Limitations are, the screening of the digested products takes time and is very difficult. Further this technique is not suitable for the larger genomes.

[0005] The second method for genotyping is random amplified polymorphic DNA (RAPD). This method is also based on polymerase chain reaction (PCR) in nature. In this method, DNA template is taken and amplified along with the specific primers in PCR. The primers used are 6-10 mer in length. As the name of the method suggests, the DNA fragments are randomly amplified. The disadvantage of this method is that, the primers may or may not bind to the DNA template in PCR reaction. Secondly the amplified DNA fragments may or may not be obtained as the target loci may not be polymorphic. Hence this method is regarded as trial and error method. The other disadvantage of this method is that the temperature for DNA fragment amplification should be maintained at 38-40 °C.

[0006] The other genotyping method is amplified fragment length polymorphism (AFLP). In this genotyping method, the template DNA is restricted and the ends of the template DNA are ligated with adapter sequences. The template DNA molecules ligated with the adapter sequences are then subjected to polymerase chain reaction (PCR). The main limitation is ligation of adapter with the template DNA is not efficient. Due to these shortcomings, the PCR profile is not consistent.

[0007] The other genotyping method is a microsatellite based technique. Microsatellites are simple sequence sets repeated number of times and distributed all over the genome. The simple sequence repeats are inherited within progenies and are variable in number within members. The genotyping protocol typically involves PCR amplification of the microsatellite region using sequence-specific fluorescent-labeled primers and sizing the amplicons by capillary electrophoresis based protocols. Genomes with limited variabilities / polymorphism would have fewer polymorphic microsatellites and thus, large number of microsatellites must be screened for genotyping such genomes. For genotyping some tomato varieties using microsatellite based protocols, screening of up to 1,000 or more microsatellites are required which makes the protocol very tedious.

[0008] The other method for the genotyping is a single nucleotide polymorphism (SNP) based method. In this method, the individual genomes are sequenced and then the specific primer sequences are designed based on the known SNPs. Hence this method consumes more time and is also tedious.

[0009] The general method of identification and genotyping of biological specimens comprising plants, microorganisms, insects etc., involves identifying phenotypic, genotypic and biological characteristics of a microorganism. This method helps in differentiating pathogenic and/or toxicogenic microorganisms from the useful/benevolent microorganism. But the identified, benevolent microorganism/plants are valid only when the active reference microorganism/plant is correctly identified, characterized and authenticated. Secondly the experienced, skilled taxonomists are required for an accurate authentication, identification and characterization of biological specimens.

[0010] The other method of authentication, characterization and identification of biological specimens involves an analysis of biochemical, physiological and metabolic characteristics. The biochemical and metabolic properties of microorganisms are studied by analyzing the growth requirements, enzymatic activities and cellular fatty acid composition. The biological tests use a specific growth media, nutrients, chemicals or growth conditions to elicit an observable or measurable biochemical response from the microorganism, thereby enabling the identification and characterization of the microorganism. These tests include an estimation of carbon and nitrogen sources utilized, growth requirements (anaerobic or aerobic), optimum temperature range, optimum pH range, preferred osmotic condition, generation of fermentation products, production of enzymes, production of antimicrobial compounds, sensitivity of metabolic inhibitors and antibiotics. But these assay techniques require biochemical and analytical skills. Further the assay techniques and isolation methods consume more time. The identification, characterization and authentication of plant specimen for taxonomic and genotypic classification are mainly done by using expert determination, recognition, comparison, keys and similar devices. The expert analyses monographs and carries out revisions and synopsis. But this method requires a valuable time of experts and generates delays in identification of specimen.

[0011] Further a new method involves a comparison of an unknown specimen with named specimen, photograph, illustration or description. This method is a reliable method, but consumes more time. This method is virtually impossible due to a lack of suitable material for comparison.

[0012] The DNA bar coding is a genotyping method which uses short genetic markers in an organisms DNA to identify a particular species to which the organism belongs. The DNA bar coding is different from molecular phylogeny in that the main goal is not to determine classification but to identify an unknown sample in terms of well known classification. The DNA barcodes are used to identify the unknown species or assess whether the unknown species should be combined or separated. The DNA barcodes are applied to identify the plant leaves, even when the flowers or fruits are not available.

[0013] The new technique developed for the identification of biological specimen includes a development of DNA barcodes. The DNA bar code is a representation of sequences of specific loci (600-800 bases) and is used to identify the polymorphism. This technique may not always give accurate results as the targeted loci might not be polymorphic enough. Hence the polymorphic loci need to be validated before developing the bar codes.

[0014] Hence there is need to develop a fluorescent probe based DNA bar coding technique that is fast, accurate and specific in nature and can differentiate two close species or varieties of the same crop. Further the technology must identify the offsprings based on "genome-wide homology" with desired characteristics within a breeding population. Also there is a need for a technique that accelerates the breeding programs to within 3-4 generations when compared to the 7-8 generations of the conventional breeding program.

[0015] The above mentioned short comings, disadvantages and problems are addressed herein and which will be understood by reading and studying the following specification.

C) OBJECTIVES OF THE PRESENT INVENTION

[0016] The primary object of the present invention is to develop a genotyping technique using the fluorescent assisted DNA bar codes for the identification of biological specimens.

[0017] Another object of the present invention is to develop the fluorescent assisted bar codes for the identification of plant, animal, insect and microbial specimens in the immature/developing stage.

[0018] Yet another object of the present invention is to develop fluorescent assisted barcode to accelerate the conventional breeding program.

[0019] Yet another object of the invention is to develop fluorescent assisted barcode to identify plants with desired characteristics within 3-4 generations of the breeding program rather than 7-8 generations.

[0020] Yet another object of the present invention is to develop the fluorescent assisted barcode which are used to distinguish very closely related species as the data is compared for more than 1000 gene loci.

[0021] Yet another object of the present invention is to develop the fluorescent assisted DNA bar codes based on the sequencing of specific loci and polymorphism.

[0022] Yet another object of the present invention is to develop the fluorescent probe based DNA bar codes to generate DNA profiles covering approximately 350 loci.

[0023] Yet another object of the present invention is to develop the fluorescent probes which are not limited to 15-25 mer to generate DNA profiles.

[0024] Yet another object of the present invention is to develop the fluorescent probes which are specific to a particular locus in genome.

[0025] Yet another object of the present invention is to develop the fluorescent probes with a melting temperature of not less than 50 - 55 °C.

[0026] Yet another object of the present invention is to develop fluorescent probes with a GC content of 50% - 60 % to increase the specificity.

[0027] Yet another object of the present invention is to develop the fluorescent probes which are labeled to specific dyes and are analyzed by capillary electrophoresis on GeneMapper.

[0028] Yet another object of the present invention is to develop the fluorescent probes that are universal and works irrespective of the source of the biological samples.

[0029] Yet another object of the present invention is to develop the fluorescent probes for analyzing single nucleotide polymorphism (SNP) and simple sequence repeat (SSR).

[0030] Yet another object of the present invention is to develop the fluorescent probes which give results for genome-wide selection of the biological specimens.

[0031] Yet another object of the present invention is to develop the fluorescent probes which are used for testing an authenticity of the biological samples, gene segregation studies, breeding program to predict heterosis, background screening, piracy testing etc.

[0032] Yet another object of the present invention is to develop a protocol to accelerate conventional breeding program combining "marker-assisted selection" and "FAB-technology based genome -wide selection testing" for secondary screening.

[0033] These and other objects and advantages of the present invention will become readily apparent from the following detailed description taken in conjugation with the accompanying drawings.

D) SUMMARY OF THE INVENTION

[0034] The various embodiments of the present invention provides a next generation genotying technology which uses a fluorescent assisted DNA bar-coding (FAB) technology for characterization, authentication and bar-coding of individual samples like plants, animals, insects, microbes etc. The fluorescent assisted DNA bar-codes enables the identification of plant, insect and microbial specimens in the immature/developing stage. Further the fluorescent probe based DNA bar-codes generate profiles covering approximately 350 loci, and the bar codes developed are universal in nature. The fluorescent probes synthesized have a GC content more than 50%. Further the melting temperature of the fluorescent probes is 55 °C. Minimum three such probe data are pooled and compared on a genetic analyzer to generate a unique bar code that is generated by comparing more than 1000 loci data for the sample. The probes used are universal and works irrespective of the source of the biological sample. A minimum of three DNA probes are used to generate 1,000 plus loci based DNA barcodes. The fluorescent probe has a melting temperature of 50-65 °C. The DNA profile generated using the fluorescent assisted DNA barcode marker/probe is reproducible.

[0035] According to one embodiment herein, the system for authenticating biological specimens/ samples through genotyping based protocol has a plurality of fluorescent assisted DNA barcode marker/probes for identification of biological specimens, and the fluorescent assisted DNA barcode identifies, authenticates and differentiates biological sample of a plant, an animal, an insect and a microbial specimens in all stages of development. The fluorescent assisted DNA barcode distinguishes and differentiates very closely related species using the data generated from more than 1,000 loci. The fluorescent assisted DNA barcode marker/probes developed are not limited to 15-25 bases to generate the DNA profiles. Further each fluorescent assisted DNA barcode marker/DNA probe generates more than 350 loci based profile. The guanine-cytosine (GC) content present in the DNA marker/probes is more than 50%.

[0036] According to one embodiment herein, the fluorescent assisted DNA barcode marker/probes are developed for a plurality of biological samples. The plurality of samples includes plurality of plant species, animal species and bacterial species. The plurality of fluorescent assisted DNA barcode marker/probes developed for the plurality of biological samples are mutually different from each other. The fluorescent assisted DNA barcode marker/probes developed is 15.

[0037] According to one embodiment herein, the fluorescent assisted DNA barcode marker/probes are developed to generate the DNA barcode for one biological samples, and the combination of the plurality of fluorescent assisted DNA barcode marker/probes are developed to generate the DNA barcode for different biological samples are mutually different.

[0038] According to one embodiment herein, the fluorescent assisted DNA markers/probes are universal and are not specific to a gene / a locus on the genome, and specific sets of probes preferentially generate polymorphic profiles for the specific types of biological samples.

[0039] According to one embodiment herein, the fluorescent assisted DNA marker is specific for analyzing a single nucleotide polymorphism (SNP) and a simple sequence repeat (SSR). The fluorescent assisted DNA markers / probes analyze the polymorphism or genome-wide selection within the genomes of the biological specimens.

[0040] According to one embodiment herein, the fluorescent assisted DNA marker is designed for testing the authenticity of the biological samples, a diversity analysis, a crop breeding program, a prediction of parental plant germplasm for heterosis breeding, a screening for homologous plant, a plant variety authenticity testing, a background screening tool and a piracy testing tool.

[0041] According to one embodiment herein, the fluorescent assisted DNA markers generate DNA profiles covering genome-wide variations with minimum 350 loci analyzed.

[0042] According to one embodiment herein, the fluorescent assisted DNA probes are designed to specifically differentiate similar biological samples differing from each other by mutations; and wherein the DNA marker/probes are sensitive to biological samples differing by one nucleotide base pair. The mutations include deletion, insertion, single nucleotide polymorphism.

[0043] The fluorescent assisted DNA marker is designed to distinguish closely related biological specimens and compare data of more than 1,000 gene loci in the closely related biological specimens.

[0044] According to one embodiment, the first step in the "fluorescent assisted bar-coding technology" (FAB) technology is to develop and validate the labeled fluorescent probes. The fluorescent probes are used in the FAB technology for genotyping different biological samples. Further the fluorescent probe assisted DNA barcodes (FAB) are sensitive to biological samples differing by one nucleotide base pair. A method of developing the fluorescent assisted DNA barcode markers/probes for genotyping biological specimens, is initiated by synthesizing a fluorescent labeled marker by a standard protocol. The fluorescent labeled marker are validated by amplifying a genomic DNA of different organism samples. The genomic DNA is isolated from the biological samples and amplified by a polymerase chain reaction (PCR) with fluorescent labeled marker/probes. The polymerase chain reaction (PCR) is performed at an annealing temperature of 50 - 55 °C. Further the amplified genomic DNA is subjected to a capillary electrophoresis; and the capillary electrophoresis produces DNA bands according to a length of the nucleotide base pairs. The nucleotide base pair are subjected to a Genetic Analyzer having a GeneMapper Software, and the Genetic Analyzer generates a genome-wide relation data. Also the GeneMapper software generates a cladogram. Developing a barcode is based on a base pair information obtained from the genome-wide correlation data, and the barcode is developed based on a plurality of nucleotide base pairs groups. The base pair groups are represented based on a number of base pairs present in the genome.

[0045] According to one embodiment herein, a method for breeding a plant variety within fewer back-cross generations using Fluorescent Assisted DNA barcode (FAB) technology, has the following steps of: transferring traits from a donor parent plant variety to a recipient parent plant variety, also called the recurrent parent, through a plant breeding program. A new plant variety is developed which resembles the recipient parent plant genetically and phenotypically but comprises the trait present in the donor parent plant through plant breeding program. A genetic distance between breeding parents, a donor plant and a recurrent parent plant is determined, using the FAB technology. The next step is screening a plurality of plant samples within back-cross populations using the FAB technology to identify progenies with maximum genetic similarity to that of the recurrent parent. The next step is performing similar screening protocols repeatedly following FAB technology within subsequent back-cross generations to obtain progenies with a genetic similarity of more than 99.50% to recurrent parent. The screening protocol results in a selection of progenies with preset percentage of homology with the recurrent parent within 3 or more generations of back-crosses. The preset percentage of homology is 99.60% or higher percentage. A breeding process is not limited to a combination of marker assisted selection process and FAB technology based genome-wide homology testing.

[0046] According to one embodiment, the fluorescent labeled primers are used in the genotyping. The "fluorescent assisted bar-coding technology has a specific protocol. The first step in the protocol is to subject the genome of the sample and fluorescent labeled probes to polymerase chain reaction (PCR) reaction. After PCR, the genome profiles are resolved with 1000 plus bands by using capillary electrophoresis. The gene bands are analyzed through capillary electrophoresis by using a GeneMapper software which runs on ABI 3500XL Genetic Analyzer. The GeneMapper software analyzes the gene bands based on the time at which the gene along with fluorescent labeled probe comes out of capillary. Further the GeneMapper analyzes the gene bands based on the fluorescent intensity of the probe associated with the gene. The GeneMapper produces the scoring matrix and produces peaks for genes with different molecular weight. This is followed by the development of the barcode.

[0047] The barcode is deduced from minimum three independent genetic profiles using three different probes. Each probe used represents more than 350 loci of the genome. For developing the barcode, the gene loci data and length of the restricted sequence is analyzed by GeneMapper. The GeneMapper generates genome-wide relation data. This data is divided into four main groups. The groups are represented based on the number of base pairs present in the genome. The four groups are 125-200bp, 201-300bp, 301-400bp, and 400-500bp. The sizes of the gene fragments from all the four categories are added to generate four sets of code for a particular probe. The GeneMapper analyzes the specific loci based on the number of base pairs and develops a cladogram. The cladogram is generated by the GeneMapper based on the similarity or dissimilarity between the nucleotide sequences (base pairs) of the biological samples. This entire procedure is called "FAB protocol" or fluorescent assisted DNA bar code protocol.

[0048] According to one embodiment, the fluorescent assisted DNA bar-coding (FAB) technology is applied to predict the impure seedlings among foundation/nuclear/breeder seeds. The first step involves selecting or acquiring hundred foundation/nuclear/breeder seeds and isolating the genomic DNA from all the hundred foundation/nuclear/breeder seeds. After the genomic DNA is isolated from seeds, the fluorescent assisted DNA barcode (FAB) protocol is followed to generate the genome-wide relation data. After the genome-wide relation data is generated, the GeneMapper converts the genome-wide relation data into cladogram. The cladogram is then analyzed to identify the impure seedlings. The identified impure seedlings are uprooted.

[0049] According to one embodiment, the fluorescent assisted DNA bar-coding (FAB) technology is applied for the prediction of parents for heterosis breeding program. The first step is the isolation of the genomic DNA from all the parent plants chosen as parents for the heterosis breeding program. After the genomic DNA is isolated from the parent plants, FAB protocol or fluorescent assisted DNA bar code protocol is followed to generate the genome-wide relation data. After the genome-wide relation data is generated, the GeneMapper converts the genome-wide relation data into a cladogram. The cladogram is then analyzed to identify the distantly related parent plant species. The distantly related parent plant species are identified and are chosen for the heterosis breeding program.

[0050] According to one embodiment, the fluorescent assisted DNA bar-coding (FAB) technology is applied for establishing the phylogenetic relationship between the samples/germplasms of sugarcane. This is also called as "authenticity/piracy testing". The first step is the isolation of the genomic DNA from all the sugarcane plant samples. After the genomic DNA is isolated from sugarcane plant samples, the FAB protocol or fluorescent assisted DNA bar code protocol is followed to generate the genome-wide relation data. After the genome-wide relation data is generated, the GeneMapper converts the genome-wide relation data into a cladogram. The cladogram is then analyzed to identify the closely related sugarcane plant species. The closely related parent plant species are identified to infer that the closely related sugarcane plant species are phylogenetically related from the time of evolution.

[0051] According to one embodiment, the fluorescent assisted DNA bar-coding (FAB) technology is also applied for generating the differential barcode for Cyanobacterial strain. The first step is the isolation of the genomic DNA from all the Cyanobacterial strains. After the genomic DNA is isolated from the Cyanobacterial strains, the FAB protocol or fluorescent assisted DNA bar code protocol is followed to generate the genome-wide relation data. The genome-wide relation data is produced based on the length of the gene fragment associated with fluorescent probes. The scoring matrix of GeneMapper produces peaks for genes with different molecular weight. This is followed by the development of the barcode. The barcode is deduced from three independent genetic profiles. The genetic profiles are generated using three different probes. Each probe used represents more than 350 loci of the genome. The GeneMapper produces a cladogram based on the scoring matrix. The cladogram is then analyzed to identify the phylogenetic relationship between the Cyanobacterial strains.

[0052] These and other aspects of the embodiments of the present invention will be better understood when conjugation with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, which indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments of the present invention without departing from the spirit thereof, and the embodiments of the present invention include all such modifications.

E) BRIEF DESCRIPTION OF DRAWINGS

[0053] The other objects, features and advantages will occur to those skilled in the art from the following description of the preferred embodiments and the accompanying drawings in which :

[0054] FIG.l illustrates a flow chart explaining a method for developing the fluorescent assisted DNA bar codes, according to one embodiment of the present invention.

[0055] FIG. 2 illustrates the graphical plots of fluorescent intensity versus time used for developing the fluorescent assisted DNA bar code according to one embodiment herein.

[0056] FIG.3A illustrates the cladogram of line purity testing of the seedling samples indicating A 12, A 24, A 33, A 39 and A 49 samples that are heterogenous/impure/off type samples, according to one embodiment of the present invention.

[0057] FIG.3B illustrates the cladogram of line purity testing of the seedling samples indicating A 12 and A 39 samples that are heterogenous/impure/off type samples, according to one embodiment of the present invention.

[0058] FIG.4 illustrates the cladogram of the heterosis breeding program and prediction of the distinctively related parents in the sample of the cotton germplams, according to one embodiment of the present invention.

[0059] FIG.5 illustrates the cladogram of the authenticity testing/piracy testing of the sugarcane plant samples, according to one embodiment of the present invention.

[0060] FIG.6 illustrates graphical plots of fluorescent intensity versus time that are used for developing the fluorescent assisted DNA bar code development for the Cyanobacterium strains, according to one embodiment of the present invention.

[0061] FIG.7 illustrates the gene flow induction/accelerated molecular breeding program using fluorescent assisted DNA bar coding (FAB) technology, according to one embodiment of the present invention.

[0062] FIG. 8 illustrates the fluorescent assisted DNA bar code for the Chili varieties, according to one embodiment of the present invention.

[0063] FIG.9 illustrates the fluorescent assisted DNA bar code for the Tomato Varieties, according to one embodiment of the present invention.

[0064] FIG.IO illustrates the fluorescent assisted DNA bar code for the Bajra Varieties, according to one embodiment of the present invention.

[0065] FIG.ll illustrates the fluorescent assisted DNA bar code for Watermelon Varieties, according to one embodiment of the present invention.

[0066] FIG. 12 illustrates the fluorescent assisted DNA bar code for Cotton Varieties, according to one embodiment of the present invention.

[0067] FIG.13 illustrates the fluorescent assisted DNA bar code for Insects, according to one embodiment of the present invention.

[0068] FIG.14 illustrates the fluorescent assisted DNA bar code for Fish types, according to one embodiment of the present invention.

[0069] FIG.15 illustrates the fluorescent assisted DNA bar code for Mice, according to one embodiment of the present invention.

[0070] FIG. 16 illustrates the cladogram of line homogeneity testing of cotton seedling samples, according to one embodiment of the present invention.

[0071] FIG. 17 illustrates the cladogram of the Ashoka tree sample genetic profile, according to one embodiment of the present invention.

[0072] FIG. 18 illustrates the cladogram of the experiment that demonstrates the specificity of FAB technology, according to one embodiment of the present invention.

[0073] Although the specific features of the present invention are shown in some drawings and not in others. This is done for convenience only as each feature may be combined with any or all of the other features in accordance with the present invention.

F) DETAILED DESCRIPTION OF THE DRAWINGS

[0074] The following detailed description, or reference is made to the accompanying drawings that form a part here of, and in which the specific embodiment that may be practiced is shown by of illustration. The embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical mechanical and other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not being taken in a limiting sense.

[0075] The various embodiments of the present invention provides a next generation genotyping technology which uses a fluorescent assisted DNA bar-coding technology (FAB) technology for characterization, authentication and bar-coding of individual samples like plants, animals, insects, microbes etc. The fluorescent assisted DNA bar-codes enable the identification of plant, insect and microbial specimens in the immature/developing stage. Further the fluorescent probe based DNA bar-codes generate profiles covering approximately 350 loci, and the bar codes developed are universal in nature. The fluorescent probes synthesized have a GC content more than 50%. Further the melting temperature of the fluorescent probes is 55 °C. Minimum three such probe data are pooled and compared on a genetic analyzer to generate a unique bar code that is generated by comparing more than 1000 loci data for the sample. The probes used are universal and works irrespective of the source of sample. A minimum of three DNA probes are used to generate 1,000 plus loci based DNA barcodes. The fluorescent probe has a melting temperature of 50-65 °C. The DNA profile generated using the fluorescent assisted DNA barcode marker/probe is reproducible.

[0076] According to one embodiment herein, the system for authenticating biological specimens/ samples through genotyping based protocol has a plurality of fluorescent assisted DNA barcode marker/probes for identification of biological specimens, and the fluorescent assisted DNA barcode identifies, authenticates and differentiates biological sample of a plant, an animal, an insect and a microbial specimens in all stages of development. The fluorescent assisted DNA barcode distinguishes and differentiates very closely related species using the data generated from more than 1,000 loci. The fluorescent assisted DNA barcode marker/probes developed are not limited to 15-25 bases to generate the DNA profiles. Further each fluorescent assisted DNA barcode marker/DNA probe generates more than 350 loci based profile. The guanine-cytosine (GC) content present in the DNA marker/probes is more than 50%.

[0077] According to one embodiment herein, the fluorescent assisted DNA barcode marker/probes are developed for a plurality of biological samples. The plurality of samples includes plurality of plant species, animal species and bacterial species. The plurality of fluorescent assisted DNA barcode marker/probes developed for the plurality of biological samples are mutually different from each other. The fluorescent assisted DNA barcode marker/probes developed is 15.

[0078] According to one embodiment herein, the fluorescent assisted DNA barcode marker/probes are developed to generate the DNA barcode for one biological samples, and the combination of the plurality of fluorescent assisted DNA barcode marker/probes are developed to generate the DNA barcode for different biological samples are mutually different.

[0079] According to one embodiment herein, the fluorescent assisted DNA markers/probes are universal and are not specific to a gene / a locus on the genome, and specific sets of probes preferentially generate polymorphic profiles for the specific types of biological samples.

[0080] According to one embodiment herein, the fluorescent assisted DNA marker is specific for analyzing a single nucleotide polymorphism (SNP) and a simple sequence repeat (SSR). The fluorescent assisted DNA markers / probes analyze the polymorphism or genome-wide selection within the genomes of the biological specimens.

[0081] According to one embodiment herein, the fluorescent assisted DNA marker is designed for testing the authenticity of the biological samples, a diversity analysis, a crop breeding program, a prediction of heterosis, a background screening tool and a piracy testing tool.

[0082] According to one embodiment herein, the fluorescent assisted DNA markers generate DNA profiles covering genome-wide variations with minimum 350 loci analyzed.

[0083] According to one embodiment herein, the fluorescent assisted DNA probes are designed to specifically differentiate similar biological samples differing from each other by mutations; and wherein the DNA marker/probes are sensitive to biological samples differing by one nucleotide base pair. The mutations include deletion, insertion, single nucleotide polymorphism.

[0084] The fluorescent assisted DNA marker is designed to distinguish closely related biological specimens and compare data of more than 1,000 gene loci in the closely related biological specimens.

[0085] According to one embodiment, the first step in the "fluorescent assisted bar-coding technology" (FAB) technology is to develop and validate the labeled fluorescent probes. The fluorescent probes are used in the FAB technology for genotyping different biological samples. Further the fluorescent probe assisted DNA barcodes (FAB) are sensitive to biological samples differing by one nucleotide base pair. A method of developing the fluorescent assisted DNA barcode markers/probes for genotyping biological specimens, is initiated by synthesizing a fluorescent labeled marker by a standard protocol. The fluorescent labeled marker are validated by amplifying a genomic DNA of different organism samples. The genomic DNA is isolated from the biological samples and amplified by a polymerase chain reaction (PCR) with fluorescent labeled marker/probes. The polymerase chain reaction (PCR) is performed at an annealing temperature of 50 - 55 °C. Further the amplified genomic DNA is subjected to a capillary electrophoresis; and the capillary electrophoresis produces DNA bands according to a length of the nucleotide base pairs. The nucleotide base pair are subjected to a Genetic Analyzer having a GeneMapper Software, and the Genetic Analyzer generates a genome-wide relation data. Also the GeneMapper software generates a cladogram. Developing a barcode is based on a base pair information obtained from the genome-wide correlation data, and the barcode is developed based on a plurality of nucleotide base pairs groups. The base pair groups are represented based on a number of base pairs present in the genome.

[0086] According to one embodiment herein, a method for breeding a plant variety within fewer back-cross generations using Fluorescent Assisted DNA barcode (FAB) technology, has the following steps, transferring traits from a donor parent plant variety to a recipient parent plant variety, also called the recurrent parent, through a plant breeding program. A new plant variety is developed which resembles the recipient parent plant genetically and phenotypically but comprises the trait present in the donor parent plant through plant breeding program. A genetic distance between breeding parents, a donor plant and a recurrent parent plant is determined, using the FAB technology. The next step is screening a plurality of plant samples within back-cross populations using the FAB technology to identify progenies with maximum genetic similarity to that of the recurrent parent. The next step is performing similar screening protocols repeatedly following FAB technology within subsequent back-cross generations to obtain progenies with a genetic similarity of more than 99.50% to recurrent parent. The screening protocol results in a selection of progenies with preset percentage of homology with the recurrent parent within 3 or more generations of back-crosses. The preset percentage of homology is 99.60% or higher percentage. A breeding process is not limited to a combination of marker assisted selection process and FAB technology based genome-wide homology testing.

[0087] According to one embodiment, the fluorescent labeled primers are used in the genotyping. The "fluorescent assisted bar-coding technology has a specific protocol. The first step in the protocol is to subject the genome of the sample and fluorescent labeled probes to polymerase chain reaction (PCR) reaction. After PCR, the genome profiles are resolved with 1000 plus bands by using capillary electrophoresis. The gene bands are analyzed through capillary electrophoresis by using a GeneMapper software which runs on ABI 3500XL Genetic Analyzer. The GeneMapper software analyzes the gene bands based on the time at which the gene along with fluorescent labeled probe comes out of capillary. Further the GeneMapper analyzes the gene bands based on the fluorescent intensity of the probe associated with the gene. The GeneMapper produces the scoring matrix and produces peaks for genes with different molecular weight. This is followed by the development of the barcode.

[0088] The barcode is deduced from three independent genetic profiles using three different probes. Each probe used represents more than 350 loci of the genome. For developing the barcode, the gene loci data and length of the restricted sequence is analyzed by GeneMapper. The GeneMapper generates genome-wide relation data. This data is divided into four main groups. The groups are represented based on the number of base pairs present in the genome. The four groups are 125-200bp, 201-300bp, 301-400bp, and 400-500bp. The sizes of the gene fragments from all the four categories are added to generate four sets of code for a particular probe. The GeneMapper analyzes the specific loci based on the number of base pairs and develops a cladogram. The cladogram is generated by the GeneMapper based on the similarity or dissimilarity between the nucleotide sequences (base pairs) of the biological samples. This entire procedure is called "FAB protocol" or fluorescent assisted DNA bar code protocol.

[0089] According to one embodiment, the fluorescent assisted DNA bar-coding (FAB) technology is applied to predict the impure seedlings among foundation/nuclear/breeder seeds. The first step involves selecting or acquiring hundred foundation/nuclear/breeder seeds and isolating the genomic DNA from all the hundred foundation/nuclear/breeder seeds. After the genomic DNA is isolated from seeds, the fluorescent assisted DNA barcode (FAB) protocol is followed to generate the genome-wide relation data. After the genome-wide relation data is generated, the GeneMapper converts the genome-wide relation data into cladogram. The cladogram is then analyzed to identify the impure seedlings. The identified impure seedlings are uprooted.

[0090] According to one embodiment, the fluorescent assisted DNA bar-coding (FAB) technology is applied for the prediction of parents for heterosis breeding program. The first step is the isolation of the genomic DNA from all the parent plants chosen as parents for the heterosis breeding program. After the genomic DNA is isolated from the parent plants, FAB protocol or fluorescent assisted DNA bar code protocol is followed to generate the genome-wide relation data. After the genome-wide relation data is generated, the GeneMapper converts the genome-wide relation data into a cladogram. The cladogram is then analyzed to identify the distantly related parent plant species. The distantly related parent plant species are identified and are chosen for the heterosis breeding program.

[0091] According to one embodiment, the fluorescent assisted DNA bar-coding (FAB) technology is applied for establishing the phylogenetic relationship between the samples/germplasms of sugarcane. This is also called as "authenticity/piracy testing". The first step is the isolation of the genomic DNA from all the sugarcane plant samples. After the genomic DNA is isolated from sugarcane plant samples, the FAB protocol or fluorescent assisted DNA bar code protocol is followed to generate the genome-wide relation data. After the genome-wide relation data is generated, the GeneMapper converts the genome-wide relation data into a cladogram. The cladogram is then analyzed to identify the closely related sugarcane plant species. The closely related parent plant species are identified to infer that the closely related sugarcane plant species are phylogenetically related from the time of evolution.

[0092] According to one embodiment, the fluorescent assisted DNA bar-coding (FAB) technology is also applied for generating the differential barcode for Cyanobacterial strain. The first step is the isolation of the genomic DNA from all the Cyanobacterial strains. After the genomic DNA is isolated from the Cyanobacterial strains, the FAB protocol or fluorescent assisted DNA bar code protocol is followed to generate the genome-wide relation data. The genome-wide relation data is produced based on the length of the gene fragment associated with fluorescent probes. The scoring matrix of GeneMapper produces peaks for genes with different molecular weight. This is followed by the development of the barcode. The barcode is deduced from three independent genetic profiles. The genetic profiles are generated using three different probes. Each probe used represents more than 350 loci of the genome. The GeneMapper produces a cladogram based on the scoring matrix. The cladogram is then analyzed to identify the phylogenetic relationship between the Cyanobacterial strains.

[0093] FIG.l illustrates a flow chart explaining the method of developing the fluorescent assisted DNA bar code, according to one embodiment of the present invention. The fluorescent assisted bar coding technology has a specific protocol. The first step in the protocol is the synthesis and validation of the fluorescent labeled probes (101). The fluorescent labeled probes are synthesized and validated by standard protocols. More than 100 sets of such probes are designed and validated by amplifying the genomic DNA of different organisms. Once the probes are developed, they can be used for the fluorescent assisted DNA bar code development. The fluorescent assisted DNA barcodes are specific to any biological sample irrespective to the developmental stage. Further the fluorescent probes are 15-25 mer in length and can withstand the melting temperature of 55 °C. The synthesized and validated fluorescent probes have a GC content of more than 50%.The next step is the isolation of the genomic DNA from the biological samples (102) for which the DNA barcode has to be developed. The biological sample ranges from the bacteria to the plant and the animal genomic DNA.

Further the genomic DNA isolated from the biological samples is subjected to polymerase chain reaction (PCR) with the fluorescent labeled probes (103). The genomic DNA is subjected to the PCR amplification for getting the fragmented and fluorescent probe associated DNA. Once the genomic DNA is amplified, it is subjected to capillary electrophoresis (104). The capillary electrophoresis is done to separate the genomic DNA according to the length of the nucleotide base pairs. The capillary electrophoresis produces bands. These nucleotide base pair bands are subjected to the ABI 3500XL Genetic Analyzer having GeneMapper software (105). The GeneMapper software generates a genome-wide relation data (106). The cladogram is generated by the GeneMapper software (107). The barcode is developed based on the base pair information obtained from the genome-wide relation data (108). The barcode is made based on length of the nucleotide base pair groups. The groups are represented based on the number of base pairs present in the genome. The four groups are 125-200bp, 201-300bp, 301-400bp, and 400-5 OObp. The sizes of the gene fragments from all the four categories are added to generate four sets of code, for a particular probe.

[0094] The list of probes and different combination of the probes that are shortlisted to generate a 1000 plus loci data to make a unique bar-code, is furnished below.

[0095] FIG.2 illustrates the graphical plots of fluorescent intensity versus time used for the development of the fluorescent assisted DNA bar code, according to one embodiment herein. The figure illustrates the procedure of development of barcode using three different probes from the biological samples. The peaks in the GeneMapper graphical plots represent the locus. Each locus is designated by molecular size based on internal size standard by software and this data is converted to barcode.

[0096] FIG.3A illustrates the cladogram for line purity testing of the seedling samples, according to one embodiment of the present invention. The first step for the line purity testing of the seedling sample is the isolation of the genomic DNA from all hundred foundation/nuclear/breeder seeds. After the genomic DNA is isolated from the seeds, the FAB protocol is followed to generate the genome-wide relation data. After the genome-wide relation data is generated, the GeneMapper converts the genome-wide relation data into a cladogram. The cladogram is then analyzed to identify the impure seedlings. Once the impure seedlings are identified, they are uprooted. FIG.3A illustrates that the seed sample A 12, A 24, A 33, A 39 and A 49 are heterogenous/ impure/ off type samples among the 100 seed sample.

[0097] FIG. 3B illustrates the cladogram of line purity testing of the chili seedling samples, according to one embodiment of the present invention. The first step is the isolation of the genomic DNA from all hundred foundation/nuclear/breeder seeds. After the genomic DNA is isolated from the seeds, the FAB protocol is followed to generate the genome-wide correlation data. From the data a cladogram is generated and analyzed to identify the impure seedlings. Once the impure seedlings are identified, they are uprooted. FIG.3B illustrates that the seed sample A 12 and A 39 are heterogenous/impure/off type samples among the 100 chili seed sample.

[0098] FIG.4 illustrates the cladogram of the heterosis breeding program and prediction of the distinctively related parents in the sample of the cotton germplams, according to one embodiment of the present invention. The first step for the heterosis breeding program is the development of bar code. The genomic DNA is isolated from all the cotton parent plants, chosen as the parents for heterosis breeding program. After the genomic DNA is isolated from the parent plants, the FAB protocol or fluorescent assisted DNA bar code protocol is followed to generate the genome-wide relation data. From the genome-wide relation data a cladogram is generated. The cladogram is then analyzed to identify the distantly related parent plant species. Once the distantly related parent plant species are identified, they are chosen for the heterosis breeding program. FIG.4 illustrates that the plant sample 955-L 14 and 955-L 9, 955-L 7, 955-L 4 and the reference 955 has the maximum genetic distance in the cladogram. Therefore these cotton plant samples are used for the heterosis breeding program. Probability of these cotton plant samples give best vigor is higher.

[0099] FIG.5 illustrates the cladogram of the authenticity testing/piracy testing in the sugarcane plant samples, according to one embodiment of the present invention. This is also known as "authenticity/piracy testing". The first step in the authenticity testing is the isolation of the genomic DNA from all the sugarcane plant samples. After the genomic DNA is isolated from sugarcane plant samples, the FAB protocol or fluorescent assisted DNA bar code protocol is followed to generate the genome-wide relation data. From the genome-wide relation data a cladogram is generated. The cladogram is then analyzed to identify the closely related sugarcane plant species. Once the closely related parent plant species are identified, it can be inferred that the closely related sugarcane plant species are closely related genetically from the time of evolution. FIG. 5 illustrates that sugarcane plant sample A2 and A4 are distantly related. The sugarcane plant sample A3 and A5 are closely associated. Hence among five sugarcane plant samples A3 and A5 are the pirated plant samples or similar samples.

[00100] FIG.6 illustrates the graphical plots of fluorescent intensity versus time that are used for developing the fluorescent assisted DNA bar code for the Cyanobacterium strains, according to one embodiment of the present invention. The first step for the development of fluorescent assisted DNA barcode is the isolation of the genomic DNA from all the Cyanobacterial strains. After the genomic DNA is isolated from the Cyanobacterial strains, the FAB protocol or fluorescent assisted DNA bar code protocol is followed to generate the genome-wide relation data. The genome-wide relation data is produced based on the length of the gene fragment associated with fluorescent probes or the genome-wide relation data is produced based on the size of the locus fragment. The barcode is deduced from three independent genetic profiles using three different probes. Each probe represents more than 350 loci of the genome. From the genome-wide relation data a cladogram is generated. The cladogram is then analyzed to identify the phylogenetic relationship between Cyanobacterial strains. FIG. 6 illustrates the fluorescent assisted DNA probe developed for the Cyanobacterial bacterial strains. Three probes, viz, probe 1, probe 2 and probe 3 are used to generate the DNA barcodes. The peaks in the GeneMappa graphical plots represent the locus. Each locus is designated by molecular size based on internal size standard by software and this data is converted to the barcode.

[00101] FIG.7 illustrates the gene flow induction/accelerated molecular breeding program using fluorescent assisted DNA bar coding (FAB) technology, according to one embodiment of the present invention. The protocol is made for accelerated conventional plant breeding program combining "marker assisted selection" and "FAB-technology based genome-wide homology testing". The protocol makes it possible to identify the plants with desired characteristics within 3-4 generations when compared to 7-8 generations of the conventional plant breeding program.

[00102] The accelerated plant variety development protocol using fluorescent assisted DNA barcode (FAB) based molecular breeding is initiated by considering the genetic distance between a donor plant variety and a recipient (recurrent) plant variety. The donor plant variety has the desired characteristics which the plant breeder wants to induce in the recipient plant variety. The desired character genes are transmitted from the donor plant variety to the recipient plant variety when cross breeding is done. The genetic distance between the donor plant variety and the recipient plant variety is determined using the standard FAB protocol (FIG. 1). The dissimilarity between the donor plant variety and the recipient plant variety is considered to be 100% or the identity is considered to be 0%. A chart is made to represent the back cross (BC) population genetic similarity with the recipient parent at each backcross cycle (FIG. 7).

The recipient plant variety is generally considered as the mother parent whereas the donor plant variety is considered as the father parent. The F1 generation is obtained after crossing the donor plant variety and the recipient plant variety. The back cross, is the cross between the F1 generation with the desired genetic trait/character and the recipient plant variety. Further the first generation obtained after crossing the donor plant variety and the recipient variety is Fl. Similarly the cross between the Fl and the recipient plant variety gives the F2 generation (back cross-1, BC-1). In the similar manner F2 is crossed with the recipient plant variety to get F3 (back cross-2, BC-2), and F3, F4, F5, F6 and F7 are obtained in the similar manner.

[00103] FIG. 7 represents the results of the different backcross performed and the similarity of the generation with respect to the recipient plant variety. The back cross -1 (BC-1) shows that the F2 generation has 75% similarity with the recipient plant variety. The back cross-2 (BC-2) shows that the F3 generation has 87.5% similarity with the recipient plant variety. The back cross-3 (BC-3) shows that the F4 generation has 93.705% similarity with the recipient plant variety. The back cross-4 (BC-4) shows that the F5 generation has 96.438% similarity with the recipient plant variety. The back cross-5 (BC-5) shows that the F6 generation has 98.438 % similarity with the recipient plant variety. The back cross-6 (BC-6) shows that the F7 generation has 99.219% similarity with the recipient plant variety. The back cross-7 (BC-7) shows that the F8 generation has 99.60% similarity with the recipient plant variety.

[00104] The fluorescent assisted barcode (FAB) technology is utilized to screen the back cross-1 plant samples (BC-1). The distribution of the alleles, for the desired trait/charater is random. There are few plants in BC-1 or F2 generation with genome more similar to the recipient plant variety. The percentage similarity of the F2 generation plant genome with the recipient plant variety is more than 75%. Hence statistically the plants similar to BC-2 or F3 generation are obtained in the BC-1 itself. These plants which have more similarity (more than 75%) in BC-1 are taken for the next back cross. Hence the plants 99% similar to the recipient plant variety are obtained in 3-4 generation rather than 7-8, as in case of the traditional breeding program. The plants obtained in the BC-1, BC-2, BC-3 and so on are constantly screened by FAB protocol to determine which are the plant species more similar to the recipient plant variety.

[00105] FIG. 8 illustrates the fluorescent assisted DNA bar code for the Chili varieties, according to one embodiment of the present invention. Three probes, i.e. probe 1; probe 2 and probe 3 are used to generate the DNA barcodes. The peaks in the GeneMapper graphical plots represents the locus. Each locus is designated by a molecular size based on internal size standard by the software and this data is converted to barcodes. FIG. 8 illustrates the three barcodes for chili variety 1, 2 and 3.

[00106] FIG.9 illustrates the fluorescent assisted DNA bar code for the Tomato Varieties, according to one embodiment of the present invention. Three probes, viz, probe 6, probe 9 and probe 10 are used to generate the DNA barcodes. The peaks in the GeneMapper graphical plots represent the locus. Each locus is designated by molecular size based on internal size standard by software and this data is converted to barcodes.

[00107] FIG.10 illustrates the fluorescent assisted DNA bar code for the Bajra Varieties, according to one embodiment of the present invention. Three probes, viz, probe 12, probe 18 and probe 21 are used to generate the DNA barcodes. The peaks in the GeneMapper graphical plots represent the locus. Each locus is designated by molecular size based on internal size standard by software and this data is converted to barcodes.

[00108] FIG.ll illustrates the fluorescent assisted DNA bar code for Watermelon Varieties, according to one embodiment of the present invention. Three probes, viz, probe 2, probe 6 and probe 8 are used to generate the DNA barcodes. The peaks in the GeneMapper graphical plots represent the locus. Each locus is designated by molecular size based on internal size standard by software and this data is converted to barcodes.

[00109] FIG.12 illustrates the fluorescent assisted DNA bar code for Cotton Varieties, according to one embodiment of the present invention. Three probes, viz, probe 4, probe 6 and probe 9 are used to generate the DNA barcodes. The peaks in the GeneMapper graphical plots represent the locus. Each locus is designated by molecular size based on internal size standard by software and this data is converted to barcodes.

[00110] FIG.13 illustrates the fluorescent assisted DNA bar code for Insects, according to one embodiment of the present invention. Three probes, viz, probe 7, probe 10 and probe 20 are used to generate the DNA barcodes. The peaks in the GeneMapper graphical plots represent the locus. Each locus is designated by molecular size based on internal size standard by software and this data is converted to barcodes.

[00111] FIG.14 illustrates the fluorescent assisted DNA bar code for Fish types, according to one embodiment of the present invention. Three probes, viz, probe 6, probe 17 and probe 18 are used to generate the DNA barcodes. The peaks in the GeneMapper graphical plots represent the locus. Each locus is designated by molecular size based on internal size standard by software and this data is converted to barcodes.

[00112] FIG.15 illustrates the fluorescent assisted DNA bar code for Mice, according to one embodiment of the present invention. Three probes, viz, probe 2, probe 6 and probe 8 are used to generate the DNA barcodes. The peaks in the GeneMapper graphical plots represent the locus. Each locus is designated by molecular size based on internal size standard by software and this data is converted to barcodes.

[00113] FIG. 16 illustrates the cladogram of line homogeneity testing of cotton seedling samples, according to one embodiment of the present invention. Five seedlings were tested using FAB and homogeneity interpreted from the cladogram generated from FAB data. FIG. 16 illustrates the line homogeneity of seven Cotton Varieties Gl to G7. The dendrogram indicates that the G7 variety is iso-line variety. The Gl, G2, G3, G4 and G6 have two types of genotype. The G5 variety has three types of genotype.

[00114] FIG. 17 illustrates the cladogram of the Ashoka tree sample genetic profile, according to one embodiment of the present invention. The FIG. 17 illustrates the experiment and the diversity of FAB technology. The Ashoka tree samples are profiled using FAB technology to generate the cladogram.

[00115] FIG. 18 illustrates the cladogram of the experiment that demonstrates the specificity of FAB technology, according to one embodiment of the present invention. The Cotton and Eggplant samples are analyzed simultaneously using Probes 4, 6 and 9. The samples cluster in the cladogram according to the crop variety. The FIG. 18 illustrates that the crop varieties E1-E6 are Eggplant varieties. The crop varieties C1-C5 are cotton varieties. The FIG. 18 illustrates that the FAB profiles are crop independent. Further the crop samples of a particular crop variety are clustered.

G) ADVANTAGES OF THE INVENTION

[00116] The various embodiments of the present invention provide fluorescent assisted DNA barcodes for genotyping the biological samples. The embodiments provide details of fluorescent assisted DNA barcodes which are specific to any biological sample. Further the fluorescent probes are 15-25 mer in length and can withstand the melting temperature of 55°C. The synthesized and validated fluorescent probes have a GC content of more than 50%. The fluorescent probe assisted DNA barcodes are universal in nature and can differentiate microbial samples to the samples of higher living organisms. Further the fluorescent probe assisted DNA barcodes (FAB) are sensitive to biological samples that differ even by single nucleotides / base pairs. Further the embodiments provide a protocol to accelerate conventional breeding program combining "marker-assisted selection" and "FAB-technology based genome-wide homology testing". The conventional breeding program is accelerated by FAB to identify the plant species with the desired genetic trait/character within 3-4 generations of breeding populations. Whereas the conventional breeding program requires 7-8 generations for identification of progenies with the desired traits/ characteristics present. The traditional marker assisted technology identifies progenies with the desired genetic traits/character in the seedling stage of the plant.

[00117] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modifications.

[00118] Although the embodiments herein are described with various specific embodiments, it will be obvious for a person skilled in the art to practice the embodiments herein with modifications.

CLAIMS:

What is claimed is:-

1. A system for authenticating of biological specimens/ samples through genotyping based protocol comprising: a plurality of fluorescent assisted DNA barcode marker/probes for identification of biological specimens, and wherein the fluorescent assisted DNA barcode identifies, authenticates and differentiates biological sample of a plant, an animal, an insect and a microbial specimens in all stages of development, and wherein the fluorescent assisted DNA barcode distinguishes and differentiates very closely related species using the data generated from more than 1,000 loci, and wherein the fluorescent assisted DNA barcode marker/probes developed are not limited to 15-25 bases to generate DNA profiles, and wherein each fluorescent assisted DNA barcode marker/DNA probe generates more than 350 loci based profile, and wherein the guanine-cytosine (GC) content in the DNA marker/probes is more than 50%.

2. The system according to claim 1, wherein a plurality of fluorescent assisted DNA barcode marker/probes are developed for a plurality of biological samples, and wherein the plurality of samples includes a plurality of plant species, and bacteria, and wherein the plurality of fluorescent assisted DNA barcode marker/probes developed for the plurality of biological samples are mutually different from each other.

3. The system according to claim 1, wherein the plurality of fluorescent assisted DNA barcode marker/probes developed is 15.

4. The system according to claim 1, wherein a combination of the plurality of fluorescent assisted DNA barcode marker/probes are developed to generate the DNA barcode for one biological samples, and wherein the combination of the plurality of fluorescent assisted DNA barcode marker/probes are developed to generate the DNA barcode for different biological samples are mutually different.

5. The system according to claim 1, wherein a minimum of 3 DNA probes are used to generate 1,000 plus loci based DNA barcodes.

6. The system according to claim 1, wherein the fluorescent probe has a melting temperature of 50 - 65 °C.

7. The system according to claim 1, wherein the polymerase chain reaction (PCR) is performed at an annealing temperature of 50 - 55 °C.

8. The system according to claim 1, wherein the DNA profile generated using the fluorescent assisted DNA barcode marker/probe is reproducible.

9. The system according to claim 1, wherein the fluorescent assisted DNA markers/probes are universal and are not specific to a gene / a locus on the genome, mitochondrial genomes, plastid genomes and wherein specific sets of probes preferentially generate polymorphic profiles for the specific types of biological samples.

10. The system according to claim 1, wherein the fluorescent assisted DNA marker is specific for analyzing a single nucleotide polymorphism (SNP) and a simple sequence repeat (SSR).

11. The system according to claim 1, wherein the fluorescent assisted DNA markers / probes analyze the polymorphism or genome-wide selection within the genomes of the biological specimens.

12. The system according to claim 1, wherein the fluorescent assisted DNA marker is designed specifically for testing the authenticity of the biological samples, a diversity analysis, a crop breeding program, a prediction of parental plant germplasm for heterosis breeding , a screening for homologous plant, a plant variety authenticity testing, a background screening tool and a piracy testing tool.

13. The system according to claim 1, wherein the fluorescent assisted DNA markers generate DNA profiles covering genome-wide variations with minimum 350 loci analyzed.

14. The system according to claim 1, wherein the fluorescent assisted DNA probes are designed to specifically differentiate similar biological samples differing from each other by mutations; and wherein the DNA marker/probes are sensitive to biological samples differing by one nucleotide base pair.

15. The fluorescent assisted DNA probes according to claim 14, wherein the mutations are a deletion, insertion, single nucleotide polymorphism.

16. The system according to claim 1, wherein the fluorescent assisted DNA marker is designed to distinguish closely related biological specimens and compare genome-wide relation data of more than 1,000 gene loci in the closely related biological specimens.

17. A method of developing the fluorescent assisted DNA barcode marker for genotyping biological specimens, the method comprising steps of: synthesizing a fluorescent labeled marker by a standard protocol; validating the fluorescent labeled marker by amplifying a genomic DNA of different organism; isolating the genomic DNA from the biological samples; amplifying the isolated genomic DNA by a polymerase chain reaction (PCR) with fluorescent labeled marker; subjecting the amplified genomic DNA to a capillary electrophoresis; and wherein the capillary electrophoresis produces DNA bands according to a length of the nucleotide base pairs; subjecting the nucleotide base pair to a Genetic Analyzer having a GeneMapper Software, and wherein the Genetic Analyzer generates a genome-wide relation data, and wherein the GeneMapper software generates a cladogram; and developing a barcode based on a base pair information obtained from the genome-wide correlation data, and wherein the barcode is developed based on a plurality of nucleotide base pairs groups, and wherein the base pair groups are represented based on a number of base pairs present in the genome.

18. A method for breeding a plant variety within fewer back-cross generations using Fluorescent Assisted DNA barcode (FAB) technology, the method comprises: transferring traits from a donor parent plant variety to a recipient parent plant variety, also called the recurrent parent, through a plant breeding program, and wherein a new plant variety is developed which resembles the recipient parent plant genetically and phenotypically but comprises the trait present in the donor parent plant through plant breeding program; determining a genetic distance between breeding parents, a donor plant and a recurrent parent plant, using the FAB technology; screening a plurality of plant samples within back-cross populations using the FAB technology to identify progenies with maximum genetic similarity to that of the recurrent parent; performing similar screening protocols repeatedly following FAB technology within subsequent back-cross generations to obtain progenies with a genetic similarity of more than 99.50% to recurrent parent, and wherein the screening protocol results in a selection of progenies with preset percentage of homology with the recurrent parent within 3 or more generations of back-crosses, and wherein the preset percentage of homology is 99.60% or higher percentage; and wherein a breeding process is not limited to a combination of marker assisted selection process and FAB technology based genome-wide homology testing.

19. A method of detecting genetically impure seeds among the breeder seeds, the foundation seeds, the certified seeds and/or the registered seeds using Fluorescent Assisted DNA barcode (FAB) technology, the method comprising steps of: selecting or acquiring a plurality of foundation seeds; germinating the seed lots and growing the saplings till 4 or more leaves emerge; subjecting the individual saplings to generate a cladogram representing the genome-wide homology data within the saplings through FAB technology; and analyzing the cladogram to identify pure and impure seedlings.

20. A method of detecting parental plant material for heterosis breeding program using fluorescent assisted DNA bar-coding (FAB) technology, the method comprising steps of: selecting a plurality of parental plant varieties / germplasm for a heterosis breeding program, and wherein a plant material which is most distantly and genetically related is known to provide heterosis events; and wherein an event exhibits a hybrid progeny having a combination of all desired traits and wherein the hybrid progeny performs better than individual parental lines; performing the FAB technology based protocol to generate the genome-wide correlation data for all parental samples; converting the genome-wide relation data into a cladogram using a GeneMapper; and analyzing the cladogram to identify and choose most distantly related parent plant material suitable for a heterosis breeding program.