Endang Tri Margawati

Quantitative Trait Loci (QTL) Analysis for Production Traits of Birth Weight and Weight 360 days in Backcross Sheep

Four half-sib families (n = 382) consisting predominantly of ITT × Merino × Merino backcross progeny, including some F2 progeny were used to analyze QTL for two production traits (Birth weight = BW1 and Body weight at 360 days = BW360). The study exploited differences in weight performance between the Merino and ITT sheep. A total of 141 informative microsatellite markers were used in a genome-wide scan covering the 26 autosomal sheep chromosomes. QTL analysis was conducted online using QTL Express. This study reports the effect of QTL for birth weight on Chromosomes 5 (p ≤ 0.05) at 112cM (0cM-128cM). Location of candidate genes for birth weight was predicted at the region of flanking markers MCM527-BMS1247. A QTL for BW360 days existed on Chromosome 18 (p ≤ 0.01) at 104cM (25.0-125cM). Location of candidate genes related to production traits for body weight 360 days was predicted at the segment of flanking markers of CSSM018-TMR1. Only the QTL on Chromosome 18 retained significance (p ≤ 0.01) under experiment-wide significance testing. This QTL region is being examined for candidate genes by investigating to the homologous human chromosomal segments.

Ovine Disease Resistance: Integrating Comparative and Functional Genomics Approaches in a Genome Information-Poor Species

In combination with goats, sheep represent the two most numerous agricultural species for which no cultural or ethical restrictions apply in their use as a source for milk, fibre and red meat. Particularly, in the developing world these species often represent the sole asset base for small-holder livestock farmers. Despite their global significance, genomic tools and approaches in disease resistance have lagged behind the efforts in the economically more influential beef and dairy cattle industries. In particular, infectious diseases have a significant economic impact on livestock production systems worldwide. The most frequently investigated diseases in sheep have focused on the economically important burdens including gastrointestinal nematodes, dermatophilosis, footrot, myiases and fasciolosis. In this study we describe the use of Indonesian Thin Tail sheep (ITT) as a resource which has been shown to have innate and acquired resistance to tropical fasciolosis (Fasciola gigantica). Using the contrast between the resistant ITT and the highly susceptible Merino in a combined functional and comparative genomics approach, we have identified putative QTL (quantitative trait loci) for an extensive panel of parasite and immune response phenotypes and putative resistance pathways and effector molecules. On refinement of candidate gene analyses and effector mechanisms we propose to map these in the economically important target species, namely cattle and buffalo. In addition we exploit the relative susceptibility of ITT sheep to temperate fasciolosis (Fasciola hepatica) to contrast parasite–host interactions and identify parasite immune evasion strategies to boost the discovery of new vaccine candidates and effector pathways, which may be amenable to exogenous control. The study highlights the power and utility of direct gene discovery in ruminant model systems. To overcome the shortage of genomic tools required for such investigations, we have drawn on the development of integrated comparative maps and alignment H.W. Raadsma Reprogen-Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, University of Sydney, Camden NSW, Australia J.P. Gustafson et al. (eds.), Genomics of Disease, 89 C

Phylogenetic analysis of Aceh cattle breed of Indonesia through mitochondrial D-Loop region

The objective of this research was to find the basic data on genetic diversity of mtDNA D-Loop in Aceh cattle and its association with Bhutanese, Chinese, and Indian cattle. There were sixty samples of DNA which had been sequenced; i.e. Banda Aceh (11), Saree (20), and Indrapuri (29). To the best of our knowledge this is the first published data on the complete mitochondrial D-Loop sequence of Aceh cattle. Results show that Aceh cattle have the closest relationship to Bos indicus and have been influenced by Bos taurus. The closest genetic ranges among Aceh cattle, Bhutanese, Chinese, Indian and Zebu were Aceh–Zebu (0.0138), Aceh–Bhutanese (0.0156), Aceh–Chinese (0.0190) and Aceh–Indian (0.0193). D-Loop mtDNA analyses showed that there were 27 haplotypes in which twenty-one samples spread in haplotype 1, two samples were in haplotype 2, and the other four haplotypes had various samples in the range of three to seventeen samples. One sample of Aceh cattle from Saree has a closest maternal genetic with B. taurus. One of the four mutations among the star-shaped clusters on median joining network was a new specific haploid-group in Aceh cattle. From this finding it could be assumed that Aceh cattle form a specific haplotype and it can be conclude that Aceh cattle are animal genetic resources from Aceh in Sumatera Island that have to be preserved.

Optimization of expression JTAT protein with emphasis on transformation efficiency and IPTG concentration

One of small accessory genes between pol and env is tat gene encoding TAT protein. This research was aimed to optimize the expression of Jembrana TAT (JTAT) protein with preparing Escherichia coli (E. coli) in advance using adopted methods of M1 (MgCl2 + CaCl2) and M2 (CaCl2 + Glycerol). The best transformation efficiency resulting from a better transformation method was used to subsequent expression of JTAT protein. A synthetic tat gene encoding protein JTAT was previously cloned into pBT-hisC. Concentration of 200; 400; 600 µM IPTG was induced to a small volume culture (200 ml; OD600 = 4), incubated for 3 h. Pellets were harvested by centrifugation (4000 rpm; 4 °C; 15 min). Buffer B (10 mM Immidazole) was added into pellets, lysed by freeze-thaw followed by sonication. Supernatant was collected by centrifugation (10,000 rpm; 4 °C; 20 min) and purified using Ni-NTA Agarose resin, released by elution buffer (E) containing 400 mM Immidazole to collect purified protein twice (E1, E2). The protein was characterized by SDS-PAGE and Western Blot (WB), quantified (at λ595 nm) with BSA standard method in prior. The result showed that transformation efficiency was better in M2 (2.53 × 106) than M1 (3.10 × 105). The JTAT protein was expressed at a right size of 11.8 kDa. Concentration of 200 µM IPTG produced a significantly better protein yield (1.500 ± 0.089 mg/ml; P < 0.05) than 600 µM IPTG (0.896 ± 0.052 mg/ml) and not different to 400 µM IPTG (1.298 ± 0.080 mg/ml). This research indicated that transformation efficiency needs to be taken account in prior of optimization of the protein expression.

PENGUJIAN PENCEMARAN DAGING BABI PADA BEBERAPA PRODUK BAKSO DENGAN TEKNOLOGI PCR: PENCARIAN SISTEM PENGUJIAN EFEKTIF [Analysis of Porcine Contamination by Using PCR Technology in Several Meat Ball Products: To Find an Effective Assessment System]

Entering globalization market, Indonesian government could not reject any import of food products from overseas. To anticipate the possibility of porcine contaminants into processed meat products of imported food such as meat or chicken ball, sausage, tin meat etc., it is important to apply laboratory research on such particular matter in regard to ethical and certain religious concern. This study was intended to identify the possibility of porcine contaminants into either processed meat products or fresh meat.A technique of polymerase chain reaction (PCR) was applied and PCR optimizing was conducted in advanced to obtain the right annealing temperature.Positive control of fresh pork meat was amplified to get porcine Leptin size which is 152bp fragment. Five samples of 4 meat balls and one fresh beef meat were individually collected for their DNA by either from minced or mashed after liquid nitrogen exposure then followed with a series of DNA extraction steps. PCR was assigned by using a specific primer of Leptin gene for porcine identification.Visualization of Leptin fragment was applied either on 1%, 2% of agarose gel or 10-20% gradient acrylamide gel.The result showed that all sample applied were not identified for containing porcine contaminants while positive control was on the right size of 152bp of Leptin gene. Specific primer used in this study was proved that there was not identified porcine Leptin gene on the negative control (fresh beef meat). This study suggests that a method of PCR is a simple analytical method for identification of porcine contaminants and visualization on 2% agarose gel is a cheaper and quicker method while by gradient acrylamide gel showing more clear band however this method is time consuming and expensive.

In Vitro Development of Ovine Embryos Following Maturation Under Limited CO2

An experiment was conducted to examine the influence of CO2 during in vitro oocyte maturation on the in vitro ovine embryo development. Three treatments of CO2 were subjected to the oocyte development. Those were 2h gasses prior to maturation in incubator (T1); without CO2 either prior to or over maturation (T2) and CO2 exposure both prior to and over 22h maturation (T3). A total of 324 oocytes were used. Putative zygotes were cultured for seven days and evaluated for their developmental stage. Presence of CO2 (T3) increased the proportion of oocytes reaching Metaphase II ( 66.50 + 3.5%; p 0.05). This study suggests that it is possible to mature ovine oocytes in the absence of CO2 without loss its potensial development. It may therefore be an effective method of maturing ovine oocytes during transportation to IVP (in vitro production) laboratory.