Simple sequence repeat primers used in polymerase chain reaction amplifications to study genetic diversity in barley

Genome ◽  
1996 ◽  
Vol 39 (1) ◽  
pp. 112-117 ◽  
Author(s):  
M. P. Sánchez de la Hoz ◽  
J. A. Dávila ◽  
Y. Loarce ◽  
E. Ferrer
Keyword(s):  
Genetic Diversity ◽  
Polymerase Chain Reaction ◽  
Simple Sequence Repeats ◽  
Genetic Relationships ◽  
Arbitrary Sequence ◽  
Chain Reaction ◽  
Barley Cultivars ◽  
Dinucleotide Motif ◽  
Polymerase Chain ◽  
Simple Sequence

In combination with oligonucleotides of arbitrary sequence, 5′ anchored oligonucleotides based on simple sequence repeats were used in polymerase chain reaction amplifications to produce barley DNA fingerprints. The aim of this work was (i) to develop a simple nonradioactive experimental procedure to reveal polymorphism in regions containing SSRs, (ii) to determine the genetic nature of polymorphisms, and (iii) to investigate the efficacy of polymorphisms contained in such fingerprints in disclosing genetic relationships between 14 European barley cultivars with known pedigrees. Different 10-mer oligonucleotides containing a dinucleotide motif were used as single primers and also in pairs with 10-mer oligonucleotides of arbitrary sequence. Further, the arbitrary oligonucleotides were used as single primers to produce RAPDs. Thirteen combinations of primers containing either GT(CA)4 or GC(CA)4 were selected on the basis of number and intensity of scorable bands in silver-stained 7% polyacrylamide gels. Of the fragments scored, 58.4% were polymorphic. Inheritance of these random amplified microsatellite polymorphic fragments (RAMP) was studied in doubled-haploid lines from the F1 of 'Steptoe' × 'Morex'. Fifty percent of the primers generated codominant markers. Genetic similarities between cultivars were estimated from RAMP and RAPD data. Principal coordinate analysis performed on RAMP data revealed a clear separation of winter six-rowed, winter two-rowed, and spring two-rowed barley. The dendograms generated faithfully reflected the genealogies of the barley cultivars. RAPD failed to show clearly the germplasm sources of the experimental cultivars. Key words : simple sequence repeats, microsatellites, amplification, genetic diversity, barley.

scholarly journals Seleção de primers polimórficos para estudo de diversidade genética em cactáceas

2020 ◽  
Vol 2 (3) ◽  
pp. 9
Author(s):  
Daniel Oliveira Jordão do Amaral ◽  
Daniel Rodrigo Cavalcante De Araújo ◽  
Juliana Gomes Freitas ◽  
Fabiane Rabelo da Costa Batista
Keyword(s):  
Polymerase Chain Reaction ◽  
Simple Sequence Repeats ◽  
Chain Reaction ◽  
Pcr Polymerase Chain Reaction ◽  
Inter Simple Sequence Repeats ◽  
Polymerase Chain ◽  
Simple Sequence

A família Cactaceae está distribuída principalmente nas Américas, apresentam uma grande importância econômica, fornecendo recursos energéticos para animais polinizadores e dispersores, podendo ser utilizadas na alimentação animal e humana, possui um grande potencial na medicina tradicional e no paisagismo. O objetivo do presente estudo foi selecionar indicadores e padronizar reações de PCR (Polymerase Chain Reaction) para analisar ISSR (Inter Simple Sequence Repeats) em estudos de variabilidade genética de Cactaceae. Foram testados 14 indicadores de ISSR com temperatura variando de 48° a 52°C, em espécies de Tacinga, e destes, 8 foram selecionados por serem polimórficos: ISSR-808, ISSR-827, ISSR-842, ISSR-845, ISSR-853, ISSR-857 ISSR-880 e ISSR-888. O número médio de sequências amplificadas por indicador foi de 11,5 bandas, com destaque para o indicador ISSR-827, que produziu 15 bandas, enquanto os indicadores ISSR-845 e ISSR-853 produziram apenas 8 bandas. Os 8 indicadores selecionados no presente estudo possibilitaram a diferenciação genética, sendo eficientes e indicando um bom nível de polimorfismo entre as espécies analisadas, dessa forma, poderão ser utilizados em futuros trabalhos para estimar a divergência genética em nível molecular em espécies da família Cactaceae.

Mapping maize microsatellites and polymerase chain reaction confirmation of the targeted repeats using a CT primer

Genome ◽  
1993 ◽  
Vol 36 (5) ◽  
pp. 884-889 ◽  
Author(s):  
M. Lynn Senior ◽  
Manfred Heun
Keyword(s):  
Polymerase Chain Reaction ◽  
Simple Sequence Repeats ◽  
Genome Mapping ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Expected Size Range ◽  
Pcr Products ◽  
Mammalian Genomes ◽  
Polymerase Chain ◽  
Simple Sequence

Microsatellites, also called simple sequence repeats (SSRs), have yielded an important class of DNA markers most notable for mapping mammalian genomes. To study the occurrence of microsatellites and their inheritance in maize, a search was made of 280 maize GenBank® sequences. Six SSRs were chosen and unique flanking primers were designed for polymerase chain reaction (PCR) amplification. Eight different maize inbreds were studied with these six primer pairs and a mean of 3.5 polymorphic patterns occurred within the expected size range. For five of these putative microsatellites, the segregation in a maize restriction fragment length polymorphism mapping population was analyzed. Four of the microsatellites cosegregated with the Adh1, Gpc1, Pdk1, and Tpi genes from which the primer sequences were derived. The fifth primer pair (MZEGPA1) showed segregating polymorphisms, but the products were larger than expected. To verify the existence of the original SSRs in the segregating PCR products, a CT primer, containing a CT SSR and an arbitrary leader sequence, was used to reamplify these products. The four microsatellites that cosegregated with the original gene were reamplified as anticipated, whereas a suspicious 230-bp product obtained when using the MZEGPA1 primers could not be reamplified. Based on these results it is concluded that microsatellites can be a valuable tool for maize mapping.Key words: maize, microsatellites, simple sequence repeats, genome mapping.

scholarly journals OPTIMALISASI PCR IKAN TONGKOL KRAI (Auxis thazard) DAN LISONG (Auxis rochei) PADA ANALISIS KERAGAMAN GENETIK

2019 ◽  
Vol 11 (2) ◽  
pp. 95
Author(s):  
Raymon Rahmanov Zedta ◽  
Bram Setyadji
Keyword(s):  
Genetic Diversity ◽  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Temperature Range ◽  
Pcr Polymerase Chain Reaction ◽  
Fisheries Resources ◽  
Pcr Product ◽  
Tuna Fishing ◽  
Polymerase Chain

Ikan tongkol lisong dan krai merupakan salah satu jenis tuna yang berperan nyata untuk usaha perikanan tangkap di Indonesia. Pengelolaan sumberdaya ikan tersebut harus selalu dapat dilakukan untuk menjaga tingkat pemanfaatannya supaya tidak lebih tangkap. Kajian keragaman genetik merupakan salah satu teknik dalam pengelolaan pemanfaatan sumberdaya perikanan dengan cara mengetahui tingkat keragaman genetik pada suatu struktur populasi. Kajian keragaman genetik ini diharapkan dapat menjadi basis kajian stok dan opsi dalam pengelolaan sumberdaya perikanan tongkol agar pemanfaatannya dapat dilakukan secara berkelanjutan. Awal mula analisis keragaman genetik dilakukan dengan memperbanyak DNA secara in vitro menggunakan teknik PCR (Polymerase Chain Reaction). Keberhasilan proses PCR dipengaruhi oleh beberapa faktor seperti suhu dan waktu penempelan oligonukleotida primer. Berdasarkan hal tersebut, penelitian ini bertujuan untuk mengetahui suhu dan waktu optimal pada primer Aro2-38. Sampel penelitian diperoleh dari hasil tangkapan pukat cincin yang didaratkan di PPN Palabuhanratu, Jawa Barat. Optimasi PCR menggunakan 12 suhu dan 2 waktu penempelan yang berbeda yaitu : 520C; 52,80C; 540C; 55,50C; 57,20C; 59,10C; 60,90C; 62,80C; 64,50C; 65,90C; 67,20C dan 680C, dan suhu penempelan 30 dan 15 detik. Hasil analisis menunjukkan bahwa produk PCR optimum (menghasilkan pita alel DNA) pada ikan tongkol krai berhasil waktu penempelan 30 detik dengan rentang suhu 52-540C. Sedangkan pada sampel ikan tongkol lisong, produk PCR yang optimum muncul pada waktu penempelan 15 dan 30 detik, dengan rentang suhu 52-60,90C.Frigate and bullet tuna constitute one of tuna species that plays a significant role in Indonesian fishing business. Management of fisheries resources must always be done to maintain the level of utilization so that it is not excessive. Genetic study is one of techniques in managing fisheries resource utilization by knowing the level of genetic diversity in a population structure. This genetic diversity study is expected to be the basis and option in the management of tuna fishing resources so that their utilization can be carried out sustainably. Genetic diversity analysis is start by multiplying fish DNA using PCR (Polymerase Chain Reaction) technique. The success of the PCR process is influenced by several factors such as temperature and time of primary oligonucleotide attachment. Based on this, this study aims to determine the optimal temperature and time in primers Aro2-38. The research sample was obtained from the catch of purse seine landed in PPN Palabuhanratu, West Java. PCR optimization uses 12 temperatures and 2 different annealing times: 520C; 52.80C; 54ÚC; 55,50C; 57.20C; 59.10C; 60.90C; 62.80C; 64,50C; 65,90C; 67.20C and 680C, and the annealing times are 30 and 15 seconds. The results of the analysis showed that the optimum PCR product (producing DNA allele bands) on the cretaceous tuna was successfully pasted for 30 seconds with a temperature range of 52-540C. Whereas in the sample of tuna lisong, the optimum PCR product appeared at the time of attachment of 15 and 30 seconds, with a temperature range of 52-60.90C.

scholarly journals Genetic diversity assessment using RAPD primers in insecticide resistant populations of diamondback moth Plutella xylostella (Linn.)

2015 ◽  
Vol 7 (1) ◽  
pp. 219-225 ◽  
Author(s):  
V. Sunitha ◽  
T.V. K. Singh ◽  
V. Ramesh Babu ◽  
J. Satyanarayana
Keyword(s):  
Genetic Diversity ◽  
Polymerase Chain Reaction ◽  
Genetic Variability ◽  
Plutella Xylostella ◽  
Rapd Markers ◽  
Diamondback Moth ◽  
Chain Reaction ◽  
Diversity Assessment ◽  
Polymerase Chain ◽  
Cluster A

Genetic diversity in acephate, spinosad and Cry2Ab resistant Plutella xylostella collected from three states of India was assessed by RAPD markers. The DNA extracted from larvae was subjected to polymerase chain reaction using 10 RAPD primers. The highest number alleles (7) were produced by primer ABA-13, followed by six alleles each by primers ABA-2, 7, 8, 11, 14; five alleles each were produced by ABA-4, 9, 10, 12. UPGMA analysis clustered the acephate, spinosad and Cry2Ab treated P.xylostella populations into two groups with overall similarity level of 33%, 27% and 34% respectively. Cluster A consisted 11 samples while Cluster B consisted only F1 of acephate and spinosad treated Karnataka population. In Cry2Ab treated population Cluster B comprised 11 samples and Cluster A had out grouped singly i.e. F0 generation from Karnataka. The genetic variability between the acephate, spinosad and Cry2Ab treated populations ranged from 33 to 69%, 27 to 56% and 34 to 69% respectively. Acephate and spinosad treated F1 population and Cry2Ab treated F0 population from Karnataka were out grouped from rest of the populations.

scholarly journals S-genotyping Supports the Genetic Relationships between Turkish and Hungarian Apricot Germplasm

2010 ◽  
Vol 135 (5) ◽  
pp. 410-417 ◽  
Author(s):  
Júlia Halász ◽  
Andrzej Pedryc ◽  
Sezai Ercisli ◽  
Kadir Ugurtan Yilmaz ◽  
Attila Hegedűs
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetic Relationships ◽  
Pcr Amplification ◽  
Prunus Armeniaca ◽  
Molecular Evidence ◽  
Gene Pools ◽  
Historical Events ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Central Turkey

The S-genotypes of a set of Turkish and Hungarian apricot (Prunus armeniaca L.) cultivars were determined by polymerase chain reaction (PCR) amplification of their S-RNase intron regions. In addition, the S-genotyping method was extended to the SFB gene to detect the non-functional SC-haplotype and hence reliably identify self-compatible apricot cultivars. We determined the complete S-genotype of 51 cultivars and the partial S-genotype of four cultivars. A total of 32 different S-genotypes were assigned to the 51 cultivars, and many of them (28) were classified into newly established cross-incompatibility groups III through XIV. Another 12 cultivars demonstrated unique incompatible genotypes and seven self-compatible cultivars were identified in the examined accessions. The fact that Turkish and Hungarian apricot cultivars carry 12 and five S-alleles, respectively, and all five alleles detected in Hungarian cultivars were also present in Turkish apricots furnished molecular evidence supporting the long-suspected historical connection between Hungarian and Turkish apricots. The connection between these two gene pools appeared to be relatively recent and associated with historical events dating back 300 years. Our results confirm that Turkish germplasm contributed considerably to the development of several desirable Hungarian apricot cultivars. Results suggest that the mutation rendering the SC-haplotype non-functional might have occurred somewhere east of central Turkey.

scholarly journals METODE EKSTRAKSI DNA TANAMAN TANPA PRESIPITASI ETANOL UNTUK KEGIATAN POLYMERASE CHAIN REACTION (PCR)

2019 ◽  
Vol 6 (1) ◽  
pp. 29
Author(s):  
Kristianto Nugroho ◽  
Rerenstradika Tizar Terryana ◽  
. Reflinur ◽  
Puji Lestari
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Simple Sequence Repeat ◽  
Sequence Repeat ◽  
Chain Reaction ◽  
Ethanol Precipitation ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Simple Sequence

A Simplified Plant DNA Extraction Protocol without Ethanol Precipitation for Polymerase Chain Reaction (PCR) Activities ABSTRACTMolecular-based research in agriculture includes DNA extraction stage involving DNA precipitation using ethanol or isopropanol which tends to take a long time. The purpose of this study was to obtain a plant DNA extraction method for Polymerase Chain Reaction (PCR) activities without going through the ethanol precipitation stage. Five important agricultural commodity crops, namely rice, corn, soybeans, chilies, and shallots were extracted by DNA using the modified Doyle and Doyle method. After the extraction phase using chloroform and isoamil alcohol solvents, the supernatant obtained was not precipitated using ethanol but was directly diluted and used as a template in PCR activities using two pairs of Simple Sequence Repeat (SSR) markers. The results showed that all samples could be well amplified, and amplicon tape visualized in both 1% agarose gel and 6% polyacrylamide gel were clearly visible. This method could save time and material, and reduce the dependence on liquid nitrogen. But this method is still limited to PCR requirements only, and cannot be used for activities that require high quality and quantity of DNA such as Next Generation Sequencing (NGS), digestion, and hybridization.Keywords: DNA extraction, ethanol precipitation, liquid nitrogen, PCR, SSR,  ABSTRAKPenelitian berbasis molekuler pada bidang pertanian mencakup tahapan ekstraksi DNA yang melibatkan presipitasi DNA menggunakan etanol atau isopropanol yang cenderung memakan waktu lama. Tujuan penelitian ini adalah untuk memperoleh metode ekstraksi DNA tanaman untuk kegiatan Polymerase Chain Reaction (PCR) tanpa melalui tahapan presipitasi etanol. Lima tanaman komoditas pertanian penting yaitu padi, jagung, kedelai, cabai, dan bawang merah diekstraksi DNA-nya menggunakan metode Doyle and Doyle yang dimodifikasi. Setelah tahap ekstraksi menggunakan pelarut kloroform dan isoamil alkohol, supernatan yang terbentuk tidak dipresipistasi menggunakan etanol melainkan langsung diencerkan dan digunakan sebagai template dalam kegiatan PCR menggunakan dua pasang marka Simple Sequence Repeat (SSR). Hasil menunjukkan bahwa seluruh sampel dapat teramplifikasi dengan baik serta pita hasil amplikon yang tervisualisasi baik pada gel agarosa 1% maupun gel poliakrilamid 6% terlihat jelas. Metode ini dapat menghemat waktu dan bahan serta mengurangi ketergantungan pemakaian nitrogen cair. Tetapi metode ini masih terbatas hanya untuk kebutuhan PCR saja dan tidak dapat digunakan untuk kegiatan yang membutuhkan DNA dengan kualitas serta kuantitas tinggi seperti Next Generation Sequencing (NGS), digesti, maupun hibridisasi.Kata Kunci: ekstraksi DNA, nitrogen cair, PCR, presipitasi etanol, SSR

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