CONSERVATION OF GENETIC RESOURCES IN INDIA Introduction India has vast animal genetic resources with a wide variety of indigenous

farm animals including cattle. The cattle breeds have evolved over generations to adapt to the agro-climatic and socio-economic needs of the people. Domestic animal diversity is defined as the spectrum of genetic differences within each breed and across all breeds within each domestic animal species, together with the species differences; all of which are available for the sustainable intensification of food and agriculture production . The domestic animal diversity has evolved over millions of years through the processes of natural selection forming and stabilizing each of the species used in food and agriculture. Over the more recent millennia the interaction between environmental and human selection has led to the development of genetically distinct breeds. Selection processes, directed by both humans and the environment, together with the random sampling processes causing genetic populations to drift over generations, have accelerated the development of the diversity within species leading to the creation of distinct genetic differences amongst breeds. GENETIC DIVERSITY IN AnGR IN INDIA Species Cattle Buffalo Yak Sheep Goat Horse Donkey Camel Pig Rabbit Fowl Quail Duck As per World Watch List 70 20 5 62 34 7 3 9 (+1 Bactrian) 8 3 19 2 6 As per Indian literature 30 10 Nil 42 20 6 Nil 8 Nil Nil 15 Nil Nil

India is the seventh largest country in the world and it is recognized as one of the 12-mega biodiversity centres of the world. It is well marked off from the rest of Asia by mountains and the sea, which gives the country a distinct geographical entity. Due to diverse agro-ecological regions and topographic conditions, India has rich repository of both flora and fauna. India has vast animal genetic resources with a wide variety of indigenous farm animals. It has 132 registered

farm animal breeds viz. 30 cattle, 10 buffaloes, 42 sheep, 20 goats, 7 camel, 5 horses/ponies and 18 chickens, besides many other non-descript and mixed populations. Livestock husbandry is an age-old important occupation for Indian farmers. The unique and rich animal biodiversity is extensively referred to in Indian scriptures.

JUSTIFICATION FOR CONSERVATION OR REASON? Different reasons for conservation of animal genetic resources include (Rao and Reddy, 1995): 1. Economic and biological reasons: A. Genetic variation both within and between breeds is the raw material with which the animal breeder works. Therefore, any loss of genetic variation will limit our capacity to respond to changes in economic forces for the exploitation of animal production in future. B. Breeds with specific qualities like disease resistance, heat tolerance, ability to survive and produce under stress and low input conditions need to be preserved for future use. C. Future requirements of type and quality of animal produce (milk, draught power) may change and this requires conservation of animals with better performance in specific production traits. D. Magnitude of heterosis depends upon the breeds crossed. For exploiting the heterosis in animal production, it is necessary to maintain breeds which are complementary to each other and on crossing result in maximum heterosis. 2. Scientific reasons: i. Breeds with unique physiological or other traits are of great value as they provide missing links in the genetic history of a livestock species by the study of blood groups or polymorphic traits. To identify the DNA sequences causing the distinctive traits, preservation of breeds with unique traits will be essential for long term research in molecular engineering. ii To evaluate the magnitude of genetic change due to selection, maintenance of a sample as control population is very much essential. iii Investigations in different areas like physiology, biochemistry, genetics immunology, etc. Require maintenance of diverse populations. iv Variety of populations are an asset for research work in biological evolution, behavioural studies, etc. v Diverse populations form an excellent teaching material for students of animal science, ecology, etc. 3. Historical and cultural reasons Conservation of historically important, culturally interesting and visually unusual and attractive population is very important for education, tourism etc. Further, conservation of breeds a. Can be a valuable material of nature and culture, b. Serve as research and teaching material in history and ethnography,

c. Will be preservation of populations with diverse sizes, colours and other morphological features, for aesthetic reasons, and d. Need be done to take care of existence of different creations of the nature for posterity. STATUS OF SPECIES -FEMALE POPULATION (Tomar et al) s,.no 1 2 3 4 5 Status Normal Insecure Vulnerable Endangered Critical Number >10,000 10,000-5,000 1,000-5,000 100-1,000 < 100

MECHANISM OF CONSERVING CATTLE GENETIC RESOURCES Once genetic resources have been identified and characterized, two basic conservation activities can be followed, 1. IN SITU CONSERVATION 2. EX-SITU CONSERVATION

IN SITU CONSERVATION 1.haploid forms a. frozen semen b. frozen eggs/oocyte

2.diploid forms

a. frozen embyo b. live animal

In situ conservation requires establishment of live animal breeding farms and their maintenance. The generation and loss of alleles is a dynamic process that should be maintained at close equilibrium through sound management. In situ conservation strategies emphasize wise use of indigenous cattle genetic resources by establishing and implementing breeding goals and strategies for animal sustainable production systems. Information for animal recording and breeding is well established in developed countries through breeding associations which zealously protect the interest of breeds including rare ones. Infrastructure appropriate to systems in

developing countries remains scarce. In India, such efforts are limited to only six breeds of cattle, in a herd registration program organized by the Central Animal Husbandry Department. Similar programs are required for the rest of the breeds and species as well. In any such program, the success depends upon the participation of the farmer for which he needs support and incentive. Therefore, it is difficult to organize the farmers for conserving the breeds which are no more economical to him. In the case of breeds which are no more economically viable, therefore, the only alternative is to bring them under government farms. In situ conservation is very costly if the entire population has to be retained for which at least 26 females and 10 males in cattle have to be maintained that would keep the inbreeding coefficient at 0.2 per cent per year. Therefore, this approach would have to be limited to those breeds which are highly endangered. Modalities for simplified animal recording, genetic development and dissemination are needed for each species for a range of national livestock structures in developing countries. MAJOR ADVANTAGES OF IN-SITU CONSERVATION: 1. Live animals can be evaluated and improved over the years. 2. Genetic defects can be detected and eliminated. 3. Live animals are always available for immediate use. 4. They are a gene bank for future use. 5. They are a constant reminder that the needs of posterity must be considered. 6. The herd may have some economic advantages (heat tolerance, disease resistance) which can be exploited and so render the enterprise economically viable. 7. The produce from live animals partly compensates the expenditure, if not entirely. 8. From aesthetic point of view, the live animals are, visible, a pleasure to look at, the people are delighted to see variety of animals and have some cultural value. THE MAJOR LIMITATION OF LIVE ANIMAL CONSERVATION While fixing the number for preservation of a breed, 1. The cost of maintenance, 2. Availability of animals and rate of inbreeding should be taken into consideration. 3. With small population size, the effective population size decreases and the genetic structure of the population is affected due to inbreeding and random drift. 4. Many models are now available which reduce inbreeding to a minimum, but random drift over long periods may lead to a population very different in genetic composition from the initial one. 5. Gene X environment interactions is another disadvantage. 6. In situ conservation involves  A large infrastructure of land,  Buildings,

 Feed and fodder resources,  Water supply,  Labour,  Technical and supervisory man-power, etc. It should be made to maintain a rate of inbreeding of less that 0.5% per generation for long term programmes while slightly higher rate could be tolerated for short term programmes. A flock/herd of 150 breeding females with 20 breeding males, the males being unrelated as far as possible, for preservation purposes. A maximum inbreeding level of 1` % per generation as tolerable and a herd size of 200 breeding animals in necessary to breed and selected successfully for a quantitative trait. EX-SITU CONSERVATION Ex-situ conservation includes cryogenic preservation. It is the storage of genetic resources, which the farmers are currently not interested in using. Ex situ conservation is based on the use of live animals populations wherever practicable, supported by cryopreservation where technology exists or can be developed, combining within-country gene banks with global repositories. Interested governments, norgovernmental organizations, research institutions and private enterprises should be encouraged to maintain in vivo samples of breeds at risk, with national inventories being established and kept up to date so that the genetic resources are readily available for use and study. Because of random drift and possible gene by environment interactions, ex situ methods are generally preferred over in situ. Ex situ conservation is comparatively more convenient, economical and easy with the application of modern reproductive technologies. Advantages 1. If the preservation is to maintain populations without genetic change, it can be best done by cryogenic storage as it is difficult to breed many generations of animals without any change in the genetic structure. 2. The resources requirement for in situ preservation is quite large as compared to cryogenic methods. Limitations 1. Ex situ preservation using frozen semen delays the restoration of a breed as it can be restored in the future only by upgrading. But this could be overcome through preservation of embryos. 2. Another important factor is the danger faced by a breed restored from cryogenic preservation from important changes in the environment like germs, climate, etc., that have taken place over the years. 3. Variability in cryogenic storage of germplasm, accessibility to their physical location, ownership, behaviour of animal, response of germplasm to freezing and thawing techniques, and poor conception rate

EX SITU/CRYOGENIC PRESERVATION INCLUDES 1. Preservation of frozen semen 2. Preservation of oocytes 3. Preservation of embryos 4. Preservation of ovaries 5. Use of embryonic stem cells or blastomeres 6. Production of chimeras 7. Production of embryos in vitro 8. Embryo splitting 9. Transgenesis 10. DNA libraries 1. Semen preservation: Semen cryopreservation and artificial insemination are important tools of animal improvement with vast scope of genetic improvement, conserving indigenous cattle resources because of their simplicity and relatively cheaper costs. Semen storage and distribution activities are being carried out in a few well-known indigenous milch cattle. Still, many breeds like Amrithmahal, Dangi, Gaolao, and Punganur lack such facilities. Spermatozoa may also be collected from the epidydimis 2. Oocyte preservation: This method provides an opportunity for conserving females in the same way as sperm is conserved. The oocyte can be recovered by surgery, laparotomy or slaughtering of donor animals. The frozen thawed oocyte can be used for IVF successfully (Schellander et al, 1988). The immature and mature oocyte from slaughtered animals could be useful in near future for cryopreservation of genetic material of endangered breeds. 3. Embryo preservation: The main advantage of cryopreservation of embryo over that of sperm or oocyte is that it contains the complete genome. The embryo transfer technique coupled with micro manipulation, embryo sexing and splitting are more useful and economical to carry out ex situ conservation of animal genetic resources. This technique is very useful in conservation of genetic resources by rapid multiplication of superior or rare germplasm. MOET can be used in resurrecting the endangered cattle breeds like Sahiwal, Punganur and Vechur. 4. Preserving of ovaries: The preservation of slices of ovaries in liquid nitrogen is a new technology which may be of great use in conservation of animal genetic resources. The ovary slices might be transferable into suitable recipients to obtain oocytes which can be fertilized.

5. Embryonic stem cell and nuclei: Preservation of embryonic stem cells could represent an important method of genome conservation and would be helpful in propagating animals from a single embryo of elite or rare animals belonging to endangered breed/species. Embryonic stem cells represent progressively growing cultures of embryonic cells which retain their pluripotential characteristics. They are derived by culturing blastocysts in vitro in such a way that the cells from the inner cell mass proliferate but do not differentiate. Embryonic stem cell lines can be isolated and then multiplied by continued culture. The importance of embryonic stem cell lines is that, if they are incorporated with normally developing embryos, they will participate in the formation of the inner cell mass and produce chimaeric animals, including germ line chimaeras which are fertile. A more direct route of regenerating animals from embryonic stem cells might be to use the nuclei from these for nuclear transplantation into enucleated oocyte and embryonic multiplication. 6. Chimaeras: Chimaeras mean the animals having body cell population with different karyotypes which have been formed from two or more zygotes with different karyotypes. Chimeric embryos have been frequently made by the aggregation of cells from two individual embryos or by injecting cells from one embryo into the blastocyst cavity of another embryo. So long as cells from both embryos are represented in the inner cell mass, the composite embryo will develop into a chimaeric animal. A more important aspect of chimaerism in genetic conservation is the potential use for inter-species embryo transfer. Using this technology sheep have been born to goat foster mother and vice versa. This would be especially important if it was a species rather than a breed of animal that was on the verge of extinction. 7. Production of embryos in vitro: This technology involves salvaging mature oocyte from ovaries of slaughtered animals and developing methods for their maturation, fertilization and in vitro culture for normal embryonic development. For conservation of rare breeds, such a method could provide an opportunity to salvage a few oocytes from the ovaries of rare and superior animals even after their normal reproductive life and to produce some blastocysts for conservation by deep freezing. 8. Embryo splitting: Embryo splitting is more advantageous in the circumstances where only few embryos of particular genotype or breed or species are available. This technique of manipulating embryos could be helpful in producing more number of animals from a few stored embryos of rare and endangered animals.

9. Transgenesis: Recent developments in molecular biology have enabled introduction of specific genes into the animal genome. A small amount of DNA is injected into the nucleus of an egg soon after fertilization. In some instances the DNA becomes integrated into the chromosomes and an embryo and foetus develops in which all the nuclei contain copies of the inserted gene. As any sequence can be spliced with any other (Anderson, 1986), transportation of genes across breeds and species is possible by recombinant DNA techniques. Successful microinjection of genes in mice embryo and their expression .the creation of transgenic forms a possibility, and for the future a viable technique for improving animal production, and for conservation and capitalize on of important genes across breeds and species.(Gordon et al) 10. DNA libraries: Theoretically, an animal can be produced from its complete DNA complement. However, at present technical developments are limited to the identification and manipulation of only a few genes. But the direction in which technical advancements are taking place gives an indication that in future breeds/species can be reconstructed from their DNA complements. This gives the hope that if complete DNA complement is stored either in lyophilised form or as cryopreserved cells, reconstruction can be taken up when techniques are standardised. Advances in biotechnology are emerging in a big way and could be of great utility in near future for animal improvement and animal genetic resource conservation programmes. THE MAIN CAUSES OF THE REDUCTION IN NUMBERS OF INDIGENOUS LIVESTOCK ARE 1. The intensive efforts being made to introduce superior germplasm to overcome the low production of the local genotypes, without any consideration to the local ecological and socio-economic situation (demand for increased milk production and introduction of exotic milk breeds of cattle like Jersey, Holstein). 2. No serious organized effort in maintaining the purity of the breeds - inter-mating among breeds located in each others’ vicinity resulting in dilution of breed characteristics and Changes in cropping pattern and increased mechanization of agriculture with neglect of indigenous draught breeds of cattle. 3. The global awareness for conservation of domestic animal diversity was touched up on at the International Conference held in Rome in 1936, which resulted in several recommendations being made in this regard. Based on these recommendations, the Government of India had initiated herd registration schemes for several established breeds of cattle, viz., Gir, Hariana, Kankrej, Ongole and Tharparkar.

4. Standards of performance were laid down for registration of animals both in registered and farmers’ herds, and also including milk recording of animals. However, this scheme was not entirely successful. Efforts were made from time to time by the Government of India/Indian Council of Agricultural Research to define the breed characteristics of important breeds of livestock and a bulletin containing breed characteristics of important breeds of cattle and a buffalo was published by ICAR in 1979.compiled information on important indigenous breeds of livestock (Acharya et al) THE OBJECTIVES OF NBAGR WITH REGARD TO CONSERVATION ARE AS FOLLOWS: 1. To develop inter-institutional co-operative programs for the genetic evaluation and conservation of animal genetic resources. 2. To develop infrastructural facilities and train manpower for the execution of the project. 3. To estimate demographical distribution of breeds in their respective tracts by undertaking proper surveys. 4. To obtain need-based information for breed description and characterisation of various special/animal types by evolving suitable formats and questionnaires to collect data through scientific surveys. 5. To obtain data on certain qualitative and quantitative morphological characteristics which are related to production and reproduction. 6. To identify superior germplasm and rare variants for the specific breeds. 7. To devise suitable strategies for the purpose of in-situ conservation and propagation. 8. To develop an inventory breeds/animal typos which are declining or are at the verge of extinction. The technical programme of the project is as follows: 1. Demographical and geographical distribution 2. Enumeration of breeds in terms of age, sex in a population. 3. Qualitative and quantitative characterisation of breeds in relation to specific phenotypes traits like type, production potential and reproductive status etc. 4. Qualitative and quantitative descriptions of unique animals, elite producers, and rare or unusual characteristics in certain specimens 5. Collection of blood samples from 100 males and 100 females of each breed/type for genetic evaluation work pertaining to blood typing and biochemical polymorphism, immunogenetics, cytogenetics, gene marker studies, restriction fragment length polymorphism of DNA and other recently developed molecular genetic techniques.

Immediate strategies would involve the Survey, Conservation of Livestock Genetic Resources, Research on Conservation and Training in Conservation and Management. Serious efforts should be made to organizing conservation and evaluation strategies. Since the major emphasis in any system of conservation is the lack of information in gene resources, it is absolutely important that evaluation and conservation should go hand in hand and can be achieved by the following: 1. All universities, educational institutions, religious trusts and organizations associated or having interested in historical preservation should be aided by the Government and encourage to maintain small groups of animals in their natural habitat or intended management systems. These groups should be under a uniform recording system, in a computer readable format with the evaluation and documentation being done at the NBAGR. 2. All the developmental programs being taken up by the state governments should have a mandatory prerequisite that breeds being used for crossbreeding or grading up should be maintained in nuclear herds in their natural habitat and subjected to continuos evaluation as described above.

Steps taken in India In India the need for conservation of animal genetic resources has now "become clear, and the lack of necessary documentation and evaluation has been recognized. A National Bureau of Animal Genetics Resources and one for fish have been sanctioned, which will have the following functions: 1. To undertake systematic cataloguing of animal germ-plasm and to establish a data bank and information service on animal genetic resources. 2. Identification, evaluation, cataloguing and conservation of herds or flocks identified as valuable for purposes of conservation consisting of important indigenous breeds of livestock and poultry in the country. 3. To undertake survey for the evaluation of merits or attributes of breeds discovered recently or threatened with extinction. 4. Formulation of criteria and parameters to enable identification of animals and flocks of superior genetic merit or worthiness for conservation. To take steps for the preservation of germplasm both as live animals or by setting up frozen semen and embryo banks. 5. Documentation of pertinent information in regard to identity of herds and flocks on a computer readable format. 6. Processing of information collected under surveys carried out for the identification of valuable animal resources material. 7. Dissemination of information in a cogent manner to enable individuals and agencies to use information in regard to the available animal genetic resources, 8. Maintenance of national/international liaison with institutions concerned with similar work. 9. Rendering financial assistance to universities, IGAR institutes and government and private bodies, where maintenance of such valuable germ-plasm is considered desirable. 10. Monitoring of the entire improvement programme and maintenance of rare breeds/herds/ flocks in the country. 11. Monitoring of new introduction of animal germ-plasm and new synthetics. 12. To stimulate programmes for improvement in the various breeds and to give adequate financial and technical support to these.

STEPS IN MOLECULAR CHARACTERISATION BEFORE YOU START Ideally, molecular characterization should be undertaken as part of a comprehensive national programme for management of AnGR, the development of which is outlined in FAO Guidelines for Preparation of National Strategies and Action Plans for Animal Genetic Resources. For maximum efficiency, molecular characterization of AnGR should be done in concert with phenotypic characterization, which is addressed in FAO Guidelines for Phenotypic Characterization of Animal Genetic Resources. KNOW YOUR BREEDS Collect and critically evaluate the available information on the breeds you want to investigate: scientific literature, breed handbooks, FAO Global Data Bank or other data banks, non-scientific literature and even anecdotal information. It is most relevant to identify the traditional rearing area and any evidence for genetic subdivision: different ecotypes, phenotypes, agroclimatic zones or isolated subpopulations. INVOLVE LOCAL EXPERTS Most of the expertise on local breeds rests with the farmers and breeding societies, who should be informed on the objectives of the study. Coordination with national breeding societies and livestock research institutions is desirable, as they are expert on the breeds, are familiar with local circumstances and may serve as liaison with the owners. Inform also FAO National Coordinators for Animal Genetic Resources, because, as noted earlier, characterization studies should ideally be undertaken as part of an over National Strategy and Action Plan for AnGR. DEFINE THE OBJECTIVES Ranging from an inventory of the pattern of diversity to a reconstruction of the history of breeds or formulation of specific guidelines for genetic management, objectives are most relevant for the sampling, choice of markers and data analysis. ACT LOCALLY, THINK GLOBALLY The data collected will invariably become more interesting if analyzed and evaluated in an international context. Combining results with other datasets requires the use of the same molecular markers, the use of common reference samples and, preferably, having one or more breeds in common. DEFINE THE SCOPE  Depending on the breeds and the objectives of the study, the following considerations may be relevant.  Breeds most likely to be distinct from other breeds are those with a long history of genetic isolation, raised in a unique environment or having unique phenotypes.

 Priority should be given to local breeds, but common or economically important international transboundary breeds should be included as a reference.  For regional transboundary breeds it will be useful to include populations across the borders or to collaborate with institutes who have studied those populations.  For breeds that are of hybrid origin by introgression, upgrading or by the planned creation of a synthetic breed, it is essential to have data from parental breeds.  For breeds having a recent history of intense selection and/or inbreeding, sampling of animals of previous generations, which may be available by cryopreservation of semen samples or from museum specimens, may be appropriate.  For mammalian species, sampling of at least 10 males allows studies on Y chromosome variation, whereas samples of poultry species preferably should contain at least 10 female birds for eventually studying W-chromosomal COLLECT SAMPLES For this most crucial step, the following considerations are relevant:  Almost all cells or tissues may be used for DNA analysis: blood, semen, hide, bone, tissue (e.g. ear tissue), plucked hair (only the root cells contain nuclei, but cut hairs can be used for mtDNA analysis) and feathers,  High quality DNA is most easily obtained from samples of peripheral blood, organs or other tissues. Most convenient are blood samples, to be collected in an anti-coagulant (EDTA or Na-citrate).  Collect enough material for present and future studies. For PCR-based applications, 10 ml of blood is adequate, but for high-density SNP typing and genomic sequencing, it is advisable to sample 50 ml or more. Note that poultry species have enucleated erythrocytes and, therefore, much less blood (~1 ml) is required.  Transport of blood samples can be at ambient temperatures, but in tropical regions samples should be processed within 36 hours.  For longer storage, samples can be placed in a room temperature preservative such as Queen's buffer (0.01. M Tris/HCL , 0.01 M NaCl, 0.01 M EDTA and 1 % nlaurosylsarcosine, pH 8.0).  Tissue samples of 1 cm squared should be minced to 1 mm squared pieces and placed in Queen’s buffer or 70% ethanol. Air-drying of ethanol-treated samples allows long term storage and the easy transport of samples. Alternatively, pieces of tissue may be directly dehydrated by placing in vials on crystals of silica gel.  Hair samples should be desiccated as soon as possible and stored dry.

 FTA cards can be used for collection of genetic material with DNA to be amplified by PCR, but special protocols are required to obtain double-stranded DNA and the stranded DNA obtained with standard isolation protocols is not suitable for all other applications.  Samples that are to be used for cloning, Southern blotting or genomic sequencing protocols require double-stranded DNA of high molecular weight.  From each animal, duplicate samples should be taken and kept separate during subsequent transport and storage.  Labelling of samples should be unambiguous and permanent. The labelling procedures should be developed and supervised by the responsible scientist of the project. • Bank it: store all samples and document all relevant information unambiguously in such a way that they can be retrieved and understood, even by persons not involved in the sampling. Also collect data Essential is recording of the following information for each sample: 1. Sample (and duplicate) number 2. Date 3. Location and GPS coordinates, 4. Name of collector 5. Breed 6. Sex of animal 7. Type of sample 8. Any relevant phenotype 9. Basic pedigree information 10. Size of herd 11. Digital photograph with measuring stick, Any interesting morphological features .Notes about any recent change in geographic location of the animal should be compiled once for each breed, to the extent that is possible based on the information available. This form addresses breed origins, farming practices, basic production information, and features of the breed such as productivity, disease resistance or adaptation to local conditions should also be recorded. LABORATORY PROTOCOL Extracting DNA Several reliable protocols for DNA extraction are available. Older protocols are based on Proteinase K/SDS lysis of cells, organic extraction and alcohol precipitation. Salt precipitation avoids the organic solvents, but the long-term stability of the DNA samples is problematic. Now, convenient commercial kits based in the specific binding of DNA to resins are available for several kinds of tissues and generally perform well. It is recommended to test out before

application to field samples any DNA extraction procedures that have not been used routinely. Consider the amount of DNA required by the different protocols. Genotyping.  Protocols for genotyping are generally available and straightforward, but the following factors should be taken into account:  Analyze in each experiment at least one reference sample in order to cross-validate successive genotyping experiments and include this in all experiments.  For microsatellites, use the FAO recommended panel and include international reference samples in order to link your data with other datasets.  Include blank extraction and amplification samples in order to check contamination of the reagents.  Outsourcing the genotyping to dedicated custom service laboratories may very well ensure high quality and cost-effective results without requiring investment in new equipment and expertise. Outsourcing does not, however, lift the requirement of analyzing reference samples and critically checking the quality of the data.  In collaborative projects with microsatellites, it is preferred that one laboratory performs all typings for a given marker in order to exclude laboratory-dependent scoring. If this is not feasible, it is most essential to share samples of reference animals in order to be able to standardize allele sizes.  Multiplexing the PCR can reduce the costs, but results should be checked carefully to ensure it does not increase the percentage of missing genotypes. This applies especially to samples with low DNA concentration. As a compromise, PCR reactions can be carried out separately and be combined on the gel (multiloading). Data analysis Check the data  Remove uncertain scores and delete markers and animals with an excess of missing data. Also check for outliers. Be aware that erroneous genotypes may distort the results of the analysis. The following checks should be carried out in order to minimize the error rate ,  Matching duplicate samples, indicating errors during sampling or processing of samples  Examining unusual alleles, this may result from clerical mistakes or incorrect interpretation of electrophoretic patterns.  Check for an excess of apparent homozygosity in samples with low DNA concentration because of allele dropout.  Standardize allele-calling with other laboratories, particularly for microsatellites.  Compare allele frequencies with data from breeds that are likely to share the most frequent alleles in order to detect inconsistent allele sizing.

 Check for absence of laboratory-dependent clustering of breeds, which may result from systematic differences in allele calling. One cause of laboratory-dependence may be the lab-dependent differentiation of microsatellite alleles that only differ by one bp in length.  Linkage disequilibrium (LD). Markers in LD in all populations are probably genetically linked.  Hardy-Weinberg (HW) equilibrium. Markers that in most breeds are not in HW may have null alleles or be linked to loci under selection, hence breaking the assumption of neutrality.  Always keep the original version of the data in which no corrections have been carried out, so data can be recovered if deleted in error. Within-breed analysis. Expected heterozygosity or allelic richness within breeds indicates the influence of drift on breed diversity, where decreased heterozygosity is associated with increased drift. Differences between expected and observed heterozygosity as well as departure from HardyWeinberg equilibrium indicate non-random mating or the existence of population substructures. The presence of inbreeding can be tested by F statistics, in particular by testing if the FIS parameter is significantly larger than zero. Genetic subdivision may be explored further by modelbased clustering Analysis of breed relationships. Breed formation has led to a partitioning of the total diversity in a within-breed and an among-breed component. These components and others, e.g. the component due to the geographic location of breeds, can be quantified by AMOVA analysis and reflect history and breeding practices. Typically, 50 to 90% of the total diversity corresponds to the within-breed component, depending upon the group of breeds sampled and the sources of variability considered. What are the data trying to tell us? Place the results in a historic perspective. Keep in mind that genetic events migration, introgression, admixture, crossbreeding, population bottlenecks, and selection have happened at different times, which may complicate the pattern of diversity. Consider alternative explanations and do not interpret according to preconceived ideas. Conservation priorities., the usefulness of the currently available algorithms is still a matter of debate . The use of genetic marker information in prioritization of breeds for conservation has been discussed in the FAO Guidelines for In Vivo Conservation of Animal Genetic Resources. PUBLISH RESULT  Publish your findings in a scientific journal. Open-access journals are recommended because of their wide diffusion and free accessibility.  Properly acknowledge contributors of samples and/or data.  After publication, deposit your data in a public database and/or comply with requests to make datasets available.

 If appropriate, formulate recommendations for genetic management and conservation and disseminate these to breeding organizations and government agencies such as the National Advisory Committee on AnGR, assuming that one exists, as well as the FAO National Coordinator for AnGR. Below are a number of examples that show how molecular observations are relevant for genetic management: CONCLUSIONS AND RECOMMENDATIONS 1. Zebu breeds are endowed with highly desirable characters like adaptability to harsh conditions and resistance to diseases. A more reliable and objective assessment of performance of indigenous breeds with special emphasis on diseases resistance and adaptability to harsh environments is essential, 2. In view of the rapidly increasing costs of conventional energy inputs, it is highly desirable to generate adequate information on draft ability of indigenous cattle and buffaloes which is very insufficient at present. 3. Precise and reliable estimates of different genetic components of variability of important economic traits of indigenous breeds of livestock should be obtained. This would be needed for evolving appropriate breeding plans for their improvement. 4. An atlas of performance of indigenous breeds and the appropriate breeding plans based on the information thus collected be drawn up. Such information would allow the best utilization of genetic resources in cattle and buffaloes and other specie 5. Properly designed selection experiments should be carried out for important indigenous breeds which are not involved in genetic improvement experiments through crossbreeding. 6. Since sizeable populations of different crossbreds are available around a number of milk shed areas, such populations should be utilized for performance recording and progeny testing of crossbred sires, in order to take full advantage of crossbreeding, progeny tested half bred bulls should be used for the development of new breeds/strains adapted to the given environment. 7. The present evaluation of different breeds and breed crosses is being done only under intensive management system. Such evaluation should be done under intensive, medium and low input (as close to the existing practices in the farmers fields as possible) so that the most efficient genotypes for each of these management levels could be identified.

8. A number of native breeds, strains or varieties are, or may be, in danger of genetic dilution through indiscriminate crossbreeding with exotic breeds. Such native breeds should be identified, so that they can be evaluated before this process leads to their essential loss. 9. Some native breeds are in danger of losing genes for high production because high-performing animals are withdrawn from breeding populations for use in units of high production and/or subsequent slaughter (e.g. city milk production in India or slaughter animals closen because of large size). Such breeds should be identified and breeding units kept intact.

1. Acharya, R.M., 1982, FAO Animal Health and Production Paper No. 30. 2. Acharya, R.M. and Bhat, P.N., 1984, Livestock and poultry genetic resources in India, IVRI, Research Bulletin No. 1, Indian Veterinary Research Institute, Izatnagar, India. 3. Anderson, G.B., 1986, Identification of sex in mammalian embryos. In Genetic Engineering of Animals; 4. Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A. and Ruddle, F.H., 1980, Genetic transformation. of mouse embryos by macroinjection of purified DNA. Proc. Nati. Acad. Sci., USA, 77: 7380 - 7384. 5. Rao, M.K. and Reddy, A.O., 1995, Livestock Genetic Resources in southern region: conservation for posterity. Symposium on ‘Breeding strategies for optimum animal production’ conducted by Dakshin Animal Breeders Society on 24 -25 February at Bangalore. 6. Tomar s.s. 2009.Conservation of animal genetics resources .chapter in :Text book of animal breeding ,kalyani publishers page no. 61-62.

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