eChapters

When there is indication of linkage from F or testcross data through significant deviation from 9: 3: 3: 1 and 1: 1: 1: 1 ratio, it is implied that independent assortment is not taking place and this is taken as an indication of linkage. The inverse of linkage strength is the recombination value and is often symbolized by ‘p’ whose maximum value is 50% because out of four strands of a tetrad, two are always parental types and two are recombinant types when all the cells show crossing over.

Translocations

Chromosomes occasionally undergo spontaneous rupture or can be induced to rupture in high frequency by ionizing radiation. The broken ends of such chromosomes behave as though they were ‘sticky’ and may rejoin into non- homologous combinations giving rise to translocations. The most common type of translocation is reciprocal translocation which involves the exchange of segments between two non-homologous chromosomes. Translocations change only the arrangement of genes in the chromosomes, not the quality or quantity of genes. For this reason, they are sometimes referred to as chromosomal rearrangements. Individuals carrying such rearrangements should be phenotypically entirely normal unless the relations of a gene or genes to adjacent genes affect the phenotypic expression (position effect). The first translocation was discovered by Bridges in 1923 in D. melanogaster.

The genetic techniques for detecting and studying translocations will be more easily understood when the cytological phenomena produced by translocations are known. Suppose two chromosomes having respectively the genes ABCDEF and GHIJKL exchange sections and give rise to translocation chromosomes ABCJKL and GHIDEF. The individual thus formed receives from one of the parents the normal and from the other parent the translocation chromosome. Such an individual is a translocation heterozygote (Fig. 36.1).

So far, we have seen the examples in which mode of inheritance of a particular character is tied to the behaviour of the nucleus or more specifically to the chromosomes. In other words, transmission of a character and its appearance in the individuals can be predicted from the knowledge of chromosome segregation and independent assortment. This analogy gets further strengthened on the ground that the DNA has been considered as the basic genetic material and practically all the cellular DNA is localized in the chromosomes. It has also been seen that DNA does not produce an organism alone but obviously depends upon an already existing medium in order to function. Thus, genotypic effects may considerably get modified by the environment. In the cell itself, one important source of environmental effect is the cytoplasm immediately surrounding the nucleus. The components of the cytoplasm may show wide variation between the individuals and therefore it cannot be ruled out that a genotype in one cytoplasm may function somewhat differently than the same genotype placed directly into another cytoplasm. It is well known fact that the cytoplasmic contribution of one of the parents in form of an egg is considerably larger than the cytoplasmic contribution of the other parent in form of sperm. For example, cross-fertilization between two strains capable of producing both sperm and eggs, leads to contribution of ‘A’ genotype and ‘a’ egg cytoplasm in one case and of ‘B’ genotype and ‘b’ egg cytoplasm in the other case. Thus, the cross-fertilized diploid zygotes have two possible genotypic-cytoplasmic combinations, namely, AB-a, and AB-b depending upon which strain provides the egg. If the egg is contributed by the A parent, then the genotype of the progeny shall be AB-a, and if the egg is contributed by the B parent, then the genotype of the progeny shall be AB-b. If each such cytoplasm produces a unique phenotype, then AB-b would have the same b phenotype as BB-b individuals. Such effects produced by the egg cytoplasm are termed as maternal effects.

In 1868, Johann Friedrich Miescher, a young Swish medical student got excited with an acidic substance that he isolated from pus cells obtained from bandages used to dress human wounds. To obtain large quantities of pus for his experiment, Miescher began purchasing used bandages from surgical clinics in Tuebingen, Germany. He separated pus cells from bandages and treated the cells with pepsin, a proteolytic enzyme that he isolated from stomach of pigs. After pepsin treatment, he recovered an acidic substance that was named as “nuclein” by him. This nuclein was unusual in the sense that it was rich in nitrogen and phosphorous, two elements known at that time to coexist only in certain types of fat. Miescher’s paper on this investigation was published in 1871. At that time, importance of Miescher’s nuclein was not fully appreciated. Even the existence of polynucleotide chains, the key component of the acidic material in Miescher’s nuclein was not documented until 1940s. The role of nucleic acid in storing and transmitting genetic information remained un-established until 1944 and the double-helix structure of DNA was not discovered until 1953 by James Watson and Francis Crick. Even in 1953, many geneticists remained skeptical to accept the role of nucleic acid as the genetic information carrier rather than proteins as nucleic acids exhibited less structural variability than the proteins (Snustad et al., 1997).

The search for the chemical basis of genetic material began about one and half century ago. Soon after discovery of Mendelian principles of segregation and independent assortment in 1865, Friedrich Miescher (1844-1895) published a method for separating cell nuclei from the cytoplasm and from these cell nuclei, he extracted an acid material termed as “nuclein” which had unusually large amount of phosphorous. Miescher concluded that this substance was unique and was just not comparable with any other group known at that point of time. Later on nuclein, now named as “nucleic acid” was found to be associated with various proteins and the combined entities were termed as “nucleoproteins”.

Watson and Crick based on their double helical DNA model, offered to explain DNA replication by a method where each single strand of DNA is template or mold for its complement and a new helix has one old strand and one newly synthesized strand. This type of DNA replication is called as semi-conservative as in this method one old strand is retained and one new complementary strand is synthesized. In contrast to semi-conservative mode of DNA replication, two more modes of DNA replication were proposed. One is conservative type where two new strands are synthesized in form of a double helix while the old double helix remains unchanged. The third type of DNA replication proposed has been dispersive mode of DNA replication which amounts to breaking down of double helical strands into small pieces along the length, their replication and random assemblage with newly synthesized pieces to form a patchwork single string of dispersed old and new pieces. These three modes of replication of DNA molecule are shown in Fig. 40.1.

According to the central dogma of molecular biology, genetic information flows (i) from DNA to DNA during its transmission from generation to generation and (ii) from DNA to protein during its phenotypic expression in an organism (Fig. 41.1).

In transcription, it has become clear as to how there is the transfer of genetic information stored in the sequence of nucleotides-pairs in DNA to the sequence of nucleotides in m-RNA molecules which carry that particular information from the nucleus to the sites of protein synthesis (ribosomes) in the cytoplasm. Further, there is the requirement of the process by which genetic information stored in the sequences of nucleotides in m-RNAs is used to specify the sequences of amino acids in the polypeptide gene products. This process known as translation takes place in the cytoplasm on complex workbenches called as ribosomes. With exception of water, proteins are by far the most prevalent component of living organisms in terms of total mass.

Proteins (= enzymes = genes) are composed of polypeptides and every polypeptide is encoded by a gene. Each polypeptide has a long sequence of amino acids linked together by covalent bonds. There are 20 different amino acids (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine, serine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine) present in most proteins. All amino acids except proline contain a free amino group (NH₂) and a free carboxyl group (COOH) as illustrated below:

Biochemical reactions are mediated by enzymes and all enzymes are proteins. Proteins are polymers of subunits (monomers) called amino acids which are often termed as ‘residues’. Each amino acid has an amino group (NH ) at one end and a carboxyl group (COOH) at the opposite end. 20 different kinds of amino acids occur naturally in proteins. Each enzyme consists of a certain number of amino acids in a precisely ordered sequence. The blueprint for making proteins is coded in the nucleotide sequence of DNA. The number of nucleotides which code for an amino acid is termed a codon. There are 20 common amino acids but only four different nucleotides. Obviously a singlet code (one nucleotide coding for one amino acid) could code only for four amino acids. A coding sequence of two nucleotides for one amino acid or a doublet code would produce 4₂ = 16 possible coding combinations or codons and this arrangement is not sufficient to take care of 20 amino acids. A codon size of three nucleotides for one amino acid or triplet code seems more likely as it produces 4₃ = 64 possible codons as illustrated in Table 43.1 (Strickberger, 1968).

Regulator Genes

Bacterial cells are capable of producing many kinds of enzymes in relatively large quantities but the kinds and amount of enzymes actually produced at any given time are usually those which are required at that particular time in response to the prevailing environment. Inducible enzymes are ordinarily absent or present in very small quantities until a specific compound appears. This compound is called as inducer. A typical example of this is production of b galactosidase (an enzyme that splits lactose into galactose and glucose, Fig. 44.1) once the lactose is introduced into the medium on which E. coli cells are grown. Here lactose acts as inducer for the enzyme b galactosidase.

The concept of gene has undergone several refinements since its discovery in 1866 by Mendel. Most genes encode one polypeptide and can be operationally defined by the complementation test. The complementation or cis-trans test provides an operational definition of the gene. It is used to determine whether mutations are in the same gene or in different genes. Intragenic complementation may occur when a protein is a multimer containing at least two copies of one gene product. The concept of gene has evolved from a bead on a string, not divisible by recombination or mutation to a sequence of nucleotide pairs in DNA encoding one polypeptide chain. The unit of genetic material not divisible by recombination or mutation is the single nucleotide pair. Historically several experimental developments have occurred to facilitate understanding of the structure and function of gene and these are briefly described in a chronological order as follows to have better understanding of the gene concept as it evolved. However, before discussing evidences supporting various gene concepts, it is better to summarize important stages in the evolution of the gene concept.

Abstract

Pigeonpea (Cajanus cajan) is an important pulse crop of India and its production is greatly affected by wilt disease. Pigeonpea wilt is a very destructive soil borne disease caused by Fusarium udum. In the experiments, ten treatments were applied viz.; Neem leaf extracts (T₁), Garlic extracts (T₂), Tulsi leaf extracts (T₃), bio-agents Trichoderma viride (T₄), Pseudomonas fluorescens (T₅), Polymer coating (polycot) @ 4 ml diluted in 5 ml of water per kg of seed (T₆), Polymer coaating + Trichoderma viride @ 6.0 g per kg seed (T₇), Polymer + Pseudomonas fluorescens @ 6.0 g per kg seed (T₈), Polymer coating (polycot) 4 ml diluted in 5 ml of water per kg seed + Vitavax power @ 3g per kg seed (T₉) and T₁₀ served as untreated. The observations were recorded six times at 10 days intervals and started from 40 days. These polymer alone @ 4.0ml per kg and in combination with bioagents and fungicides were applied to seed were tested against Fusarium udum. The highest per cent disease control and per cent wilt disease incidence were recorded 96.09 to 64.09 and 2.74 to 33.32, respectively in T₉ (Polymer + Vitavax power @ 3g per kg seed) at 10 days intervals followed by T₈ (Polymer + P. fluorescens) gave 94.46 to 53.06 per cent inhibition, T₇ (92.39 to 52.74) and T₆ (90.97 to 49.73) however, the lowest per cent disease control was noticed as 76.19 to 40.13 per cent in T₁ (Neem leaves extract) amongst all treatments except untreated.

Abstract

Pigeon pea is an important pulse crop of India and its production is greatly affected by wilt disease. Pigeon pea wilt is a very destructive soil borne disease caused by Fusarium udum. In present research work plant extracts (Garlic, Neem, Tulsi), bio-agents (Trichoderma viride and Pseudomonas fluorescens) and fungicides (Vitavax, Bavistin and Thiram + Carbendazim) were tested against Fusarium udum. Among them fungicides were found most effective in inhibiting mycelial growth of Fusarium udum recorded 100% inhibition followed by garlic bulb extract (52.12 to 56.33%). Among bioagents, Pseudomonas fluorescens inhibit the growth up to (46.00 to 51.79%) followed by T. viride (44.32 to 48.79%) amongst all treatments except untreated.

Keywords: Pigeon pea, Fusarium udum, bio-agent, fungicide

Pigeon pea (Cajanus cajan L. Millsp.) is one of the major pulse crops grown in the tropics and subtropics belonging to family Leguminaceae. It has wide adaptability and low input requirements mostly grown in kharif season.

Abstract

Lentil is an important pulse crop and play very important role in the supply of the protein to under nourished vegetarian population of the country. Fusarium wilt of lentil caused by Fusarium oxysporum f. sp. lentis is one of the wide spread and destructive diseases in India under rice-based cropping system. Use of synthetic fungicides has led to the emergence of several problems like environment pollution, residual effect in grains and killing of non-target organism(s). Hence, for minimizing the losses caused by Fusarium wilt, four plant extracts such as Garlic, Neem, Eucalyptus, Tulsi and three antagonistic bio-agents viz. Trichoderma viride, Trichoderma harzianum and Pseudomonas fluorescence were evaluated in vitro. The results clearly showed that all plant extracts were more or less inhibitory to mycelial growth of Fusarium oxysporum f. sp. lentis. The effectiveness of extracts increased with increase of their concentration and maximum growth inhibition was obtained in Eucalyptus followed by Garlic and Tulsi. While minimum per cent inhibition was recorded in Neem. Among bio-agents, T. viride showed maximum growth inhibition of Fusarium oxysporum f. sp. lentis followed by T. harzianum at both 4 and 7 days incubation period.

Abstract

Fusarium wilt is major disease in Central India, the major lentil growing region of country. The disease is soil borne causing heavy loss, development of wilt resistant varieties is most effective means of controlling this disease. Highly resistant sources of wilt in lentil have not been reported from the Indian lentil breeding programme. One hundred thirty lentil genotypes including one susceptible check were evaluated in control conditions of green house against wilt, based on controlled condition screening- LL 1370, IPL 333, PL4, RLG 195, RVL 14-4, L 4737, RKL 607-1, WBL 77, LL 1320, LL 1397, VL 525, LL1379, VL526, RVL14-5, L4717, VL126, L4076, RLG191 and VL150 have been identified as highly resistant genotypes. These can be used in hybridization programme for wilt resistance breeding and studying the inheritance of wilt resistance in lentil.

Abstract

Chickpea is one of the major grain legume crops of the tropics and subtropics. It is the second most important pulse crop after pigeon pea in India and is commonly known as gram or Bengal gram is a versatile crop among the grain legumes with a total production of 11.6 (Mt) from 13.2m ha. However, it ranks fifth in the productivity after Fababean, pea, lupin, and lentil and ranks first among the pulses both in acreage and production. Whereas, area under pigeon pea 3.90 mha with a total production and productivity of 2.89 mt and 741 kg/ha, respectively and is commonly known as red gram or Arhar is a very old crop of India. The chromosome number of pigeon pea is 2n = 22 chromosomes. They are the rich source of protein and supplies a major share of the protein requirement of the vegetarian population of the country. Chickpea accounts for 70.7% of the world Chickpea production n India followed by Australia, Pakistan, Turkey, Myanmar, Ethiopia, Iran USA, Canada and Mexico. Both crops are used as an important source of protein in human nutrition and cattle feed. Chickpea is considered to have medicinal effects and it is used for blood purification. Chickpea contains 21% protein, 61.5% carbohydrate, 4.5% fat. Its seed is also rich in protein, starch, fiber, calcium, iron and niacin, malic and oxalic acid, which makes it one of the best nutritionally balanced pulses for human consumption. However, like any other pulses, the chickpea seed also contains antinutrional factors, which can be reduced or eliminated by cooking. Both crops enrich the soil fertility by fixing atmospheric nitrogen in the root nodules and improve the soil structure. It is also to improve soil fertility by biological nitrogen fixation which helps reduce the input cost for the existing crop. These crops usually receive few inputs other than labor, insecticides and seed. Chickpea is a crop of both tropical and temperate regions. Low yield of chickpea attributed to its susceptibility to several fungal, bacterial and viral diseases. In general, estimates of yield losses by individual insects and diseases range from 5 to 10% in temperate regions and 50 to 100% in tropical regions. Whereas, Pigeon pea can be attacked by more than 100 pathogens. These include fungi, bacteria, viruses, nematodes, and mycoplasma like organisms. Fortunately, only a few of them cause economic losses and the distribution of the most important diseases is geographically restricted. The diseases of considerable economic importance at present are Fusarium wilt, sterility mosaic, phytopthera blight and alternaria blight.

Abstract

The major losses of banana at present by Panama wilt disease pathogen of banana and its management is very typical because it is caused by a soil-borne pathogen (Fusarium oxysporum f.sp. c u b e n s e ). The micronutrients are used to manage the disease as well as it provides the healthiness of plant and increases the fertility of the soil that’s why the plant is free from the disease or less infected. An experiment of micronutrients was conducted in the laboratory of plant pathology BAU, Sabour for Invitro evaluation of the micronutrients viz. Zinc Sulfate, Ferrous Sulfate, Ammonium Sulfate, Copper Sulfate, Calcium Nitrate, Borax and Potassium Chloride against Fusarium oxysporum f.sp. cubense causing Panama wilt of banana. The results of the experiment revealed that among the micronutrients used, Borax @ 500 and 750 ppm completely inhibited the mycelial growth of FOC followed by Zinc Sulfate (45.7, 42.6, 39.2 and 18.5 mm at 100, 250, 500 and 750 ppm respectively). The incorporation of borax in the solid medium at higher concentrations viz., 500 and 750 ppm is very effective for the management of Panama wilt disease of banana. This approach leads to maintain our ecosystem sustainability for a longer time and manage the disease.

Abstract

Mungbean [Vigna radiata (L.) Wilczek] is an important pulse crop grown in India. It is a short duration annual with yellow flowers and fuzzy brown pods. Majority of the mungbean grown in India is under rainfed conditions and the productivity of mungbean is not upto the mark to meet the daily dietary requirement of a populous country like India. The biotic and abiotic stresses in mungbean, majorly MYMV, powdery mildew, drought, salinity, water logging etc leaves a severe yield loss in mungbean crop. Among them, MYMV and powdery mildew diseases show yield losses accounting upto 85% and 40%, respectively in severe conditions. The present review paper is based on the study of both biotic and abiotic stresses and the recent advances in breeding for resistance to stresses. A detail explanation of major diseases and insect pests affecting mungbean are discussed along with the information of genomic resources and research done in mungbean in recent years. Recently SSR’s have been using widely for all molecular level research work like genetic diversity, constructing linkage maps for identifying QTL’s, marker assisted breeding etc. in mungbean. The resistant varieties developed against these biotic and abiotic stresses are listed which are very important for attaining a prominent yield in mungbean.

Abstract

Lentil (Lens culinaris Medik) is an important rabi leguminous annual herb with rich source of protein and other balanced nutrition. Lentil is susceptible to many insect-pests and diseases which causes significant yield loss which may range from 50 to 100% leading to one of the most important biotic constraints in productivity. The major fungal diseases in lentil are Fusarium wilt, Ascochyta blight and rust which causes severe losses in lentil production. Other minor diseases like botrytis grey mould, anthracnose, alternaria, stem rot, collar rot and some viral diseases also infect the lentil field under condusive environment conditions. Coming to major insect pests of lentil viz. helicoverpa, aphids, leaf weevils and lygus bugs with several noteworthy minor field pests such as thrips and cutworm with few storage pests that infest the crop. With an integrated blueprint of disease management, one can minimize risk of yield loss by following modified package and practices with need based use of pesticides, thus maximizing profit in lentil production.

Abstract

A field experiment was carried out during rabi 2012-13 at Bihar Agricultural College farm, Sabour, Bhagalpur to assess the effect of different herbicides on microbial population dynamics of soil in winter maize (Zea mays L.). The experiment comprised of ten weed management treatments involving various herbicides (acetochlor @ 1.125, 2.25 and 4.5 kg a.i. ha^-1, atrazine @ 1.0 kg a.i. ha^-1, pendimethalin @ 0.5 kg a.i. ha^-1 + atrazine @ 0.5 kg a.i. ha^-1, alachlor @ 0.5 kg a.i. ha^-1 as pre-emergence and 2,4-Diethyl ester @ 0.6 kg a.i. ha^-1and topramezone @ 0.04 kg a.i. ha^-1+ atrazine @ 0.25 kg a.i. ha^-1 as post-emergence including weed free and weedy check laid in randomized block design with three replications. Results indicated that all the herbicides’ treatment showed significant adverse effect on microbial population in rhizospheric soil of winter maize up to 45 days after sowing. Further, there was reduction in soil microbial population occurred in all the herbicides treated plots up to said time period as compared with weed free and weedy check. Initially the soil microbial population in weed free plot was much higher than that of weedy check, however, it was found in reverse trends in rest of the time intervals as well as harvest. Thereafter, soil microbial population was gradually increasing at different time intervals and at harvest, their population increased considerably in herbicidal treated plots though the weed free and weedy check showed higher soil microbial population than other herbicide treated plots.

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.