eChapters

Abstract

Spot blotch of wheat is devastating disease as one of the yield limiting factor especially in north eastern plains zone and also in north western plains zone of India. Chemical fungicides play an important role in controlling the spot blotch in India due to non availability of spot blotch resistant commercial varieties. To identify the suitable chemicals method and time of application, various fungicides thiram 37.5% WP, carboxin 37.5% WP +, propiconazole 25% EC, tebuconazole 25% EC and mancozeb 75% WP) were evaluated either as seed treatment or foliar sprays or in combination under artificial epiphytotic in vivo conditions for two crop seasons. Seed treatment (ST) by thiram + carboxin @ 2.5 g/kg of seed along with Foliar spray (FS) of propiconazole @ 0.1% at boot leaf stage on Flag-1 leaf followed by second spray at 20 days interval (two sprays) resulted in best management of spot blotch in severe conditions. It was found that the fungicide treatment not only reduced the spot blotch disease but also increases the grain yield.

Abstract

The present investigation was carried out in okra consisting of 11 diverse genotypes (8 lines & 3 testers) and their 24 F1 hybrids. Phenotypic and genotypic correlation indicated positive association of average fruit length with average fruit weight while negative association between days taken to first flower with number of fruits per plant. The maximum positive direct effect on yield was due to number of fruits per plant followed by average fruit weight. The F₁ hybrid VRO-6 × Parbhani Kranti exhibits highest negative heterobeltiosis for days taken to 1^st flowering while D-1-87-5 × Pusa A-4 and Arka Abhay × Parbhani Kranti are earliest heterobeltiotic hybrids for days taken to 1st harvest. The hybrid Arka Abhay × Pusa A-4 represents highest heterobeltiosis for average fruit length, number of fruits/ plant and average yield. Among all parents, only Akra Abhay exhibits better GCA effect. The highest SCA effect for average yield observed in VRO-6 × Arka Anamika, while VRO-6 × Pusa A-4 for per cent infestation of YVMV. The cross Arka Abhay × Pusa A-4 incurred the highest net returns/ ha and B: C ratio (3.21) followed by that of D-1-87-5 × Parbhani Kranti and the parent Hisar Unnat gave the higher net returns/ ha with B: C ratio of 2.02 among the parents. These elite hybrids may be tested for yield and other quality traits under different agro-climatic conditions for commercial exploitation of hybrid vigour.

Abstract

The agricultural scientists particularly those who are in advisory service, irrespective of specialization, are often approached by the farmers with diseased samples of their crops and sought advice on plant protection strategy. The strategy includes components of control measures, doses, mode and time of application. In the present article different steps of formulation of plant disease control strategy are discussed. To further elaborate the contents, examples from integrated management strategy of mango malformation disease is cited.

Keyword: Mango Malformation

Introduction

Mango malformation, disease was first time reported from Darbhanga District of Bihar State in 1891 by G. Watt. The disease is currently prevalent in almost all the mango growing country and is thus designated as the plant disease of International importance and previously considered to be a serious problem in the northern parts of the country, has now started to appear in central India too in severe form. It is well known, the mango malformation is the most serious disease, rendering mango cultivation non-lucrative in subcontinent and other regions of the world (Ahmad et al., 2002; Ploetz, 2001). Mango malformation caused by fungus Fusarium moniliforme var. Subglutinans, are proved (Freeman 1999; Zheng and Ploetz, 2002). Recently, these isolates were described as members of a new species, F. mangiferae Britz, Wingfield and Marasas sp nov. (Britz et al., 2002). The casual organism of mango malformation grows well at lower temperature and higher relative humidity. The disease is quite common in both grafted and the seedling plants (Kumar and Chakrabarti, 1997). The symptoms manifest as bunchy top (BT) in 4-5 month old seedlings in the nursery, vegetative malformation (VM) of twigs arising from axillary buds and malformation of inflorescence (MF). Normally floral malformation is more prevalent in bearing trees than vegetative malformation. Flowers in the malformed panicle are much enlarged and crowded with hypertrophied axes of the panicle. Malformed inflorescences/ panicle of different varieties were variable in respect of their shapes, sizes, growth and compactness, depending upon the cellular structure and physiology under environmental factors (Chakrabarti et al., 1990). Generally malformed inflorescences produce no fruit, or abort already stages (Schloser, 1971) and is directly responsible for reduction in yield (Kumar et al., 1993 and Singh et al., 1998). In the present study the incidence of Floral malformation on a large number of mango cultivars was recorded over a period of 5 years with a view to identify the entries of future resistant breeding programme.

Abstract

Mushroom production is emerging as an added dimension in agriculture diversity; its cultivation lies on the utilization of agricultural wastes rich in proteinaceous materials. Five different substrates such as wheat straw, maize cob, paddy straw, banana pseudo stem, and lentil straw were used with suitable supplements to evaluate the average period of spawn run, pin head initiation, time taken for harvesting of flushes, fruit yield during both crop season 2014 and 2015. In 2014, highest fruit yield (380.3 g/ 500g) were recorded when wheat straw supplemented with rice bran (2% of the substrate weight), corn flour @ 25 g/kg prepared straw and gypsum @ 20 g/kg straw among various used substrates. This treatment also resulted better in respect of time taken for spawn run (9.3 days), pin head initiation (11.7 days), time taken for harvesting of flushes (I^st flush- 20.5, II^nd flush-26.7, III^rd flush- 32.9). The second best treatment was paddy straw when supplemented with rice bran (2% of the substrate weight), corn flour @ 25 g/kg prepared straw, and gypsum @ 20 g/kg. The lowest fruit yield (193.6 g/500g), time taken for spawn run (15.9 days), pin head initiation (22.4 days), time taken for harvesting of flushes (I^st flush- 27.9, II^nd flush-37.1, III^rd flush- 44.7) were recorded with banana pseudostem (chopped) when supplemented with rice bran (2 % of the substrate weight), corn flour @ 25 g/kg prepared straw and CaCO₃ gypsum @ 20 g/kg straw. The same experiment was repeated during the year crop season 2015. Surprisingly wheat straw supplemented with rice bran (2% of the substrate weight), corn flour @ 25 g/kg prepared straw and gypsum @ 20 g/kg prepared straw was found again superior over other treatments with more or less similar performance. Similarly more or less same performances were resulted in rest of the treatment too. The incidence of mould infection in mushroom was also noticed in accordance with the kinds of substrates used during both crop season 2014 and 2015. Lowest incidence of mould i.e. green mould (5.13 %), black mould (3.10 %) and blue mould (2.42%) was observed on wheat straw when supplemented with rice bran (2% of the substrate weight), corn flour @ 25 g/kg prepared straw and gypsum @ 20 g/kg straw, green mold (3.13 %), black mold (3.10 %) and blue mold (2.42 %). Similarly more or less same pattern of mould incidence was reported in the crop season 2015.

Abstract

Potato (Solanum tuberosum L.), an important cash crop of India, is prone to many diseases and amongest these, late blight [Phytophthora infestans (Mont) de-Bary] is one of the highly destructive diseases and a major constraint in the profitable cultivation of potato. After realizing the significance of controlling the devastating disease for the sustainability in potato production and thus ensuring more profitability, the present investigation has been conducted with an objective to select the most effective fungicides and botanicals for the management of the disease. The present study was carried out by KVK, Saharsa during rabi season for consecutive two years (2015-16 and 2016-17) as On-Farm Trial mode at farmers fields of an adopted village of Saharsa district in Bihar. In each trial, there were four treatments including one control i.e. farmers’practice. The study concludes that the best treatment option to combat late blight disease is Seed treatment with Trichoderma viride @ 5g per kg of seed and spray of Ridomil @ 2g per litre of water starting from 30 DAP alternating with Dimethomorph 1g + ridomil 2g per litre of water at 10 days intervals. The results of economic analysis reveal that the net return as well as benefit cost ratio was also highest in Seed treatment with Trichoderma viride @ 5g per kg of seed and spray of Ridomil @ 2g per litre of water starting from 30 DAP alternating with Dimethomorph 1g + ridomil 2g per litre of water at 10 days intervals.

Introduction

The term biological control was first used in scientific literature in 1914. Baker and Cook (1974) described biological control as “reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state, by one or more organisms, accomplished naturally or thoroughly manipulation of environment, host, or antagonists or by mass introduction of one or more antagonists.”

Certain genera of fungi have greater potential for being effective bio-control agents eg. Trichoderma, Gliocladium, Chaetomium; entomogenous fungi as Entomophthora, Beauveria, Metarrhizium, Hirsutella; Nematophagous viz., Catenaria, Zoopage, Stylopage, Paecilomyces and predaceaus fungi like Arthrobotrys, Dictylella, Dactylaria and Nematoctonus (Narain, 2018). Biopesticide formulations based on these fungi are also called as “Microbial pesticides”.

Abstract

Palms are generally grown commercially in tropical climate. But some of the palms like coconut, areca nut and palmyra palm are cultivated successfully in West Bengal, Orissa, Assam, and some pockets of Bihar that lies under sub tropics of India.Due to unsuitable climatic factor their production is more affected by abiotic and biotic stress including different kinds of diseases. The important disease are bud rot, basal stem end rot, nut shedding,stem bleeding in coconut, mahali disease, foot rot, stem breaking in arecanut and palmyra palm in the region have negligible incidence of diseases. These destructive diseases are the major constrain in production and causes severe yield loss in these palm fruits. Therefore effective management strategies are required by continuous monitoring the incidence and severity of these diseases caused by different pathogens.

Integrated disease management is the most accepted strategies for sustainable crop production that involves the holistic approach integrating cultural, physical, mechanical, chemical and biological means to control the disease. However, results of the disease management trials depend upon soil condition, age of the crop, stage of disease development and weather parameters of that particular area. Although it is impossible to manage a field free of pathogen but considerable reduction of the disease can be achieved through a proper integrated disease management system. Thus the causes and remedy of the diseases prevalent mainly in palms particularly in subtropical conditions has been reviewed for better management and sustainable production.

Abstract

Fungal rot and wilt caused by Sclerotinia sclerotiorum is a devastating disease of vegetables causing huge loss. The fungus has a broad host of more than 400 species including diverse vegetable crops, viz., carrot, potato, tomato, green pepper, eggplant, lettuce, peas and beans, cole crops, bottle gourd, pointed gourd, etc. Being able to overwinter in the soil in form of sclerotia upto 5 years and having such a wide host range, management of the disease becomes very difficult. Thus development of resistant varieties can be the only long term strategy for control of the pathogen. The screening of the existing genotypes and identification of resistant genotypes forms the base of any resistant breeding programme. The widely available genotypes of different crops have been screened by several researchers and resistant genotypes have been identified in cauliflower, vegetable soybean, peas, tomato, etc. These genotypes are further used in breeding programmes for as donors for resistance to Sclerotinia sclerotiorum.

Keywords: Vegetables, Sclerotinia, Resistance breeding, Genotypic screening

Abstract

India ranks first among world’s mango producing countries accounting for about 50 % of the world’s total mango production. In Bihar it is the most important fruit crop as well as occupies a large area affecting socio economic conditions of the region. The crop suffers from over 140 diseases all over the world. From young seedlings to mature trees and fruits in their all stages from start to storage may suffer from a number of biotic and abiotic stresses. All parts of the plant namely, trunk, branch, twigs, leaf, petiole, flower and fruits are prone to attack by a number of pathogens The changes in climatic patterns and shifting towards commercial cultivation of this crop has seen the emergence of some diseases which can cause huge losses to the production of mango over the years. One such problem, decline in mango is reported to be a complex problem involving multitude of factors. Decline disorders are recognized in virtually all mango-producing regions of the world. In most cases, the disease has been characterized by leaf drooping and drying; bark splitting, gum secretion from branches and main trunk, bud necrosis, dieback, vascular discoloration and death of the whole tree. The bark beetle Hypocryphalus mangiferae is closely associated with trees affected by mango sudden decline disease. Report suggests the causal agent to be Botryodiplodia theobromae, Ceratocystis manginecans, Fusarium spp, Colletotrichum sp, Pestalosiopsis maniferae. In most of the cases Botryodiplodia theobromae, Ceratocystis spp are associated with it. Although, none of the pathogens isolated could singly incite the complete decline symptoms under artificial inoculation on mango seedlings indicating towards complex etiology of the disease. Since pathogens are endophytic in nature; mangement of the pathogen after infection is difficult and relies more upon prohytalctic measures. Over all plant health, its nutritional status and abiotic conditions of flood and drought are also the pre disposing factors to decline in Mango. Effective management of decline in mango needs holistic approaches using optimum nutrition, general sanitation, routine crop protection schedule, weed management and appropriate intercropping practices.

Abstract

Onion (Allium cepa L.) and garlic (Allium sativum L.) are important members of the family Amaryllidaceae. Bulbs, which are the edible component in both onion and garlic grow underground and are of nutritional and medicinal importance. These underground bulb vegetables are affected by many diseases caused by different microorganisms like fungi, bacteria, nematode and viruses causing huge economic loss. Stemphylium leaf blight caused by Stemphylium vesicarium, is the major fungal disease affecting their productivity of the edible bulbs adversely.The fungus primarily attacks the leaves but can affect bulbs in storage. Initial infection is characterised by appearance of small greyish white oval spots on the leaves. These spots are often surrounded by a yellowish halo with irregular margins. Fungal pseudothecia may over-winter in contaminated plant material or may survive in the soil having plant debris and ascospores are the primary inoculums in the following season. Spores require rain or persistent dew to cause infection. This disease can spread rapidly when environmental conditions are favourable for spore development. Suitable integration of more efficient treatments like fungicides, bio agents in combination with appropriate cultural control provide better management of the Stemphylium blight disease

Abstract

Bacterial diseases of solanaceous crops like tomato, brinjal, chilli, capsicum and potato are the most serious and destructive diseases affecting both under field and greenhouse conditions. Under moist field conditions they can cause epidemics to affect total crop loss, quality of the produce and reduce market value. These diseases can be distinguished from each other on the basis of their symptoms appearance of the various parts of the plant as well as by using advanced techniques of diagnosis. Diagnosis of the disease is important step to detect pathogen at early stage of disease and subsequently manage it effectively in efficient manner.

Keywords: Bacterial diseases, solanaceous crops, symptoms, diagnosis.

Introduction

Bacterial diseases of solanaceous crops like tomato, brinjal, chilli, capsicum and potato are the most serious and destructive diseases affecting both under field and greenhouse conditions. Under moist field conditions they can cause epidemics to affect total crop loss, quality of the produce and reduce market value. These diseases can be distinguished from each other on the basis of their symptoms appearanceofthe various parts of the plant as well as by using advanced techniques of diagnosis. Diagnosis of the disease is important step to detect pathogen at early stage of disease and subsequently manage it effectively in efficient manner. Therefore, the early detection of the bacterium from asymptomatic plants, soil, and water and plant residues could facilitate elimination and certainly reduce the risk of crop loss. Powerful tools for the identification and detection of the bacterium in diverse substrates (plant, seed, water and soil) are available for diagnosis of the bacterial diseases. DNA amplification of pathogen offers many advantages over classical techniques; neither purification nor cultivation of the pathogen is required and the specificity, sensitivity and response time of tests are improved. Nevertheless, the PCR method has not yet become a routine diagnostic tool for many laboratories, mainly because of the inhibition of the amplification reaction by compounds contained in crude bacterial extracts, which give false negative results or low detection sensitivity.

Abstract

Information and Communication Technology (ICT) played and continue to play pivotal role in better decision making of farmers for plant health management under the climate change scenario. For judicious plant health management by farmers through the use of ICT tools (viz. interactive Kiosk, Mobile Apps, Whatsapp, Webportal), information need and information access network to be assessed. Based on farmers’ information need and network, information delivery tree should be designed in ICT tools. Under this backdrop this research work was carried out among the farmers of Bhagalpur district of Bihar. The sample size was 120. From the study it was noted that the major information needs of the farmers were seed availability, crop disease management, insect pest management, weed management and weather information and pesticide application. Farmers were enthusiastic to improve their knowledge on insect pests and disease identification and management. Specifically, farmers need information on insect pest management of wheat (aphid), maize (stem borer), vegetables (fruit borer), pulses (pod borer) and oilseeds (aphid) and also disease management of wheat (leaf blight and spot blotch disease), maize (Turcicum leaf blight), vegetables (wilt and leaf spot) and pulses (wilt disease). Farmers decision making was positively and significantly correlated with crop disease management(0.196*),insect pest management of crop(0.208*) and weed management(0.239**). Through the Path Analysis it was noted that farmers decision making on disease management was mediated by the variables - Bihar Agricultural University-Kisan Diary, Mobile Apps(Bihar Krishi Apps) and Kisan Salahkar. So, it can be concluded that these much needed information should be provided to the farmers though different ICT tools and extension personnel should be trained to operate these ICT tools for accelerated use of ICT tools by the farmers.

1. pH meter

pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples.

What is DNA

DNA is a simple molecule

DNA is negatively charged

DNA has lots of ring structures; ring structures are generally quite hydrophobic.

DNA is a very robust molecule.

DNA work has lots of toys.

Although it can be argued that due to its overall trendiness, they have been a lot of recent developments in making it more fun to work with. As a molecule, it is very similar to DNA (the most notable chemical difference is its flexibility), but suffers because the nucleases (RNases) that can destroy it are notoriously very stable and very difficult to get rid of.

Electrophoresis is an analytical method frequently used in molecular biology and medicine. It is applied for the separation and characterization of proteins, nucleic acids and subcellular-sized particles like viruses and small organelles. Its principle is that the charged particles of a sample migrate in an applied electrical field. An electric current is passed through a medium containing the molecules, and each molecule travels at a different rate depending on its electrical charge, size and shape. Separation by electrophoresis is based on these differences. In electrophoresis, agarose and acrylamide gels are used for electrophoresis of nucleic acids and proteins.

What is Gel Electrophoresis?

A gel is a solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule’s properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

Two types of molecular weight (MW) standards are routinely used. The Lambda/HindIII and PhiX174/HaeIII MW standards provide a useful reference for calculating molecular weights of large and small DNA fragments, respectively, after electrophoretic separation; the “internal MW standards” provide a means for normalizing fragment migration distances within each lane to facilitate comparisons between lanes on the same or different luminographs in fingerprinting studies.

Polyacrylamide

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymer- ization process, they must be poured between glass plates (or cylinders).

Proteins

Proteins are of central importance in the structure and function of all living organisms. Some proteins serve as structural components while others function in communication, defense, and cell regulation. Some proteins serve as enzymes that act as biological catalysts which control the pace and nature of essentially all biochemical events. Although DNA serves as the genetic blueprint of a cell, none of the life processes would be possible without the proteins.

Electrophoresis of Proteins

Proteins can be separated and purified. Methods for separating proteins take advantage of properties such as charge, size, and solubility, which vary from one protein to the next. Because many proteins bind to other biomolecules, proteins can also be separated on the basis of their binding properties. The source of a protein is generally tissue or microbial cells. The cell must be broken open and the protein must be released into a solution called a crude extract. If necessary, differential centrifugation can be used to prepare subcellular fractions or to isolate organelles. Once the extract or organelle preparation is ready, a variety of methods are available for separation of proteins. Ion-exchange chromatography can be used to separate proteins with different charges (similar to the way amino acids are separated). Other chromatographic methods take advantage of differences in size, binding affinity, and solubility. Nonchromatographic methods include the selective precipitation of proteins with salt, acid, or high temperatures. In addition to chromatography, another important set of methods is available for the separation of proteins, based on the migration of charged proteins in an electric field, a process called (gel) electrophoresis. Gel electrophoresis is especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of proteins in a mixture or the degree of purity of a particular protein preparation. Also, gel electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight.

Hunter and Mohler discovered the isozymes. In 1959 Markert and Moler introduced the concept of isozymes, which they defined as the different molecular forms in which proteins may exist with the same enzymatic specificity (Buth 1984). This means that different variants on the same enzymes have identical or similar functions and are present in the same individual. As such, their importance for understanding gene action in development and differentiation was exploited during the1960s in both animals and plants. Nevertheless, isozymes played a minor role in research on plant biochemistry until 1966 when genetic polymorphism for isozymes within the same population was discovered (Stebbins 1989, Wendel 1989). That revealed the possibility for population genetics to make precise quantitative estimates of genetic variability based upon one parameter of the molecular structure of the primary products of the genes themselves.

Plant population genetics were not long in following their zoological colleagues and the investigation of both animals and plants increased explosively. This application, however, necessitated a partitioning of the isozyme concept, since the relevant isozyme subset, defined by Prakash (Buth 1984) as the variant proteins produced by allelic forms of the same locus, to avoid the now common confusion with isozymes which are the various polymer produced from monomers specified by different loci. The development of genetic distance or similarity coefficients (Lukasová 1985) allowed the summarization of isozyme data for intersample comparisons and so quantified isozyme data were soon applied to comparative studies of taxa.

There are many different methods of extracting DNA bands from an agarose gel. Which one you choose will probably depend on the consumables you have available in your lab. Another important consideration is the yield/ purity of the DNA after extraction. Agarose contains various impurities which may inhibit downstream reactions if not efficiently removed from the DNA. We used to give the generic term ‘hoonose’ to the unspecified carbohydrates that co-purified along with the DNA and inhibited enzyme reactions. Modern spin-column kits are very good at removing impurities, old-fashioned ‘kitchen sink’ methods are less good.

Cutting Out the DNA Band

Most of the methods require you to cut out the band. You must visualise the band, in an ethidium bromide stained gel, in a dark-room on a UV light- box (a trans-illuminator). UV is dangerous, wear gloves, long-sleeves and face protection. Never look at UV with unprotected eyes. If possible, set the trans-illuminator to long-wavelegth UV (or low-power) and minimise the amount of time the DNA is exposed. This is because the UV mutagenises the DNA at a measurable rate. Use a scalpel blade to cut around the band of interest. Switch off the transilluminator, switch on the white light and carefully remove the band from the gel and place it on the glass. It is good to trim off as much empty agarose as possible so go back to UV illumination briefly to do this. Place the excised band in a 1.5mL microfuge tube.

Agriculture Scenario

It has been seen since last few decades there is huge decrease in the production of the agricultural production. Three factors which can be held responsible for the increase of agricultural production per hectare:

1. Overall increase in efficiency due to the merger of farms and a more efficient redistribution of farm land.
2. More efficient cultural practises and better quality and availability of inputs (fertilizer, pesticides).
3. Genetic improvement of crops.

At this moment over 6 billion people need to be fed and it is expected that in the year 2050 more than 10 billion people will inhabit the earth (FAO, 1996). Moreover, demands per capita will rise when standards of living in developing counties improve. As a consequence there will be a huge increase in the demand for food, and production will need to triple in the coming 40 years. A continued effort to secure agricultural production in the future will therefore be vital. Reduction of losses caused by a-biotic and biotic stress will be a key issue. Scenario studies on world food security generally assume that a large increase in production will be achieved by genetic improvement of crop species and biotechnology. History has indeed shown a continuous increase in crop yields, resulting from plant breeding efforts. However, in the light of the speed at which the human population develops, and taking into account the expected reduction of available arable land due to climate change and human intervention, it may be necessary to accelerate the rate at which genetic improvement is achieved. Modern biotechnology provides new tools that can facilitate development of improved plant breeding methods and augment our knowledge of plant genetics. The knowledge that is obtained with these new tools can be used to contribute to an enhanced food security throughout the world.

Restriction Enzymes

Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. Another application of restriction enzymes is to map the locations of restriction sites in DNA.

Restriction endonucleases, often called restriction enzymes, cut DNA within the molecule by hydrolyzing the phosphodiester bonds between the nucleotides. Restriction enzymes were discovered in bacteria and there are now more than 1200 known restriction enzyme types. These enzymes are named using a simple system. EcoRI, for example, was isolated from E. coli and was the first enzyme isolated from a particular strain, hence the designation of I. HaeIII was isolated from Haemophilus aegyptius. HindIII was isolated from Haemophilus influenzae, and was the third enzyme discovered in a particular strain.

Usually, restriction enzymes only cut the DNA at or near a very specific nucleotide sequence known as a recognition site. This “restrictive” nature of these enzymes allows molecular biologists to pin-point exactly where the DNA is to be cut. In many cases, the recognition site of a restriction enzyme consists of a palindromic sequence where the order of the bases is read the same on each side of the helix. The two sides of the helix are antiparallel and the recognition sequence reads from 5’ to 3’ on either side of the helix. For example the recognition site for EcoRI is as follows;

Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) technique, invented in 1985 by Kary B. Mullis, allowed scientists to make millions of copies of a scarce sample of DNA. The technique has revolutionized many aspects of current research, including the diagnosis of genetic defects and the detection of the AIDS virus in human cells. The technique is also used by criminologists to link specific persons to samples of blood or hair via DNA comparison. PCR also affected evolutionary studies because large quantities of DNA can be manufactured from fossils containing but trace amounts.

Kary Mullis invented the PCR technique in 1985 while working as a chemist at the Cetus Corporation, a biotechnology firm in Emeryville, California. The procedure requires placing a small amount of the DNA containing the desired gene into a test tube. A large batch of loose nucleotides, which link into exact copies of the original gene, is also added to the tube. A pair of synthesized short DNA segments, that match segments on each side of the desired gene, is added. These “primers” find the right portion of the DNA, and serve as starting points for DNA copying. When the enzyme Thermus aquaticus (Taq) is added, the loose nucleotides lock into a DNA sequence dictated by the sequence of that target gene located between the two primers.

The test tube is heated, and the DNA’s double helix separates into two strands. The DNA sequence of each strand of the helix is thus exposed and as the temperature is lowered the primers automatically bind to their complementary portions of the DNA sample. At the same time, the enzyme links the loose nucleotides to the primer and to each of the separated DNA strands in the appropriate sequence. The complete reaction, which takes approximately five minutes, results in two double helices containing the desired portion of the original. The heating and cooling is repeated, doubling the number of DNA copies. After thirty to forty cycles are completed a single copy of a piece of DNA can be multiplied to hundreds of millions.

Allele-Specific PCR

This diagnostic or cloning technique is used to identify or utilize single- nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.

Amplification Refractive Mutation System (ARMS) - PCR

In ARMS, the primer pair is designed so that one of the 3' ends coincides with a variant nucleotide in the target sequence. When the primer mismatches the template the frequency of extension is very low and consequently the effective number of sequence copies available for amplification is greatly reduced. ARMS PCR exploits a thermostable polymerase that lacks 3' exonuclease activity (usually Taq polymerase). Such enzymes extend primers bound to their target sequences very inefficiently when the 3' base is mismatched. Because the 3' exonuclease activity required for mismatch repair is not present, the extension of such primers in PCR is a rare event and amplification is retarded. However, once mismatch extension has occurred amplification proceeds normally from the newly synthesised target strand. Thus an important principle of ARMS is that non-matching (i.e. non-matching at only the 3' end of one primer) template may be amplified. The factors that contribute to this apparent ‘failure’ of ARMS are that when very high numbers of template molecules are added to the reaction, amplification is likely to proceed because the small proportion of mismatched primer extensions reach the sensitivity threshold of the reaction. Second, certain mismatches are extended more efficiently than others so that proportion extended in each PCR cycle varies.

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.

No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD is used to characterize, and trace, the phylogeny of diverse plant and animal species.

In 1985, Sir Alec Jeffreys developed restriction fragment length polymorphism (RFLP), which quickly became the standard technique for DNA testing throughout the 1980s. RFLP provided the world with the first form of genetic testing based on DNA, the body’s genetic material.

Definition : RFLP analysis is a difference in homologus DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the samples in question with specific restriction endonucleases (RE).

A restriction fragment length polymorphism , or RFLP, (commonly pronounced “rif lip”), is a variation in the DNA sequence of a genome that can be detected by breaking the DNA into pieces with restriction enzymes and analyzing the size of the resulting fragments by gel electrophoresis. It is the sequence that makes DNA from different sources different, and RFLP analysis is a technique that can identify some differences in sequence (when they occur in a restriction site). Though DNA sequencing techniques can characterize DNA very thoroughly, RFLP analysis was developed first and was cheap enough to see wide application. Analysis of RFLP variation was an important tool in genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting, and paternity testing.

Introduction

The AFLP amplified fragment polymorphism also known as selective restriction fragment amplification (SRFA) technique is used to visualize hundreds of amplified DNA restriction fragments simultaneously. The AFLP band patterns, or fingerprints, can be used for many purposes, such as monitoring the identity of an isolate or the degree of similarity among isolates. Polymorphisms in band patterns map to specific loci, allowing the individuals to be genotyped or differentiated based on the alleles they carry. produces highly complex DNA profiles by arbitrary amplification of restriction fragments ligated to double-stranded adaptors with hemi-specific primers harboring adaptor-complementary 5' termini.The technique has been widely used in the construction of genetic maps containing high densities of DNA marker loci.

The AFLP protocol amplifies restriction fragments obtained by endonuclease digestion of target DNA using “universal” AFLP primers complementary to the restriction site and adapter sequence. However, not all restriction fragments are amplified because AFLP primers also contain selective nucleotides at the 3' termini that extend into the amplified restriction fragments. These arbitrary terminal sequences result in the amplification of only a small subset of possible restriction fragments. The number of amplified fragments can therefore be “tailored” by extending the number of arbitrary nucleotides added to the primer termini. Alternatively, the use of endonuclease combinations that vary in their restriction frequency can also be used to tune the number of amplicons. Generally, the abundant restriction fragments produced from complex genomes require of AFLP primers with longer selective regions. Conversely, analysis of small genomes require of only few arbitrary nucleotides added at the primer 3' termini. The resulting AFLP fingerprints are usually a rich source of DNA polymorphisms that can be used in mapping and general fingerprinting endeavors.

Also Referred to as Microsatellites

SSR markers are based short sequences of nucleotides (2-6 units in length) that are repeated in tandem:

Di-nucleotide repeat: CACACACA

Tri-nucleotide repeat: ATGATGATGATG

It is believed that when DNA is being replicated, errors occur in the process and extra sets of these repeated sequences are added to the strand. Over time, these repeated sequences vary in length between one cultivar and another.

Minisatellite is a section of DNA that consists of a short series of bases 10– 60bp. These occur at more than 1000 locations in the human genome. Some minisatellites contain a central (or “core”) sequence of letters “GGGCAGGANG” (where N can be any base) or more generally a strand bias with purines (Adenosine (A) and Guanine (G)) on one strand and pyrimidines (Cytosine (C) and Thymine (T)) on the other. It has been proposed that this sequence per se encourages chromosomes to swap DNA. In alternative models, it is the presence of a neighbouring cis-acting meiotic double-strand break hotspot which is the primary cause of minisatellite repeat copy number variations. Somatic changes are suggested to result from replication difficulties (which might include replication slippage, among other phenomena). When such events occur, mistakes are made, this causes minisatellites at over 1000 locations in a persons genome to have slightly different numbers of repeats, thereby making them unique. The most highly mutable minisatellite locus described so far is CEB1 (D2S90) described by Vergnaud.

Since the mid 1980s, genome identification and selection has progressed rapidly with the help of PCR technology. A large number of marker protocols that are rapid and require only small quantities of DNA have been developed. Three widely-used PCR-based markers are RAPDs (Williams et al., 1990), SSRs or microsatellites (Tautz, 1989), and AFLPs (Vos et al., 1995). Each marker technique has its own advantages and disadvantages. RAPD markers are very quick and easy to develop (because of the arbitrary sequence of the primers) but lack reproducibility (Karp et al., 1997; Hansen et al., 1998; Jones et al., 1999; Virk et al., 2000). AFLP has medium reproducibility but is labour intensive and has high operational and development costs (Karp et al., 1997). Microsatellites are specific and highly polymorphous (Karp et al., 1997, Jones et al., 1999), but they require knowledge of the genomic sequence to design specific primers and, thus, are limited primarily to economically important species.

The choice of a molecular marker technique depends on its reproducibility and simplicity. The best markers for genome mapping, marker assisted selection, phylogenic studies, and crop conservation have low cost and labour requirements and high reliability. Since 1994, a new molecular marker technique called inter simple sequence repeat (ISSR) has been available (Zietkiewicz et al., 1994). ISSRs are semiarbitrary markers amplified by PCR in the presence of one primer complementary to a target microsatellite. Amplification in the presence of nonanchored primers also has been called microsatellite-primed PCR, or MP-PCR, (Meyer et al., 1993).

SCARs are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific 15-30 bp primers, designed from nucleotide sequences established in cloned RAPD (Random Amplified Polymorphic DNA) fragments linked to a trait of interest. By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered with RAPDs. Obtaining a codominant marker may be an additional advantage of converting RAPDs into SCARs. However, SCARs may exhibit dominance when one or both primers partially overlap the site of sequence variation. SCARs are locus specific and have been widely applied in gene mapping studies and marker assisted selection (Paran & Michelmore, 1993).

Synonym : PCR-RFLP (PCR- Restriction Fragment Length Polymorphism)

The Cleaved Amplified Polymorphic Sequence or CAPS method is a technique in molecular biology for the analysis of genetic markers. It is an extension to the Restriction Fragment Length Polymorphism (RFLP) method, using polymerase chain reaction (PCR) to more quickly analyse the results.

Like RFLP, CAPS works on the principle that genetic differences between individuals can create or abolish restriction endonuclease restriction sites, and that these differences can be detected in the resulting DNA fragment length after digestion.

In the CAPS method, PCR amplification is directed across the altered restriction site, and the products digested with the restriction enzyme. When fractionated by agarose or acrylamide gel electrophoresis, the digested PCR products will give readily distinguishable patterns of bands. Alternatively, the amplified segment can by analyzed by Allele specific oligonucleotide (ASO) probes, a process that can often be done by a simple Dot blot.

Single strand conformation polymorphism (SSCP), or single strand chain polymorphism, is defined as conformational difference of single stranded nucleotide sequences of identical length as induced by differences in the sequences under certain experimental conditions. This property allows to distinguish the sequences by means of gel electrophoresis, which separates the different conformations.

Background

A single nucleotide change in a particular sequence, as seen in a double stranded DNA, cannot be distinguished by electrophoresis, because the physical properties of the double strands are almost identical for both alleles. Contrarily, after denaturation, single stranded DNA undergoes a 3- dimensional folding and may assume a unique conformational state based on its DNA sequence. The difference in shape between two single-stranded DNA strands with different sequences can cause them to migrate differently on an electrophoresis gel, even though the number of nucleotides is the same. Predictability, however, is a shortfall of SSCP - since conformational states are subject to many experimental conditions and sequence differences that cause minimal changes in strand conformation may not be detected.

What are Retrotransposons?

The presence of mobile elements, capable of moving from one region of the genome to another, has been known and thoroughly studied since the 1950’s. These elements are capable of exciding and inserting themselves from one region of the chromatin to another, through their peculiar terminal repeat regions. This process is mediated by enzymes encoded by the transposon itself.

Retrotransposons are a class of mobile elements, widespread in plant genomes, where they account for a significant part of noncoding DNA. Barley, for example, contains thousands of copies of the Bare-1 element, dispersed throughout the euchromatic regions of chromosomes and exhibiting sequence identity higher than 95% to each other.

Unlike other classes of mobile elements, retrotransposons insert into the genome via an RNA intermediate, synthesized by their own reverse transcriptase, without the loss of the parental copy. Unique sequence properties show that retrotransposons of the Ty1-copia group are close to retroviruses, and their mode of integration in the host genome is also very similar.

The number and exact point of insertion of retrotransposons may differ from one individual to another and this can be exploited to characterize individuals, varieties or species, depending on how recent the transpositional event is. Furthermore, insertion of a retrotransposon is an irreversible event, a characteristic that can be helpful when tracing an ancestry.

Silver Staining for Protein

Silver staining is one of the procedures, in addition to Coomassie Blue, R and G types, and fluorescent dyes that are available for detecting proteins separated by gel electrophoresis. Switzer introduced silver staining in 1979, a technique that today provides a very sensitive tool for protein visualization with a detection level down to the 0.3-10 ng level.

The basic mechanisms underlying silver staining of proteins in gels are relatively well understood. Basically, protein detection depends on the binding of silver ions to the amino acid side chains, primary the sulfhydril and carboxiyl groups of proteins, followed by reduction to free metallic silver. The protein bands are visualized as spots where the reduction occurs and, as a result, the image of protein distribution within the gel is based on the difference in oxidation-reduction potential between the gel’s area occupied by proteins and the free adjacent sites. A number of alteration in the silver staining procedure can shift the oxidation-reduction equilibrium in a way that gel- separated proteins will be visualized either as positively or negatively stained bands. Silver staining protocols can be divided into two general categories: 1) silver amine or alkaline methods, and 2) silver nitrate or acidic methods. In general, the detection levels’ using the various procedures is determined by how quickly the protein bands develop in relationship to the background (e.g. signal-to-noise ratio). The silver amine or alkaline methods usually have lower background and as a result are most sensitive but require longer procedures. Acidic protocols, on the other hand, are faster but slightly less sensitive. Clearly, each protocol has different advantages regarding timing, sensitivity, cost, and compatibility with other analytical methods, especially mass-spectrometry (MS) a tool that is being used in combination with gel electrophoresis or chromatographic methods for rapid protein identification. Until recently most of the silver staining protocols used either glutaraldehyde- of formaldehyde-based sensitizes in the fixing and sensitization step, thus, introducing the chemical modifications into proteins. The utilization of those chemicals causes the cross-linking of two lysine residues within protein chain, thus, strongly affecting two most crucial steps for the subsequent MS analysis: hampering the trypsin digestion and highly reduces the protein extraction from the gel .

Deoxyribonucleic acid (DNA) is a molecule made up of pairs of building blocks called nucleotides. The four kinds of nucleotides that make up DNA are adenine (abbreviated as the single letter A), guanine (G), cytosine (C), and thymine (T). The DNA molecule has the shape of two intertwined spirals, referred to as a double helix. DNA is packaged into chromosomes that are located within the nucleus of all cells. These chromosomes are the same in every cell of an organism and together make up the organism’s genetic information, its genome. Chromosomes contain stretches of DNA called genes that code for amino acids that make proteins. It is the proteins that are the foundation of life for all organisms. The interaction and structure of proteins determine the visible characteristics or phenotype of an organism, while the genetic makeup of an organism is called its genotype.

The sequence of nucleotides that make up a gene can differ among individuals. The different forms of a gene are called alleles. The alleles are the result of nucleotide differences in a gene that affect an amino acid sequence of a protein. This can result in a change, addition, or deletion of a protein that can affect the phenotype.

Genetic Mapping

Perhaps one of the most powerful applications of markers is genetic mapping. This technology allows researchers identify markers located within or very close to genes of interest. With this it is possible to trace these genes through generations of crosses.

If the trait in question is complex and multigenic, mapping will also be of tremendous help to dissect the various genes or loci that contribute to the overall characteristic. Determining the amount of impact each locus delivers is a process called Quantitative Trait Loci or QTL mapping.

QTL Traits

Polygenic inheritance, also known as quantitative or multifactorial inheritance refers to inheritance of a phenotypic characteristic (trait) that is attributable to two or more genes and their interaction with the environment. Unlike monogenic traits, polygenic traits do not follow patterns of Mendelian inheritance (qualitative traits). Instead, their phenotypes typically vary along a continuous gradient depicted by a bell curve. Though not necessarily genes themselves, quantitative trait loci (QTLs) are stretches of DNA that are closely linked to the genes that underlie the trait in question. QTLs can be molecularly identified (for example, with PCR or AFLP) to help map regions of the genome that contain genes involved in specifying a quantitative trait. This can be an early step in identifying and sequencing these genes.

Introduction

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

Introduction

The western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against tens of thousands of different proteins. Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.

Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).

The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting and the detection of post-translational modification of protein is termed Eastern blotting.

Northern Blot

The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.

Eastern blotting is a technique to detect protein post translational modifications (PTM) and is an extension of the biochemical technique of western blotting. There are multiple versions of the method, but in general proteins or lipids are blotted from one or two-dimensional SDS-PAGE gel on to a PVDF or nitrocellulose membrane are analyzed for post-translational protein modifications using probes that may detect lipids, carbohydrate, phosphorylation or any other protein modification. Eastern blotting should be used to refer to methods that detect their targets through specific interaction of the PTM and the probe, distinguishing them from a standard Far-western blot. The most common application is lectin blotting, however, a variety of other probes can be used for detecting different PTMs.

History and Multiple Definitions

Definition of the term Eastern blotting is somewhat confused due to multiple sets of authors dubbing a new method as Eastern blotting, or a derivative therof. All of the definitions are a derivative of the technique of Western blotting developed by Towbin in 1979. The current definitions are summarized below in order of the first use of the name, however all are based on some earlier works. In some cases, the technique had been in practice for some time before the introduction of the term.

DNA Sequencing

DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. Sequencing can be done on a short fragment of DNA to cover the entire genome. The maps thus constructed using the nucleotide sequences in the correct order is called as sequence map.

In 1977, twenty-four years after the discovery of the structure of DNA, two separate methods for sequencing DNA were developed: the chain termination method (Sanger et al., 1977) and the chemical degradation method (Maxam and Gilbert, 1977). Both methods were equally popular to begin with, but, for many reasons, the chain termination method is the method more commonly used today. In chemical degradation method, chemicals specific to each base remove the last base, which has been labelled. The method uses toxic chemicals which required high care while handling.

In chain termination method, DNA polymerase extends a DNA strand in which dideoxynucleotide becomes incorporated terminating the chain extension (phosphodiester formation) at the 3’OH site making an array of fragments differing in length. This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis.

The transfer foreign gene to the plant cell is a very crucial method to production of transgenic plant. So improvement of plant quality requires to transfer our interest gene or we can say useful for production of our interest product. To achieve genetic transformation in plants, we need the construction of a vector (genetic vehicle) which transports the genes of interest, flanked by the necessary controlling sequences i.e. promoter and terminator, and deliver the genes into the host plant. The two kinds of gene transfer methods in plants are.

1. Vector-mediated or Indirect Gene Transfer

Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used. This bacteria is known as “natural genetic engineer” of plants because these bacteria have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The foreign gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted sequences.

To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected and infected with Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The infected tissue is then cultured (co-cultivation) on shoot regeneration medium for 2-3 days during which time the transfer of T-DNA along with foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are transferred onto selection cum plant regeneration medium supplemented with usually lethal concentration of an antibiotic to selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred to root-inducing medium, and after another 3-4 weeks, complete plants are transferred to soil following the hardening (acclimatization) of regenerated plants. The molecular techniques like PCR and southern hybridization are used to detect the presence of foreign genes in the transgenic plants.

What is FISH ?

Fluorescent in situ hybridization is a technique in which single-stranded nucleic acids (usually DNA, but RNA may also be used) are permitted to interact so that complexes, or hybrids, are formed by molecules with sufficiently similar, complementary sequences. Through nucleic acid hybridization, the degree of sequence identity can be determined, and specific sequences can be detected and located on a given chromosome. Fluorescent in situ hybridization (FISH) is a powerful technique for detecting RNA or DNA sequences in cells, tissues, and tumors. FISH provides a unique link among the studies of cell biology, cytogenetics, and molecular genetics.

Principle of FISH

FISH involves the preparation of short sequences of single-stranded DNA, called probes, which are complementary to the DNA sequences the researchers wish to paint and examine. These probes hybridize, or bind, to the complementary DNA and, because they are labeled with fluorescent tags, allow researchers to see the location of those sequences of DNA. Unlike most other techniques used to study chromosomes, which require that the cells be actively dividing, FISH can also be performed on non dividing cells, making it a highly versatile procedure.

The biological diversity of each country is a valuable and vulnerable natural resource. Sampling, identifying, and studying biological specimens are among the first steps toward protecting and benefiting from biodiversity. Knowledge about biodiversity and the ability to identify organisms that comes with it are global public goods. That is, this knowledge can create benefits for all countries - benefits that no one country can create by itself.

DNA Barcoding: Documenting Biodiversity with a Gene Sequence

“DNA barcoding” is an exciting new tool for taxonomic research. The DNA barcode is a very short, standardized DNA sequence in a well-known gene. It provides a way to identify the species to which a plant, animal or fungus belongs. The Consortium for the Barcode of Life (CBOL) is promoting international partnerships that will enable people in all countries to better understand and protect their biodiversity.

DNA barcoding is a taxonomic method that uses a short genetic marker in an organism’s mitochondrial DNA to identify it as belonging to a particular species. It is based on a relatively simple concept: most eukaryote cells contain mitochondria and mitochondrial DNA (mtDNA) has a relatively fast mutation rate, which results in significant variance in mtDNA sequences between species and, in principle, a comparatively small variance within species.

In cell biology, a mitochondrion is a membrane-enclosed organelle found in most eukaryotic cells These organelles range from 0.5–10 micrometers (µm) in diameter. Mitochondria are sometimes described as “cellular power plants” because they generate most of the cell’s supply energy in form of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction, and may play a role in the aging process.

Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria; whereas in Murinae (rats), 940 proteins encoded by distinct genes have been reported. The number of proteins encoded in plant mitochondrial DNA is probably not much higher than the number encoded in mammalian mitochondria.Experiments have shown that most of the protein synthesis in isolated maize mitochondria can be accounted for by some 18-20 polypeptidesThe mitochondrial proteome is thought to be dynamically regulated. Although most of a cell’s DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

In genetics, microRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (i.e. they are non-coding RNAs); instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression. They were first described in 1993 by Lee and colleagues in the Victor Ambros lab .

MicroRNAs can be encoded by independent genes, but also be processed (via the enzyme Dicer) from a variety of different RNA species, including introns, 3' UTRs of mRNAs, long noncoding RNAs, snoRNAs and transposons.

The genes encoding miRNAs are much longer than the processed mature miRNA molecule; miRNAs are first transcribed as primary transcripts or pri- miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. These pre- miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). This complex is responsible for the gene silencing observed due to miRNA expression and RNA interference. The pathway in plants varies slightly due to their lack of Drosha homologs; instead, Dicer homologs alone effect several processing steps. The pathway is also different for miRNAs derived from intronic stem-loops; these are processed by Dicer but not by Drosha. Either the sense strand or antisense strand of DNA can function as templates to give rise to miRNA.