scholarly journals Development of SSR Databases Available for Both NGS and Capillary Electrophoresis in Apple, Pear and Tea

Plants ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2796
Author(s):  
Sogo Nishio ◽  
Miyuki Kunihisa ◽  
Fumiya Taniguchi ◽  
Hiromi Kajiya-Kanegae ◽  
Shigeki Moriya ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Simple Sequence Repeat ◽  
Computational Pipeline ◽  
Chain Reaction ◽  
Breeding Programs ◽  
A Value ◽  
New Varieties ◽  
Polymerase Chain ◽  
Generation Sequencing ◽  
Simple Sequence

Developing new varieties in fruit and tea breeding programs is very costly and labor-intensive. Thus, establishing a variety discrimination system is important for protecting breeders’ rights and producers’ profits. Simple sequence repeat (SSR) databases that can be utilized for both next-generation sequencing (SSR-GBS) and polymerase chain reaction–capillary electrophoresis (PCR-CE) would be very useful in variety discrimination. In the present study, SSRs with tri-, tetra- and pentanucleotide repeats were examined in apple, pear and tea. Out of 37 SSRs that showed clear results in PCR-CE, 27 were suitable for SSR-GBS. Among the remaining markers, there was allele dropout for some markers that caused differences between the results of PCR-CE and SSR-GBS. For the selected 27 markers, the alleles detected by SSR-GBS were comparable to those detected by PCR-CE. Furthermore, we developed a computational pipeline for automated genotyping using SSR-GBS by setting a value “α” for each marker, a criterion whether a genotype is homozygous or heterozygous based on allele frequency. The set of 27 markers contains 10, 8 and 9 SSRs for apple, pear and tea, respectively, that are useful for both PCR-CE and SSR-GBS and suitable for automation. The databases help researchers discriminate varieties in various ways depending on sample size, markers and methods.

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

scholarly journals Application of retrotransposon-based Inter-SINE Amplified Polymorphism (ISAP) markers for the differentiation of common poplar genotypes

2021 ◽  
Author(s):  
Birgit Reiche ◽  
Anja Koegler ◽  
Kristin Morgenstern ◽  
Marie Brueckner ◽  
Beatrice Weber ◽  
...  
Keyword(s):  
Simple Sequence Repeat ◽  
Genetic Background ◽  
Plant Material ◽  
Standard Laboratory ◽  
Chain Reaction ◽  
Laboratory Equipment ◽  
High Identity ◽  
Poplar Trees ◽  
Polymerase Chain ◽  
Simple Sequence

On the basis of retrotransposon-insertion polymorphisms, molecular markers were developed for the identification and differentiation of poplar (<i>Populus</i> spp.) genotypes. For this purpose, short interspersed nuclear elements (SINEs) in the genome sequence of <i>Populus tremula</i> were identified and assigned to different SINE-families. For families with high copy number and high identity values, primers were derived to amplify Inter- SINE Amplified Polymorphisms (ISAPs) with polymerase chain reaction (PCR). The resulting fragments produce genotype-specific fingerprints. This molecular approach utilizes standard laboratory equipment and is therefore easy to use for the verification of plant material. We demonstrate the functionality of three distinct ISAP primer combinations by comparison to ten simple sequence repeat (SSR) markers to differentiate 23 poplar genotypes. Already by using a single ISAP primer combination all genotypes can be clearly discriminated. Furthermore, the cluster analysis based on three primer combinations divides clones according to their genetic background into two subclusters (by a bootstrap value of 98). Our results clearly demonstrate the usability of ISAP markers to differentiate genotypes and trace progenies of poplar trees.

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