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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access GENOME-WIDE SEARCH FOR GENETIC DIVERSITY, SELECTION SIGNATURES AND BREED SPECIFIC MARKERS IN NATIVE GOAT BREEDS OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2020-12-16) MARYKUTTY THOMAS; Radhika G.Population genomics study was carried out in Attappady Black and Malabari goats, the two native goat breeds of Kerala with objectives of (1) genome wide assessment of genetic diversity (2) genome wide scan for selection signatures (3) detection and validation of breed specific markers (4) identification, validation and association analysis of prolificacy linked candidate SNP markers. Whole genome SNP genotypic data obtained by high throughput genotyping of reference population (24 each of Attappady Black and Malabari goats) using goat SNP50 BeadChip were used for genome wide scans. After quality control, 47,100 polymorphic SNP markers were available for downstream analysis, demonstrating low influence of ascertainment bias. Analysis of autozygosity and demographic history using runs of homozygosity (ROH) revealed mean ± S. E. of ROH length (Mb) as 4.02±0.38 and 6.00±0.53 in Attappady Black and Malabari goats respectively. The higher proportion of short ROH segments (2 to 4 and 4 to 8 Mb) in both breeds denoted ancient inbreeding due to common ancestors between 6 to 25 generations ago. Genomic inbreeding coefficient based on ROH was extremely low and was 0.07 and 0.52 per cent in Attappady and Malabari goats respectively. Investigation of linkage disequilibrium (LD), considering 0.794 million SNP pairs showed low and comparable level of LD (r2=0.07) between adjacent SNP markers (marker interval, 0.17 Gb) in both breeds, indicating the high genetic diversity and low selection pressure in native goats. The LD estimates also denoted the requisite for a denser SNP array to capture useful genomic information for selection and association analysis. Historical effective population size estimated from LD decay was 518 and 371 in Malabari and Attappady Black goats respectively at 17 generations back. Other population genetic indices like minor allele frequency (0.30), observed heteroygosity (0.38), expected heterozygosity (0.39) and FIS (0.03) also supported genetically diverse nature of native goats. Though genetic differentiation among two breeds was low (FST, 0.018), on Principal Component Analysis, first two principal components which accounted for 0.09 proportion of variance, grouped the sampled animals in two distinct breed clusters. Model based clustering using STRUCTURE showed that relative proportions of Attappady Black and Malabari ancestral blocks at k=2 (based on least cross validation error and k =number of sub populations) were 0.93 and 0.07 respectively in the genome of Attappady Black and 0.12 and 0.88 respectively in the genome of Malabari goats. This implied the distinct ancestral influence of two breeds. Selection signature analysis that facilitates detection of genomic regions under natural and artificial selection was performed using outFLANK and hapFLK analysis. The outFLANK analysis that detects ancient selection signals, detected 104 numbers of highly differentiated SNP loci at genome wide levels of significance (p<0.001). Gene ontology analysis using DAVID identified important genes that are related to coat colour (MITF and WNT2) and mammary gland development (FGFR2 and GLI2) in ancient sweep regions. The hapFLK analysis that exposes recent selection signatures yielded seven significant (p<0.005) sweep regions including the genomic area that harbours casein cluster and quantitative trait loci for milk production on chromosome 6. Gene ontology enrichment analysis of 166 putative selective genes that was linked to recent sweep regions using Panther listed significantly (p≤0.05) over-represented 13 Panther pathways that included TGF-beta signalling pathway and GnRH receptor pathway. Development of breed genetic traceability for Attappady Black and Malabari goats could enhance breed profitability and sustainability of its production system. Breed genetic traceability for Attappady Black goats was developed by adopting deterministic approach using breed specific markers. High throughput genome wide SNP genotypic data of reference goat population was used to identify candidate breed specific markers. Nine putative Attappady breed specific markers were selected and were validated for breed specificity by converting them into PCR-RFLP markers and genotyped in large sample goat population. On validation, four markers retained their breed specificity with probability of identification (Pi) ranging from 0.24 to 0.66. Panel of four Attappady Black breed specific markers yielded Pi of 0.92. Similar protocol was adopted for genetic breed traceability of Malabari goats as well. Seven candidate Malabari breed specific markers were selected and further genotyped in large sample goat population by PCR-RFLP. Three candidate markers confirmed their breed specificity on validation, with Pi ranging from 0.21 to 0.56. Panel of three Malabari breed specific markers had Pi of 0.73. Genome wide screening of high throughput genotypic data of reference goat population for candidate SNP markers useful for litter size trait selection in native goats of Kerala identified 49 putative candidate SNP markers. Population genetic analysis of putative candidate markers based on genotypic data of reference goat population revealed that five candidate SNPs were either significantly (p≤0.05) deviated from Hardy Weinberg equilibrium (HWE) or with significant (p≤0.05) genotype frequency differences among two breeds. These five candidate markers that were on SETDB2, SOX6, AHCTF1, FAT4 and USP16 genes were genotyped in large population by PCR-RFLP method and analysed for their association with prolificacy trait. Population genetic analysis based on genotypic data in large population showed that SETDB2, SOX6 and USP16 were significantly (p≤0.05) deviated from HWE denoting potential selection. Association analysis illustrated that genotype of USP16 gene variant had highly significant (p≤0.01) influence on litter size trait of native goats of Kerala. The USP16 gene variant could be a useful SNP marker for litter size enhancement in native goats of Kerala.ThesisItem Open Access CHARACTERISATION AND EVALUATION OF THE DWARF CATTLE OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 1994) C. R. GIRIJA; Sosamma lypeThe native cattle of Kerala have been treated as non descript animals always eventhough they possess some special features. The dwarf cattle often called as Vechur were very popular in Central Travancore until 35 years back. with the emergence of the crossbred population of cattle the traditionally reared local cattle have gradually suffered genetic erosion. Under this circumstance, the present work was undertaken to characterise and evaluate the germplasm of local dwarf cattle of Kerala by studying (a) the karyotype and morphology of chromosomes using G-banding (b) the population structure by means of gene frequencies of different blood proteins (c) the growth and production performance. The characterisation and the evaluation would help in finding out the genetic differences of the dwarf cattle which will help in deciding about the conservation of their germplasm as a reserve for the future. The dwarf cattle maintained under the ICAR scheme on "Conservation of germplasm of Vechur cattle of the coastal area and the dwarf cattle of the high ranges of Kerala" formed the material for the study. The characterisation and evaluation was carried out through the cytogenetic, immunogenetic and polymorphism studies as well as through the description of the growth and production traits. Karyotype analysis was carried out using peripheral blood leukocyte culture technique described by Hainan (1977) and Hainan (1989) with suitable modifications. G-banding of chromosomes were done by the method described by Thiagarajan, ^19 9!^. Blood protein polymorphism systems^ such as Haemoglobin and transferrin were studied by poly acrylamide gel electrophoresis in horizontal dimension ' y(Cahne et al. ,1977) with suitable modifications. The statistical analysis of the growth and production data were done as suggested by Snedecor and Cochran (1967). The diploid chromosome number of the dwarf cattle was found to be 60, with 29 pairs of autosomes and one pair of sex chromosomes. All the autosomes and the 'Y' chromosome were acrocentric. The X chromosome was submetacentric. The relative length of the autosomes ranged from 1.757 to 5.431 per cent. The relative length of the X and Y chromosomes were found to be 5.591 per cent and 2.875 per cent respectively. In the karyological array, the X chromosome occupied the first position. The X chromosome was biarmed and the arm ratio and centromere index obtained were 2.182 and 0.314 respectively. The karyotype and morphometric measurements resembled the finding in Bos indicus group of cattle. The G-banding pattern of chromosomes revealed 72 regions and 314 G-bands. The Y chromosome had 7 G-bands in the 'q' arm which resembled the 'q' arm of Bos taurus described in the international system for cytogenetic nomenclature of domestic animals. A B There were two haemoglobin variants Hb and Hb and three phenotypes viz. Hb^^, Hb^® and Hb®^, in the population. The heterozygocity was found to be 0.4815. The population was found to be in genetic equilibrium with respect to the Haemoglobin locus. Six transferrin phenotypes controlled by three alleles Tf^, Tf*^ and Tf^ were observed. The frequency of Tf^ (0.359) allele in the dwarf cattle was as high as the frequency of the allele reported in the zebu cattle. The absence of F H N G transferrin variants like Tf , Tf , Tf and Tf and higher frequency of Tf allele are probably indicative of the genetic isolation of the population from exotic breeds. The absence B F of Tf and Tf allele which is present in Gir, Hariana, Kankrej, Kangayam, Ongole, Red Sindhi, Sahiwal and Tharparkar also indicates that the dwarf cattle has not inherited genes from the above cattle breeds. The body weights and measurements of calves at birth studied showed that the male calves had a higher body weight (12.55 + 0.311 kg with a CV of 7.86 per cent) than female calves (10.78 + 0.404 kg with a CV of 15.02 per cent). The same trend was observed with regard to the birth body measurements also. The heart girth measurement and body weight showed a positive correlation from birth to the 24th fortnight. There is a 100 per cent increase in the birth weight by the 5th fortnight and a three-fold increase by the 10th fortnight. The average daily gain in weight for the four periods i.e., fortnights 0-6, 7-12, 13-18 and 19-24 were 0.160 + 0.011, 0.167 + 0.018, 0.212 + 0.011 and 0.139 + 0.015 respectively for female calves, and 0.188 + 0.023, 0.145 + 0.016, 0.116 ^ 0.025, 0.242 + 0.049%respectively in male calves. During the period from birth to 6th fortnight the growth rates in males and females were similar. The gain in body weight per day during the periods from 7 to 12th and 13 to 18th fortnight was comparatively less for males but the trend reversed during the period of fortnights for 19 to 24th. The average body weights of adult females and males were 126.90 + 3.56 kg (CV 16.39%) and 210 + 15.75 kg (CV 14.95%) respectively. The body measurements such as length, heart girth and height (in cms) in females were 97.5 + 1.12 (CV 5.85%), 115.60 + 1.32 (CV 5.82%) and 87.53 + 0.82 (CV 4.82%) respectively. The corresponding figures in males were 111.5 + 3.77 (CV 6.76%), 146.0 + 2.92 (CV 3.99%) and 107.5 + 1.35 (CV 2.50%) respectively. The average body weights and measurements were lesser than those reported in other Indian breeds and crossbred cattle. The total lactation milk production performance of the dwarf cattle was 471.68 + 38.72 kg (cv 45.29%) in an average lactation length of 217 + 16.50 days (CV 32.20%). The average daily yield was 2.17 + 0.11. kg (cv 29.48%). • The dwarf cattle attained a peak yield of 3.71 + 0.16 kg (cv 21.5%) in 23.23 + 1.703 days (CV 37.38%). The milk production performance eventhough was lesser than crossbreds or some recognised Indian breeds, the milk production in comparison with the body size was reasonable. Considering the morphology of the Y chromosome, the Hb as well as Tf polymorphism and their allelic frequencies, it is to be summarised that the stock of dwarf cattle of Kerala maintained at Kerala Agricultural University is gehetically isolated from the other cattle breeds of the country and world. The body size and milk production of the cow indicates its suitabMity for a farmer who requires milk just for home consumption. The study strongly confirms the necessity of conservation of the dwarf cattle of Kerala which is the smallest variety available in India and perhaps in the world itself.ThesisItem Open Access STUDY OF GENETIC DIVERSITY IN MALABARI GOATS (Capra hircus) UTILIZING BIOCHEMICAL AND IMMUNOLOGICAL MARKERS(College of Veterinary and Animal Sciences, Mannuthy., 2006) K. A. BINDU; Dr.K.C.raghavanABSTRACT Goat populations of Tanur, Thalassery and Badagara were studied for biochemical polymorphisms, immunological and microsatellite markers to investigate the similarities and differences between these populations. With regard to biochemical markers tested, polymorphism was observed only for haemoglobin, transferrin and glutathione loci. Two variants were observed for haemoglobin, Hb and Hb with a frequency of 0.987 and 0.012, respectively, suggestive of three phenotypes, viz. Hb aa, Hb ab and Hb bb, and indicating the predominance of Hb in the pooled population. Hb ^ variant was observed only in the Thalassery population (gene frequency 0.038). Two variants for transferrin (Tf^ and Tf were detected with a predominance of TC in the population. All the goats from Thalassery population belonged to Tf aa type. In the present study only two phenotypes as regards transferrin locus could be observed, (Tf AA and Tf bb) with,the notable absence of Tf ab- No polymorphism was observed for albumin, cerruloplasmin, amylase and carbonic anhydrase loci in all the animals tested. The animals were classified as low and high glutathione types based on the values obtained for blood glutathione concentration. In the pooled population, majority of the animals belonged to low GSH type (53.68 per cent). The least square analysis of glutathione concentrations showed significant variation between populations. With regard to potassium loci, all the animals in the present study belonged to low potassium type, with the mean potassium concentration of the pooled population recorded at 4.18 :L0.09 meq/1. The least square analysis of variance of potassium concentrations showed that there existed significant difference between different sub-populations. Genetic distance was calculated as described by Balakrishnan and Sanghvi (1968), using the allelic frequencies of protein polymorphic loci. Genetic distance between Tanur and Badagara was found to be 0.1249 , while that between Tanur and Thalassery was 0.6690 and between Badagara and Thalassery was 0.3351. The only possible conclusion that could be arrived at from the above studies is the existence of a relationship between these populations. Hence an attempt was made to study the different populations at molecular level, using microsatellite markers. Three markers, viz. INRA 063, HUJ 1177and ILSTS 030 were found to be polymorphic. Based on the genetic distances, it was found that Thalssery and Badagara were closely related than Tanur population.. This finding, much in agreement with biometrical traits, reiterates the close relationship between the Thalassry and Badagara populations. On an average, the goats of Thalassery and Badagara were heavier in comparison to Tanur goats, though the prolificacy remained higher in Tanur animals than the other two populations. The different sub-populations under the present study were also screened for the antibody response to SRBC. The highest concentration of antibody was observed on day seventh after primary immunization. The titre gradually reduced by the 15"^ day, reaching the lowest values on IT"^' day of post immunization. The effect of antibody response to SRBC on the 7"^, IS"*" and 2C days post immunization was not found to be significant for the occurrence of diseases like diarrhoea and pneumonia. The cutaneous response to intradermal injection to Phytahaemagluttinin - M (PHA-M) was also studied to find out the differences, if any in and between the various sub- populations under study. The values for skin thickness were maximum at 24 hours post-intradermal injection of PHA-M and were recorded as 3.24±0.05, 3.23±0.06 and 3.33±0.06 mm in Tanur, Thalassery and Badagara, respectively. The skin thickness reduced considerably after 48 hours and reached 1.61±0.02, 1.62±0.02 and 1.65±0.02 mm, respectively at 72 hours. The least square analysis of variance revealed that the values for pre and post immunization skin thickness at 0, 24, 48 and 72 hours were non significant between different sub-populations. Total protein, albumin and globulin concentrations also were estimated. The highest mean concentration for globulin was detected in Badagara population (3.28±0.22g/dl) and the lowest in Tanur (2.340 ± 0.3]g/dl). The least square analysis of variance of globulin concentration revealed significant difference between populations. Though all populations under study had all predominant physical characteristics of the Malabari breed, the Tanur population stood apart as regards the biometrical characteristics, like litter size and body weight and charecteristics perceivable at the molecular level. It could well be inferred that this population might have evolved through mixing up of the local nondescript Tanur goats with original Malabari goats. The study reiterates the need for more research activities directed at exploring the chances of conserving and developing such unique populations within a breed.ThesisItem Open Access PORCINE IMMUNE RESPONSE AS MARKER TRAITS FOR SELECTIVE BREEDING(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2002) . RAJAN, M. R; RAGHUNANDANAN, K.V.Survivability and better performance of pigs under tropical stress have been reported to be significantly influenced by immune responses. Immune response traits under genetic control offer potential possibilities for exploited in commercial pig production. The present research project on the utilisation of porcine immune responses by estimating the magnitude of humoral and cell mediated immune responses in Desi and Large White Yorkshire attempted to evaluate the genetics of immune responses and to identify the association between the immune response traits and economic traits. The immune response traits were studied in 150 piglets aged between two to three months, 75 each belonging to Desi and Large White Yorkshire of both sexes and sired by eight sires each. The immune response traits studied were antibody response to sheep red blood cells (SRBC), delayed type hypersensitivity (DTH) to intradermal injection of PHA-M and lymphocyte transformation response to BCG. The economic traits recorded were litter size at birth, litter weight at birth, litter size at weaning, litter weight at weaning, weaning mortality and the occurrence of diarrhoea and pneumonia. Naturally occurring antibodies to SRBC could not be detected in both the breeds. Peak antibody response to SRBC was obtained at day 7 post immunisation with a mean titre of 4.830. Heritability estimates of antibody response to SRBC were 0.8969 + 0.4235. 0.9187 ± 0.4893 and 0.8174 ± 0.4893 respectively at 7*^ day, 15"' day and 21" day post immunisation. Litter size at birth and weaning had no significant influence on antibody response. Similarly, antibody response to SRBC among piglets was not influenced by the incidence of diarrhoea, pneumonia and pre-weaning mortality to a significant level. DTH responses to intradermal injection of PHA peaked at 24 hours post injection with a mean value of 3.39 mm. The mean pre injection skin thickness was 3.508 mm and 3.012 mm among Large White Yorkshire and Desi pigs respectively. This difference was found to be significant (P<0.05) and this difference was due to the significant breed difference confounding with body weight classes. The effect of breed on PHA responses at 24, 48 and 72 hours were not significant. Sex of the pig also did not influence the PHA responses significantly. The body weight classes did not influence the DTH response to PHA significantly. Sire effect was not significant on the pre injection skin thickness. But the DTH response at 24 hours was influenced by the sires in both Large White Yorkshire and Desi pigs to a highly significant level (P<0.01). At 48 and 72 hours post injection also DTH responses were influenced by sires to a significant level (P<0.05). The heritability estimates for pre injection skin thickness and DTH responses at 24, 48 and 72 hours were 0.5173 + 0.4179, 0.8136 + 0.5643, 0.6816 + 0.5187 and 0.7134 ± 0.5283 respectively. The litter size at weaning was not influenced by the initial skin thickness. DTH responses to PHA at 24, 48 and 72 hours had no significant influence on the litter size at birth and weaning. PHA responses at 24, 48 and 72 hours were not influenced significantly by the incidence of diarrhoea, pneumonia and pre weaning mortality. The analysis of lymphocyte transformation and stimulation index to BCG on zero day was around one indicating that there was not marked increase in the lymphocyte multiplication in PPD stimulated samples. The stimulation index on 15"'' day was 6.0161 in Large White Yorkshire and 6.3340 in Desi. This index further increased to 6.1070 and 6.5920 on 30'^' day and began to decline from 45"' day with a mean value of 6.0020 in Large White Yorkshire and 5.9890 in Desi pigs. The effect of breed, sex and body weight class of piglets was not found to influence the stimulation index significantly. Sire effect was not significant on the pre inoculation index while it was highly significant on 15*^. and 45*^ day in Large White Yorkshire and Desi. The estimates of heritability on 30"^ and 45*^ day stimulation index were 0.5171 ± 0.2893, 0.6289 + 0.3817 and 0.4983 ± 0.2583 respectively. Litter size at birth and weaning was not found to have any significant influence on the LT response to BCG. Correlation analysis among different immune response traits revealed that antibody response to SRBC at 7, 14 and 21'' day had highly significant positive correlation. Similarly, the correlation between PHA responses at 24, 48 and 72 hours were also highly significant and positive. PHA responses at 24 hours and LT responses at 15*^ day was also positive and significant. LT responses at 15*^ and 30th day were also significant and positive. The association between LT responses during different time intervals were always positive and significant. PHA responses were always negatively correlated with initial skin thicknesss to a significant level. Antibody response at 7, 15 and 21" day had a significantly high negative influence on the body weight at weaning. There was a significant decrease in pre weaning mortality associated with LT response at 15th day.ThesisItem Open Access DELINEATION OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS IN CROSSBRED CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2003) ANILKUMAR, K.; RagiTunandanan, K. V.A study was conducted to delineate the Random Amplitied PoKmorphic DNA markers of crossbred cattle of Kerala. DNA was isolated from whole blood, fresh semen or frozen semen samples for this study. Three panels of DNA samples were used in this stud)'. The)' were DNA samples of 84 unrelated crossbred cattle, which was used for assessing the RAPD polymrophic patterns. DNA samples from 52 offspring, dam and sire combinations for assessing the possibility of using the technique in parentage identification and DNA samples ot 108 dwarf cattle for microsatellite analysis. The average yields of DNA obtained from whole blood was 256 ± 2.12|ig/ 5 ml. fresh semen was 241.23 ± 8.42pg/ 400 million sperms and trom frozen semen was 91.32 ± 6.01 pg/ 0.25 ml straw: The optical density ratio calculated as an indicator of purity of the sample were 1.72 ± 0.14. 1.81 ± 0.26 and 1.61 ± 0.34 for the DNA obtained from whole blood, frozen semen and fresh semen respectively. Twenty random oligonucleotide primers from Operon kit A and six custom svnthesised primers were used for the stud). Based on intensit). clarity and polymorphism of bands. 19 primers were selected for amplification of DNA samples of crossbred cattle. Number of bands obtained from different primers ranged from 2 to 16. The acerage number of bands produced by different primers ranged from 3.78 ± 0.15 in OPA 4 to 8.15 ± 0.25 in ILO1127. The size of amplified products ranged from -230 base pairs to -3580 base pairs. Percentage of polymorphism represented by indi\ idual primer caried from 66.66 to 100 in different groups. Se\en primers namely 0PA2. OPA 4. OPA 17. OPA 18. OPA 20. ILO 1127 and ILO 876 yielded 100% polymorphic bands. Frequencies of the bands and their allelic frequencies were worked out. The largest average number of bands was produced by the primer ILO 1127 ( 8.15 ± 0.25)-and the lowest average number of bands was observed for the primer OPA 4 (3.78 ±0.15). The bands having frequency less than 0.25 were classified as rare bands. The primer OPA 15 produced 7 rare bands, the primer OPA 2 and OPA 19 five bands each, the primers for OPAl. 0PA4. OPA 8. OPA 9.OPA 16 and ILO 1127 four each and primers 0PA7. OPA 14. OPA 17 and ILO 876 three each.ILO 526oduced two rare bands, four primers namely OPA 10. OPA 12. OPA 18 and OPA 20 produced one rare band each . and no rare bands were observed for the primer Gl. Twelve RAPD primers were selected to study 52 offspring, dam and sire combinations. Non parent bands were observed in offspring to the extent of 2 to 13.6%. Three of these primers nameh' OPA 14. OPA 4 and 01 did not produce any non parent bands in offspring. It was concluded that due to presence of the non parent bands in offspring. RAPD-PCR technique cannot be the method of choice for parentage verification. Phenol chloroform procedure was used to isolate DNA from whole blood of 24 Vechur. 25 Highrange dwarf. 24 Vatakara and 35 Kasargode animals for microsatellite studies. The average yield of DNA obtained per 5 ml ot blood samples was 210.1 ±9.4 pg with the ratio of optical density at 260 nm and 280 nm as 1.86 ± 0.23. PGR conditions for the four microsatellite loci namely DRB3. ETH 131. HEL 6 and FSHp were standardised. The size of the alleles ranged from 176 to 236 bp for DRB3 locus. 160 to 184 bp in ETH 131 locus and 263 to 295 base pairs in HEL-6 locus. The number of alleles identified in different loci were 21. 14 and 13 respectively for DRB3. ETHI31 and HEL 6 loci. The PIC \ alue of the primers, direct count heterozygosity and unbiased heterozygosit) were worked Ill out. The identification ot new alleles in this stud) was attributed to the tact that dwarf cattle of Kerala are Bos indiciis . The possibilit> of using the microsatellite marker analysis for genetic characterization of dwarf cattle ot Kerala and their phylogenic studies are indicated in this work. Parentage \eritication tacilities can be established, where this technique can be employed in disputed cases.
