DNA Bands Research Articles

Molecular Diversity Analysis of Somaclonal Variants of Potato (Solanum tuberosum L.) by Random Amplified Polymorphic DNA Markers

Aims: An experiment was conducted to analyze the DNA fingerprinting and genetic diversity of nine potato (Solanum tuberosum L.) somaclonal variants and three check varieties.
 Place and Duration of Study: The experiment was carried out at the Biotechnology laboratory of the Department of Biotechnology, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh during November, 2013 to December, 2014.
 Methodology: The somaclonal variants investigated were SIP-3, SIP-5, SVP-6, SVP-18, SVP-19, SVP-25, SVP-55, SVP-56 and SVP-68, while the check varieties were Cardinal, Diamant and Asterix. Six RAPD primers were used to perform PCR reaction after genomic DNA was extracted from young leaves.
 Results: The selected 6 primers produced total 54 distinct and differential amplified DNA bands of size range 88 bp to 3265 bp, where 47 bands (~87%) were polymorphic and 7 bands (~13%) were monomorphic. The pair-wise inter-genotype similarity indices were ranged from 61.59% to 93.55% with an average of 74.31%. The Nei’s genetic distance among 12 potato genotypes was estimated from 0.0972 to 0.6217 whereas, genetic identity was between 0.5370 and 0.9074. Here, the distantly linked accessions among the somaclonal variations with check varieties were SVP-6 (to Cardinal and Diamant) and SVP-25 (to Asterix). In addition, the UPGMA dendrogram segregated the 12 potato genotypes into two broad clusters containing 8 and 4 genotypes, respectively. Furthermore, the dendrogram also displayed the highest genetic distance between SVP-6 vs SVP-68 genotype pair.
 Conclusion: Significant relationship and diversity were found among the studied 12 genotypes. This genetic diversity among the potato genotypes would be utilized for further potato improvement.

Cloning and Expression of Vacuolating Cytotoxin A (VacA) Antigenic Protein in Nicotiana benthamiana Leaves a Potential Source of the Vaccine against Helicobacter pylori

Background: The applications of transgenic plants in the healthcare system are immense. They offer an alternative strategy for the fabrication of antigenic determinants of medically important pathogens. Objectives: Cloning and transient expression of the vacuolating cytotoxin A (vacA) gene of Helicobacter pylori in Nicotiana benthamiana is undertaken in the present study. Methods: The vacA gene of H. pylori was amplified. The vacA and pBI121 vectors were digested with BamHI and SacI and the vacA gene was cloned in pBI121 by T4 ligation. The vacA-pBI121 construct was transformed into Escherichia coli DH5α and the transformants were confirmed by isolation and sequencing of vacA-pBI121. Further, the vacA-pBI121 was transformed into Agrobacterium tumefaciens EHA105 by electroporation. The transformants were used for agroinfection of N. benthamiana by agroinjection technique and the transgenic plant was screened for vacA gene expression by Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE). Results: The vacA gene amplification was confirmed by observing an intense DNA band in agarose electrophoresis. Sequencing of vacA gene of E. coli DH5α transformants indicated a gene size of about 2877bp which revealed 99.82% sequence similarity with online available H. pylori vacA gene sequence. The A. tumefaciens EHA105 transformants were confirmed by amplification of the vacA gene. The screening of transgenic leaves of N. benthamiana for vacA gene expression by SDS-PAGE showed VacA protein with a molecular weight of 105kDa. Conclusion: A novel transgenic plant expressing VacA protein was developed as a source of eco-friendly-based synthesis of antigenic determinants for various medical applications.

Role of Fok I VDR polymorphism in TB risk assessment; A Study in Central India population

PurposeStudy aim to investigate the possible role of Fok I VDR polymorphism in TB risk assessment. MethodsTo investigate the role of Fok I VDR polymorphism in TB risk assessment, we collected a blood sample from 106 healthy individuals including both gender. The genomic DNA was isolated and the VDR gene was amplified using gene-specific primers using polymerase chain reaction. The Genotyping of the Fok I polymorphism was performed using Fok I restriction enzyme. The distribution of genotype frequencies of VDR gene polymorphisms was calculated using Hardy-Weinberg equilibrium calculator. ResultsIn the present investigation, the VDR gene was amplified with an undigested DNA band of 265 bp as FF homozygote. Additionally, 169 bp and 96 bp as ff homozygote and three bands ((265 bp, 169 bp, and 96 bp) were observed in the heterozygote. As per our finding, we report a significantly low frequency of susceptible genotypes in Ff (27.4%) and ff (6.6%) compared to average frequency Ff = 41.80% and ff = 8.25% of India. We also reported here the low frequency of mutant allele f in India's central part, i.e., Bhopal division, Madhya Pradesh. Considering our finding, the mutant allele's low frequency in central India, Madhya Pradesh, shows a negligible contribution of Fok I VDR polymorphism in TB prevalence. ConclusionsIn a comparative analysis with the Northern and Southern India population based on previous reports, the involvement of FoK I polymorphism in the VDR gene was non-significant in Central India. All the studied populations were found in Hardy-Weinberg equilibrium except North India for the Fok1 VDR genotypes.

English

Macrophomina phaseolina is a highly destructive pathogen of more than 500 plant species. It is difficult to eradicate it through chemical means as no patented fungicide is available against this pathogen. Biological control is the possible alternative method for its suitable management. The present study was carried out to evaluate the biocontrol potential of five Trichoderma spp. against M. phaseolina and the possible mechanisms of action. Identifications of all the Trichoderma spp. viz. T. hamatum, T. harzianum, T. koningii, T. longipile and T. viride were confirmed on molecular basis by using two universal primer pairs namely ITS and EF1. Their biocontrol potential was evaluated in dual culture plate method where T. viride showed the highest inhibitory efficacy (63%) against M. phaseolina. T. koningii, T. hamatum and T. longipile showed akin effects by arresting growth of the pathogen by 46–47% followed by T. harzianum (28%). To find out the mechanisms of action, secondary extrolites of the best biocontrol fungus T. viride were tested against the pathogenic genomic DNA where all the concentrations partially degraded DNA bands after 24 h of incubation and a complete DNA band disappearance was noted after 48 h incubation. In addition, T. viride culture filtrates were partitioned with chloroform and ethyl acetate and subjected to GC-MS analysis for identification of potential antifungal constituents. The most abundant identified volatile compounds in the two organic solvent fractions were 9,12-octadecadienoic acid (Z,Z)- (44.54%), n-hexadecanoic acid (24.02%), hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (14.25%), 9-tricosene, (Z)- (10.43%) and [1,1'-bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester (10.43%). To conclude, T. viride was the best biocontrol agent against M. phaseolina and acts against the pathogen by DNA disintegration and production of antifungal secondary metabolites. © 2021 Friends Science Publishers

Molecular bird sexing of sulphur‐crested cockatoo (Cacatua galerita) by poly

Sex identification of endangered and protected birds in captivity is very important for conservation programs. Half of the world’s bird species are monomorphic, where male and female are difficult to distinguished morphologically, including cockatoos. Sex identification using molecular bird sexing is more accurate and applicable because it directly targets the sex chromosomes. The purpose of this study was to determine the sex of Sulphur‐crested cockatoo (Cacatua galerita) by detecting differences in the intron size of the chromodomain helicase DNA‐binding 1 (CHD1) gene on the Z and W chromosomes by polymerase chain reaction (PCR) method and to compare of plucked feathers and blood samples as DNA sources for molecular bird sexing. DNA was extracted from feather and blood samples from four C. galerita. Extracted DNA was amplified on the CHD1 gene by PCR method with P2, MP, and NP primers, which were visualized using agarose gel 1.5% under UV transilluminator with a wavelength of 280 nm. The resulting PCR product was detected at 392 bp for the CHD1 Z gene segment and 297 bp for CHD1 W gene segments, where males showed a single DNA band (ZZ) and females showed a double DNA band (ZW). Four C. galerita were 100% successfully determined, consisting of one female and three males. Electrophoresis results showed DNA bands from blood samples were thicker and brighter than DNA bands from feather samples.

FILOGENI MOLEKULER BAKTERI DARI MEDIA PEMELIHARAAN ROTIFER YANG DIBERI OLAHAN LIMBAH IKAN SEBAGAI SUMBER NUTRISI

This study aims to identify and construct molecular phylogeny of an isolate bacteria from culture media of rotifer Brachionus rotudiforis supplied with processed fishery waste feed as nutritional source. The use of fish waste-based food for rotifer showed positive effects on growth and nutrient content of the rotifers. Genomic DNA of the isolate bacteria BRLI- 01 was extracted and the 16S rRNA gene was amplified using primers (8F and 1492F) and further sequenced using Sanger sequence technique. The 16S rRNA gene was analysed using SeqScanner® and MEGA® followed with BLAST (Basic Local Alignment Search Tool) analyses in the NCBI (National Centre for Biotechnology Information). Amplification result of 16S rRNA gene bacteria s NCBI site as a reference for identification and phylogeny of bacterial species. BRLI-01 was successfully cultured on rotifer rearing media. The results of the 16S rRNA gene amplification of the isolate bacteria showed a DNA band with a length of 1400 bp. The BLAST result on the NCBI showed that the isolate bacteria BRLI-01 had a percent identity (98.46%) and is in the same phylogony branching position with Vibrio rotiferianus Keywords: Rotifers, Bacteria, Fish waste, 16S rRNA Genes, Phylogeny identification

Molecular Identification of T. mentagrophytes and M. canis Isolated from Children Infected with Tinea Capitis in Basra Governorate by Using AP-PCR Technique

The current study included molecular identification of dermatophytes causing Tinea capitis in children inBasrah governorate, including T. Mentagrophytes&M. canis, using AP-PCR or RAPD –PCR polymerasechain reaction technique. By using EZ-10 Spin Column Fungal Genomic DNA Mini –Preps Kit used toextract DNA from dermatophytes fungi, where DNA was obtained from these two dermatophytes fungi.The DNA bands appeared clearly after the electrophoresis of the DNA extract showed the results of DNAamplification .The efficiency of the primeres used in the diagnosis of these two types of dermatophytes, called: randomprimeres OPAA11, OPAA17, OPU15, OPD18, was obtained by the positive results obtained when amplifiedusing these primeres.Used for both dermatophytes The results obtained by using OPAA17 showed successin DNA amplification - obtained from M.canis and T. Mentagrophytes, with six bands for M.canis. As forT. Mentagrophytes, the number of bands obtained was 13, and the amplification results obtained usingOPAA11 showed the efficiency of this primer.Five bands were obtained when used to amplify the DNA of the dermatophytes. While seven bands belongedto T. Mentagrophytes, The amplification results of OPU15 indicated that there were six bands for M.canis,while only one bande for T. Mentagrophytes showed the results of amplification using OPD18 used indiagnosis of dermatophytes to four bands for M.canis while the number of bands for T. Mentagrophytes hasten bands. This method for identifecation is fast and has a high diagnostic efficiency of dermatophytes, asthe diagnosis based on genetic characteristics is more accurate than resulting on phenotypic characteristics .

Halal Food Safety: PCR Based Detection of Porcine DNA in Imported Chocolate

Halal food and food products consumption is a major part of living in Muslim community. Pork meat or meat items are not considered halal in Muslim countries and consumers. Ensuring pork-free food items is a challenge for the food industry and exporters to Muslim nations. This study aims to detect porcine DNA in imported chocolate products in Bangladesh for halal safety assurance. The imported chocolate samples were collected from various multi shops in Dhaka. Polymerase chain reaction (PCR) method is used in our research to detect the porcine DNA. Two primer sets are used for the detection of porcine mitochondrial cyt-b (cytochrome-b) gene fragments in chocolate samples. To visualize the amplified DNA, agarose gel (1%) was used. After electrophoresis, DNA band in agarose gel indicated that the gene fragments are amplified properly. In our research, out of 42 chocolate samples, only 2 samples were found positive. The chocolate samples were branded as Wild Berry Flavor Chocolate and Cadbury Milk Tray Chocolate. In comparison with the positive pork sample, these two samples also containing the 165bp and 359bp fragment of the porcine cytochrome b gene. We reported that chocolate products contain the pork contamination were not labeled as halal. While other samples that did not have any halal logo originated from outside Bangladesh and imported also showed negative result. The present study established the DNA-based porcine detection system based on mitochondrial cyt-b that is viable in highly processed products. It can be used in the halal certification process to determine the pork items presence in food and halal safety. Our research also reported that imported chocolates should halal certify before release into the market.

Variabilidad genética de 29 accesiones de quinua (Chenopodium quinoaWilld) peruana mediante marcadores AFLP y análisis multivariante

Aiming to determine the genetic variability of accessions of Chenopodium quinoa Willd. "Quinoa" from the Germplasm Bank of INIA - Ayacucho, analyze 29 accessions, which were characterized using AFLP molecular markers. The quality of the DNA extracted by the Doyle and modified Doyle method was optimal for all samples, obtaining modifications in a range of 100 - 400 ng / μl. In the standardization of the AFLP technique, it was determined that the proteins between 100 and 200 ng / μl of digested DNA were adequate to efficiently develop the AFLP technique, and it was also determined that the preamplification products that allowed the resolution amplification profiles variables were higher with a 1:4 ratio. A total of 7 primer combinations generated 220 DNA bands, being 201 polymorphic loci. Molecular data grouped the accessions into seven groups at a similarity of 0.81 ultrametric units, of which a group is made up of 23 accessions and the remaining six made up of a single accession. It was found that the mean genetic variance was 0.306 ± 0.011, with a Shannon information index of 0.463 ± 0.015, these results showed that in the 29 quinoa accessions it presents a high degree of genetic variability and there is no duplicate accession. The study determines the genetic relationship and is essential to raise the genetic improvement and conservation of quinoa.

DNA Profiling of Biotypes of Trichosanthes Tricuspidata; an Important Ethno medicinal Cucurbit Using RAPD, ISSR and Scot Molecular Marke

Trichosanthes tricuspidata Lour., is an important ethnomedicinal plant belonging to the Cucurbitaceae family. It is known by various vernacular names like Red ball snake gourd in English, Lal Indrayan in Hindi, Kalayar in Malayalam, and Avaduta in Telugu. There are several studies about the medicinal applications of Trichosanthes tricuspidata, but there is scanty information about molecular level identification of different biotypes of Trichosanthes tricuspidata. Hence, the present study was undertaken to develop a protocol for genetic analysis of three biotypes of Trichosanthes tricuspidata collected from wild areas of Warangal, Karimnagar and Adilabad districts of Telangana State, India by using Randomly Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Start Codon Targeted (SCoT) primers-based PCR method. Genomic DNA was isolated from 1-gram fresh leaf material of three biotypes of T. tricuspidata. The primers OPA-2 of RAPD, S-5 of ISSR and S-8 of SCoT has shown good amplification with generation of monomorphic DNA bands ranging from 250 bp to 1000 bp by OPA-2 of RAPD primer; 500 bp to 1500 bp by S-5 of ISSR primer and 1450 bp by S-8 of SCoT primer. In conclusion, no genetic variation was detected in three biotypes, as the generated DNA bands from OPA-2 of RAPD, S-5 of ISSR and S-8 of SCoT primer-based amplifications were monomorphic. The developed protocol of genetic analysis of three biotypes of T. tricuspidata by RAPD, ISSR and SCoT primers could be used in near future for molecular level studies.

Analysis of Vannamei shrimp DNA fragment resistant to White Spot Virus Syndrome

The attack of WSSV in Vannamei shrimp cultivation is still common. Shrimp quality improvement can be made through selection with the help of markers (marker-assisted choice). This study aimed to evaluate the DNA fragment profile of white shrimp that was resistant to WSSV disease. The analysis was performed using the PCR-RAPD method. WSSV challenged four groups of 100 Vannamei shrimp, then DNA was extracted from live and dead shrimp. The results showed that 2 of the 17 primers tested had high potential as markers, namely OP-09 and OPD-2. PCR products with OPC-09 primers had specific DNA bands measuring about 1.2 kb in all post-challenge WSSV resistant shrimp individuals. The amplification results using OPD-02 primers showed a particular band of DNA with a length of about 1.0 kb, with 60 % of the appearance in WSSV-resistant shrimp. In contrast, the WSSV-susceptible shrimp group did not have specific DNA fragments. Thus, the two RAPD primers had a high chance of being used in the selection with the help of markers to produce WSSV resistant shrimp.

Genetic diversity of Some Indonesian Local Rice Varieties based on Simple Sequence Repeat (SSR) marker related to Aromatic Genes

Rice plant (Oryza sativa L.) has various varieties. People generally prefers to rice with good taste and aroma. The aroma is part of the physicochemical properties of the rice plant. The aroma of rice arises because of the gene that controls the nature of the aroma. PCR (polymerase chain reaction) is an in vitro method for generating a large amount of specific DNA fragments with defined lengths and sequences in a small number of complex templates. The objective of this study was to analyze the genetic diversity of some Indonesian local rice varieties based on SSR marker related to aromatic genes. The selected Simple Sequence Repeat (SSR) primers associated with aromatic genes were: RM 484, RM 410, RM 251, RM 247, RM 223, and RM 9. Based on the DNA band pattern, 6 primers were derived, in which there were 14 loci related to the aromatic gene. The resulting dendrogram showed that two main groups of rice achieved a 57% similarity. Group A consisted of aromatic rice accessions, i.e. Rojolele, Rabeg, Rumbah, Cere Lintang, Manikam, Cao, Jawara Hawara, Jalawara, Pare Racik, Godok, Mayang, Caragol, Segubal, Konjal, Beuruem Batu, Tambleg, Parajaketra, Pondok Leger, Seungkeuhan, Waren, Pare Emas, Kapundung, and Cireh Hudang. Group B consisted of non-aromatic rice accessions, i.e. Ciherang, Care Wari, Care Beuruem, Cokrom, Kewal Bulu Hideung, Maninjau, Seren, Sidenuk, Tampai Beureum, Mira, and Pare Caok.

Establishment of Glyoxalase I gene transformation and regeneration of cotton (Gossypium hirsutum L.)

Aim: The current study was carried out to develop transgenic cotton plantlets with glyoxalase I (gly I) gene using Agrobacterium-mediated transformation. Methodology: Seeds of cotton were inoculated on MS medium and the explants such as shoot tip (3-5 mm), hypocotyl and leaf were aseptically removed from in vitro plantlets. The pre-cultured and infected explants with Agrobacterium harboring gly I gene and the shoot tip were inoculated on MS media for shoot initiation and subcultured on elongation medium with growth hormones, and antibiotics. Healthy and well-grown shoots were subcultured on medium with indole butyric acid (IBA) (0.3 mgl-1) for root formation and the plantlets were hardened in plastic cups with sterile soil. The putative transgenic plantlets were analyzed histochemically for gus gene and the PCR analysis was performed for gly 1 gene. Results: The transformation efficiency of cotton ranged 48.57 to 64.53 %. The regenerated plantlets showed the presence of gus gene in terms of blue coloration in shoots, whole leaf and leaf segments. The PCR was performed in putative transgenic plant lets with both gus gene as well as gly I gene primers. The PCR results showed the presence of 1031 bp DNA band with gus gene primers and 800 bp DNA band with the gly I gene primers. Interpretation: The current study has proven the reproducible procedure for the Agrobacterium-mediated gene transfer and regeneration of Indian cotton varieties. The PCR results revealed the presence of glyoxalase I gene in the transformants. Key words: Cotton varieties, Glyoxalase I gene, PCR analysis, Regeneration, Transformation

Survey of Banana bunchy top virus on non-cultivated bananas in West Java

Banana is a horticulture crop originated from Southeast Asia. Therefore, the genetic diversity of bananas is presumably high in Indonesia. Banana bunchy top disease caused by Banana bunchy top virus (BBTV) has been reported occurred in several regions in Indonesia, especially in the banana cultivation. A survey was conducted in April 2019 in West Java to make documentation on the types of bananas that grow unattended and to monitor BBTV infection. Banana cultivars with various types of genomes was found during the survey, i.e. “Mas”, “Uli”, and “Rejang” (type AA); “Ambon”, “Muli”, and “Urang” (type AAA); “Kapas” and “Raja” (type AAB). Besides, wild bananas were also found, i.e.“Caukole” in Cianjur and “Tongka Langit” in Tasikmalaya with genome types AAw and T, respectively. Leaf samples from infected plants were collected and taken to the laboratory for detection of BBTV using polymerase chain reaction (PCR) method. Specific DNA band of BBTV around 240 bp was successfully amplified from all leaf samples using mRepF/mRepR specific primer pairs. The results of this survey indicated that BBTV has spread in the field and has the potential to be a source of disease inoculum if no immediate control efforts are made.

Molecular Characterisation of the GdhA- Derivative of Pasteurella multocida B:2

Pasteurella multocida B:2 is an important veterinary pathogen causing fatal and acute haemorrhagic septicaemia (HS) in bovine. A live vaccine candidate, P. multocida B:2 GDH7 was reported to enable protection in cattle and buffaloes via intranasal (i. n.) administration. This potential vaccine was also reported to be self-transmitted from the vaccinated animal to the free-ranging animals allowing wider vaccination coverage. Prior to commercialisation, this potential vaccine requires further characterisation in accordance with the authoritative guidelines from the World Organisation for Animal Health (OIE). Hence, in this study, the potential vaccine strain, P. multocida B:2 GDH7 and the virulent parent strain were characterised through genomic and proteomic profiling. A crucial first step was to develop a sensitive yet simple and robust identification test to differentiate both strains which has been achieved by the development of a precise yet straightforward PCR method. In genomic profiling, Repetitive Extragenic Palindromic sequence-PCR (REP-PCR) was manipulated and both strains have a different display of genomic DNA band patterns. Some of the major OMPs were observed and prominent immunogens of P. multocida, OmpA and OmpH were observed to be expressed differently between these strains through SDS-PAGE analysis. In conclusion, a reproducible PCR detection method has enabled differentiation of both strains. Further characterisation of these strains shows a significantly different profile through genomic and proteomic profiling.

Sensitivity of enrichment-PCR method for Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis analysis in chicken carcasses

Salmonella spp. is Gram negative-pathogenic bacteria that usually found as a contaminant in chicken carcasses. This study was aimed to increase the sensitivity of PCR enrichment step and apply the enrichment-PCR combination to detect Salmonella in chicken carcasses. In this study were used Salmonella enterica serovar Hadar, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis with the target genes were invA, STM4497, and respectively. A total of 25 g of the chicken carcasses were artificially contaminated by approximately 0.96 and 3.33 MPN/mL for each serovar separately. Samples were incubated in pre-enrichment and enrichment media for 8 hrs prior to the DNA extraction. The pre-enrichment and enrichment media was Buffered Peptone Water and Rappaport-Vassiliadis-soya. The result showed that the target genes of S. enterica ser. Hadar, S. enterica ser. Typhimurium and S. enterica ser. Enteritidis were detected in chicken carcasses, indicated by the presence of DNA band with the size was 429 bp, 311 bp and 135 bp respectively. These result in line with analysis using ISO method and BLAST-comparison analysis of DNA amplicon sequences with GenBank references. Application of this method for Salmonella detection in chicken carcasses sold in the traditional market showed a higher prevalence than the previous result without enrichment. All samples (n = 100) from unsanitary practice sellers were positively contaminated by Salmonella spp. and also high prevalence for S. enterica ser. Typhimurium and S. enterica ser. Enteritidis. It can be concluded that enrichment is an important step to increase the sensitivity detection of PCR method.

ANALISIS KEKERABATAN GENETIK TANAMAN PADI (Oryza sativa L.) DI KABUPATEN KAMPAR DENGAN MENGGUNAKAN PENANDA Random Amplified Polymorphic DNA (RAPD)

Rice is a cereal crop that belongs to the graminae family of high economic value, and is the main food for more than a portion of the world's population. This study aims to determine the relationship genetic of rice in Kampar by RAPD marker. Eight genotypes of rice from district in Kampar were amplified using eight primers (OPA 5, OPB 7, OPC 19, OPD 2, OPD 3, OPD 8, OPD 11, and OPD 13). The analysis of molecular data was performed by using NTSys pc -2.02 and GenAlex 6.1 . The results of the amplification of rice produced 48 loci with DNA band sizes obtained ranging from 350-1700 bp. The average percentage of polymorphic loci of the eight primers was 49.80%, where the highest percentage of polymorphic loci produced by OPA-7 primer was 75%, while the lowest polymorphic locus percentage was observed in OPD-13 primer which was 14.28%. The value of genetic distance between rice from Kampar in this study is in the range of 0.06 - 0.37. At a genetic distance of 0.79, rice plants can be grouped into two groups, namely the first group consisting of sokan and coku. The second group consisted of suntiong, korean , cupak putio, kuniong, jangguik, and cupak tenggi. The results of this study are expected to be taken into consideration in developing rice breeding strategies in the future.

DETECTION AND IDENTIFICATION OF YELLOW MOSAIC STUNT DISEASE ON Petunia sp. USING NESTED PCR METHOD

Yellow mosaic stunt disease was found at some nurseries of Petunia in Sleman, Yogyakarta, also in Muntilan and Magelang Central Java. The disease was very important due to its ability reducing the quality and quantity of Petunia seedlings. The causal agent of the disease may be carried over to imported seeds and necessary to identify as a basic information for developing control strategies. This research was done by mechanical transmission on indicator plants. The observation of the causal agents was conducted using electron microscope with quick dipping method and the molecular detection was done using nested PCR with TobRT up1-TobRT do2 as the external primers and TobN up3-TobN do4 as the internal primers. Mechanical inoculation showed chlorosis symptoms that developed into local spot on Chenopodium amaranticolor as well as mosaic and vein banding on Nicotiana benthamiana. The observation using electron microscope showed rod-shaped virus particles sized approximately 300 nm and by PCR method produced around 568 bp and 400 bp DNA band. Based on the sequence analysis, the disease was caused by Rehmania mosaic virus. This type of Tobamovirus has 96% similarity with ReMV-Japan. ReMV, a plant pathogen which was a member of Tobamovirus that has never been reported in Indonesia. This research was the first report of ReMV in Indonesia infecting Petunia as ornamental plant.

Analysis of the Protein Profile of Cassava Plantlets (Manihot esculenta Crantz.) Resistance to Fusarium Wilt Disease

Fusarium wilt disease is still a production constraint in Cassava (Manihot esculenta Crantz.) cultivation. The disease is caused by the fungus Fusarium oxysporum (Fo), which to date has not been cured. It is expected that the use of varieties of cassava that are resistant to Fusarium wilt is an important alternative for disease control. Induced Resistance cassava research on Murashige and Skoog medium containing Fusaric Acid (FA) selective concentration has been done before and there were indications FA concentration tolerant to the selection of resistant plantlets in vitro. It is hoped that cassava plantlets that are resistant to FA will also be resistant to Fo. In previous studies, the inoculation of Fo fungal isolates on resistant cassava plantlets was carried out in vitro, followed by DNA pattern analysis compared to controls. The results of the DNA pattern analysis, in the form of a new (specific) DNA band that has a size of 550 bp (OPA_1) and 300 bp (OPA_10), are predicted to be candidates for RAPD markers for cassava resistance to Fo. Based on the results of these previous studies, it is necessary to study more deeply in this study to ascertain whether the new DNA strand is really a peroxidase protein that causes cassava plantlets to be resistant to Fo by protein profile analysis. The results of protein profile analysis showed that the appearance of new protein bands (around 98 kD) indicated the formation of PR-protein (peroxidase) in cassava plantlets that were resistant to Fo and missing protein bands (around 65 kD) in cassava plantlets.

Virulence of five anthracnose Colletotricum acutatum isolates from West Java against the resistance of hot pepper

The most dominant disease attacked hot pepper plantations is anthracnose (Colletotrichum acutatum). This disease can attack fruit, causing substantial losses in quality and quantity of 20% - 90%. The purpose of this research is to select lines or varieties of hot pepper that are potentially resistant to Colletotricum acutatum. This research was carried out at the Indonesia Vegetable Research Institute from January to December 2018. Samples of anthracnose-infected plants were taken from several locations in West Java. Identification of C. acutatum isolates was done by PCR method. The results showed that: (1). The intensity of anthracnose disease is different in five districts (Bandung, Garut, Tasikmalaya, Ciamis and Sukabumi) ranging 50% - 73%. The high intensity of anthracnose symptoms depends on cropping patterns, used of varieties, age and plant conditions. (2). The morphology of the isolate the top view white and gray, and beige, white, peach for the colony the bottom. (3). C. acutatum isolates from West Java were identified by PCR, DNA band measuring 490 bp. (4). The isolates from Sukabumi were the most virulent. (5). Inoculation of 5 isolates from West Java on hot pepper were not obtained resistant lines / varieties.