Molecular markers for exploring biodiversity

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Credit Seminar On Molecular Markers for Exploring Biodiversity in Livestock of India:

Credit Semina r On Molecular Markers for Exploring Biodiversity in Livestock of India Presented By : Manas Kumar Das M.V. Sc. 1 st year (AGB ) NDRI, Karnal

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What are molecular markers? Molecular markers are those parameters those are capable of detecting genetic characters, variation & abnormalities at DNA sequence level. Introduction Molecular markers are those parameters those are capable of detecting genetic characters, variation & abnormalities in any living organisms at DNA sequence level.

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What do you mean by Biodiversity? It is the degree of variation of life forms within a given species, ecosystem, biome or, an entire planet. Biodiversity Species diversity Ecosystem diversity Genetic diversity

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It is the effective number of different species that are represented in a collection of individual The level of biodiversity refers to the total number of genetic characteristics in the genetic make up of a species Species diversity Genetic diversity

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Breed diversity It is the differentiation that belongs to a group of individuals within a species which separates them from the others on morphological, geographical & qualitative basis.

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Basis Of Biodiversity Among Livestock

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Procedures to Estimate Biodiversity Previously done by the phenotypical characters, morphological differences among breeds Quantitative characters was also used for diversification of the livestock of various breeds Molecular markers which are being used in new array proved to be the most promising for estimation of biodiversity

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iat Evolution Of Molecular Markers

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Subjective view of the changing relative importance of different molecular markers ( Schlotterer C, 2004 )

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World Context on Diversity Study 87 different projects in 93 different countries for genetic distance studies mostly on ruminants ( Baumung et al., 2004 ) Species Specific Standards by ISAG in 1995 based on microsatellite loci as about 30 per species for cattle, chicken, sheep & swine ( Erhardt & Weimann, 2007 ) Study on 57 European sheep breeds from 15 European countries ( Peter et al., 2004 )

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High levels of genetic diversity obsesrved in Argentina ( Mate’ et al., 2005 ) European Cattle Diversity Consortium (2006) investigated genetic distance between 69 European cattle breeds Cont... Genetic diversity study on Northern European cattle breeds by Tapio et al. in 2006 Pig Biodiversity Project II found significant clustering of lines within main breeds of European pigs ( SanCristobal et al., 2006 )

Molecular Markers For Biodiversity Estimation:

Molecular Markers For Biodiversity Estimation

Molecular Markers For Biodiversity Estimation:

Molecular Markers For Biodiversity Estimation Nuclear markers DNA based markers e.g. Minisatellites, RFLPs PCR based markers e.g. Microsatellites, RAPDs, SSCPs, ISSRs, AFLPs etc. DNA Sequencing

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Protein polymorphisms were the first markers used for genetic studies DNA polymorphisms have become the markers of choice for molecular based genetic variation study Mostly used DNA marker for biodiversity study is the Microsatellites Molecular Markers For Biodiversity Estimation

DNA Based Markers:

DNA Based Markers

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Microsatellite Markers A) Microsatellites are Single Sequence Repeats (SSR) or, Simple Tandem Repeats (STR) of 2 to 6 bp long nucleotide e.g. -CACACACACACACACA B) High mutation rate & co dominant nature allow more detailed analysis of mutation pattern C) Highly polymorphic, abundant & evenly distributed through out euchromatic chromosome

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D) Reconstruction of phylogenetic tree through Microsatellite analysis by N-J ( Neighbor Joining Method) ( Saitou & Nei , 1987 ) E) Multivariate analysis & Bayesian Clustering approach also useful for Microsatellite data analysis ( Pritchard et al., 2 000) Cont...

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Cont... G) Microsatellites gain & lose repeat units by DNA replication slippage specific for tandem repeats F) Microsatellite data used to assess genetic relationship mainly by Nei’s Standard genetic Distance ( Beja -Pereira et al., 2003; Joshi et al., 2004; Sodhi et al., 2005 )

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RAPDs ( Randomly Amplified Polymorphic DNAs) ISSRs ( Inter Simple Sequence Repeats) or, Retrotransposons IRAPs ( Inter Retrotransposon Amplified Polymorphism) AFLPs ( Amplified Fragmented Length Polymorphism) RAPDs (Randomly Amplified Polymorphic DNAs) ISSRs ( Inter Simple Sequence Repeats) or, Retrotransposons IRAPs ( Inter Retrotransposon Amplified Polymorphism) AFLPs ( Amplified Fragmented Length Polymorphism) Other PCR Based Markers

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The principal advantage of RAPDs, ISSRs, IRAPs & AFLPs - do not require prior knowledge about Primer Sequence in target species MNA ( Mean Number of Alleles) per population & H 0 & H E - Most common parameters for assessing within breed diversity Simplest parameter for among breed diversity are indices e.g. F ST & G ST Other PCR Based Markers

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AFLPs are biallelic markers with dominant mode of inheritance AFLP profiles are highly informative in assessing the relationships between breeds ( Ajmone- Marsan et al., 2002; Negrini et al., 2006) F ST measures the degree of genetic differentiation by std. variance in allele frequency Cont...

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High potential for marking on a single nucleotide position of genome & introduce mutations in expressed sequence SNPs Alternative of Microsatellite markers in diversity study Most SNPs located in Non Coding regions which are single base mutations in DNA Variations at single nucleotides don’t change all length DNA sequence

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mtDNA Polymorphism The polymorphisms in the sequence of the hyper variable region of the D loop or, control region of mtDNA have contributed greatly to the identification of the wild progenitors of domestic species, the establishment of geographical patterns of genetic diversity, & the understanding of livestock domestication. ( Bruford et al., 2003 )

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mtDNA In World Context 4 maternal lineages has been found in Bos taurus in Europe Multiple maternal origins with 3 mtDNA lineages in Goats ( Luikart et al., 2001 ) 3 rd mtDNA in native sheep, 4 th in native goat & 5 th in native cattle has been discovered in China ( Guo et al., 2005; Chen et al., 2005; Lai et al., 2006 ) 9 mtDNA classes found in Asian chicken ( Liu et al., 2006 )

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• Y and mtDNA does not recombine with each meiosis • This means that the Y and mtDNA remains constant from generation to generation –Except for mutations • Therefore Y and mtDNA are known as lineage markers • Lineage markers are passed down from generation to generation without changing • They can help determine the lineage (family tree) of an individual • Y Chromosome Markers –Determine Paternal Lineage Lineage Markers

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• Y chromosome has two parts: –Non-Recombining Portion –Y only –PAR –recombine with the X Chromosome • Third smallest chromosome • More than half of chromosome is heterochromatin –no genes • Contains many repeats and palindromes • For this reason primers sometimes may bind to more than one region of Y Y Chromosome

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Y Chromosome STRs

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Y-STRs •Y chromosome contains same sort of variation as autosomes: – STRs – SNPs – Alu repeats – Minisatellites • STRs give most information – More alleles per marker – High mutation rate Mutation Rates • Mutation rates calculated for Y-STRs is in same range as autosomal STRs: – 1-4 per 1 thousand meiosis

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Of all Y chromosome polymorphic STRs DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393 and YCAII have more data accumulated, being the most used in population genetics. Additional Y-STRs have been introduced: DYS434, DYS435, DYS436, DYS437, DYS438 and DYS439 ( Ayub et al. ) DYS460 (GATA A7.1), DYS461 (GATA A7.2), GATA-10, GATA-C4 and GATA-H4 ( White et al. )

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DNA Sequencing Sequence determination for a given genomic region provides the most fine grained genetic information DNA sequencing offers the advantage of no ascertainment bias like the SNPs Species with low polymorphism, sequencing a large fraction of invariant sites could be informative One more advantage of sequencing is the analytical framework for demographic analysis is well developed

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Markers Advantages Disadvantages SNPs Low mutation rate High abundance Easy to type Cross study comparison easy Expensive Ascertainment bias Low information content Microsatellites Highly informative Low ascertainment bias Easy to isolate High mutation rate Complex mutation behavior Not abundant enough RAPDs & derivatives Cheap Produce large number of bands that can be analysed individually Low reproducibility Mainly dominant Difficult to analyze Cross study comparisons difficult Comparison of Different Molecular Markers

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Markers Advantages Disadvantages DNA sequencing Highest level of resolution possible Not biased Cross study comparisons are easy More expensive mtDNA polymorphism High mutation rate Rapid detection of hybridization between species No recombination No paternal lineage can be analyzed Comparison of Different Molecular Markers ( Schlotterer C , 2004 )

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India is one of the mega biodiversity centers for the world & rich in farm animal diversity Diversity Study on Indian Context

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Diversity Study on Indian Context Species Population ( million) Cattle 199 Buffalo 105 Sheep 71.55 Goat 140.53 Horse 0.611 Camel 0.517 Pig 11.134 Total Livestock 529.698 Livestock Population of India

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Diversity Study on Indian Context Diversity of Species in India

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Species Diversity in India compared to the World Group No. of species in India No. of species in World % of the world in India Mammals 350 4629 7.6 Birds 1224 9702 12.6 Reptiles 408 6550 6.2 Amphibians 197 4522 4.4 Fishes 2546 21730 11.7

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Most diversity studies in India is done by the Microsatellite markers Diversity Study on Indian Context The Food and Agriculture Organization (FAO) suggests that five different alleles per locus are required for estimation of genetic differences between breeds. Various diversity estimates, mean number of alleles (MNA); effective number of alleles; gene diversity, and observed heterozygosity used for the existence of substantial within-breed diversity through microsatellite data

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Study on Zebu Cattle A) 7 microsatellite loci namely ETH225 , CSRM60 , HEL5 , INRA005 , INRA035 , ILST002 , & ILST006 studied for Gir, Deoni & Kankrej breed suggesting high polymorphism of the 7 loci & high genetic variation among them ( Kale D S et al., 2010 ) B) Genetic polymorphism in the two cattle breeds, Ongole and Deoni were analyzed by using 10 microsatellite marker, where 8 microsatellite markers showed polymorphism while the remaining two markers ARO23 and PZE46 were monomorphic in both the breeds suggesting moderate genetic differentiation ( Nagaraju J et al., 2004 )

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C) Hallikar cattle were assessed using 5 microsatellite markers (ETH10, ETH225, ILSTS006, ILSTS011 and TGLA122) revealing the polymorphic nature of those microsatellite loci. ( Chandra Sekhar M et al., 2011 ) D) 25 microsatellite markers in Krishna Valley cattle were screened in which the percentage of polymorphic loci obtained was 96, since all microsatellite loci, except ILSTS030, exhibited polymorphism. ( Karthickeyan S M K et al., 2006 ) Cont...

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E) 6 native cattle breeds Rathi, Tharparkar, Nagori, Mewati, Gir, and Kankrej were studied based on microsatellite loci to understand the existing genetic diversity and structure. ( Sodhi M et al., 2011 ) Cont... F) A set of 20 microsatellite markers namely ETH225, ETH10, ILSTS006, HEL1, ILSTS011, MM8, BM1818, INRA005, INRA063, INRA035, CSRM60, HAUT27, ILSTS030, ILSTS054, ILSTS033, ILSTS005, HEL9, BM1824, ILSTS034 recommended for cattle in FAO’s DADIS MoDAD programme were utilized for generating microsatellite genotyping data. ( Sharma R et al., 2009)

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Study on Buffalo 27 microsatellite loci BMS4012, BMS4016, BMS2519, BMS2847, ILSTS089, MSBQ 27, AFR227, CA004, BMS1724, TGLA159, BM1352, BM4513, RM372, BMS1226, 0TGLA36, BMS1316, ILSTS058, BL1029, CSSM047, BL1036, BL1134, BMS518, BMS462, BM7579, BMS2325, BMS2116, BMS1747 were used to define genetic variation and relationships among 8 Indian riverine buffalo breeds. ( Kumar S et al., 2006 )

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Parameter Bhadaw-ari Nagpur- i Surti Pandhe-rpuri Toda Mehsa-na Murra -h Jaffarba-di Mean no. of alleles 7.15 6.60 6.63 6.74 6.15 7.19 7.19 6.56 Observed Heterozygosity 0.71 0.70 0.69 0.63 0.66 0.67 0.69 0.70 Expected Heterozygosity 0.77 0.75 0.75 0.75 0.71 0.76 0.78 0.76 HW deviated loci BMS462 BMS4012 BMS1724 BMS4012, BMS1724, BMS462 BMS4012, MSBQ, CA004, BMS1724, BL1029, BMS518,BM757 ILSTS089 BMS4012, BMS1724, BMS1747 BMS1724, BL1134, BMS462 BMS1724, BL1134, BMS1747 Statistics of Variation in Buffalo Species ( Kumar S et al., 2006 )

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DNA fingerprinting & RAPD markers were used for studying genetic relationship among 6 Indian Goat breeds namely- Barbari, Black Bengal, Jamunapari, Marwari, Sirohi & Jhakrana . ( Yadav & Yadav , 2008 ) 25 microsatellite markers each for Spiti & Zanskari breed of Horses were studied for their genetic characterization respectively ( Chauhan M et al., 2004), ( Behl R et al., 2006 )

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Polymorphism of 13 microsatellites in two Indian domesticated pig types ( North Indian and Northeast Indian ) was studied. The evaluated microsatellites exhibited a very high heterozygosity and polymorphism information content that may be used with reliability for studying the genetic diversity and for identification of individuals in Indian pig types. ( Kaul R et al., 2001 ) 25 microsatellite loci for Magra breed of Sheep was studied ( Bhatia et al., 2006 )

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16 microsatellite markers each for Bikaneri & Kachchhi breed of Camels were studied for their genetic characterization respectively ( Mehta & Sahani, 2007 ), ( Mehta S C et al., 2007 ) Ankamali pigs, the domesticated native pigs of Kerala province of India were genetically characterized using 23 FAO recommended microsatellite markers. ( Behl R et al., 2006 )

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Conclusion Although several works have already been done for this study, but still there is huge necessity of biodiversity assessment in the context of huge proportion of livestock animal in this sub continent. Microsatellite markers are surely promising but shifting to SNPs for the same is on the way with prospective availability & high throughput technology for further knowledge of genetic relationship, phylogeny, demographic characterization & origin of domestication & above all genetic improvement of livestock.

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Dr. Avtar Singh, Principal Scientist, DCB Division, NDRI, Karnal Dr. B. R. Yadav , Principal Scientist, DCB Division, NDRI, Karnal Dr. I. D. Gupta, Principal Scientist, DCB Division, NDRI, Karnal Dr. A. K. Chakraborty , Principal Scientist, DCB Division, NDRI, Karnal Acknowledgement