Screening of blast resistance genes in rice breeding samples

Rice is one of the most widespread and cultivated crops in the world. It is necessary to increase the yield of crops or expand their sown areas to resolve a food security problem in Russia. Current impossibility of expanding rice cultivated areas in the Rostov region and the need to maintain and increase its yield require developing new disease-resistant varieties. Rice genotypes with multiple blast resistance genes avoid significant yield losses. Since pyramiding and selection of resistance genes in the same genotype through traditional selection methods are complicated, it is urgent to search for homozygous samples using marker-assisted selection methods. This study was aimed to identify Pi-1, Pi-2, Pi-33 and Pi-ta blast resistance genes in breeding rice samples by MAS-methods. The study used CTAB-method for DNA-isolation, PCR, electrophoresis on agarose and polyacrylamide gels. The resulting gels were stained in a solution of ethidium bromide and photographed in ultraviolet light. To control the presence of blast resistance genes the following parental cultivars were used: C104LAC for the Pi-1 and Pi-33 genes, C101-A-51 for the Pi-2 gene, IR36 for the Pi-ta gene; Novator and Boyarin as controls of non-functional alleles of all studied genes. The 446 selection samples of the seventh generation were analyzed. As a result of the research, 127 rice samples that combine 2 or 3 different blast resistance genes were identified. The Pi-2 and Pi-33 genes combination was identified in 43 samples (1128/1, 1149/3, 1171/2, 1177/3, 1177/4, 1186/4, et al.). Samples with three resistance genes are the most interesting for selection and further breeding. For developing new blast-resistant varieties, we recommend using rice samples with the following combinations of resistance genes Pi-1+Pi-2+Pi-33 (1197/1, 1226/2, 1271/1, 1272/2), Pi-1+Pi-2+Pi-ta (1197/4, 1304/2, 1304/3, 1482/3, 1482/4, 1486/1) and Pi-2+Pi-33+Pi-ta (1064/1, 1064/3, 1281/2, 1281/3, 1281/4, 1282/2, 1283/1, 1283/2, 1284/3).

POLYMORPHISM OF TAGLGAP MICROSATELITE LOCUS AND ITS CONNECTION WITH ALLELELIC VARIETIES OF GLIADINS OF BREAD WHEAT

Introduction. Gliadins are monomeric and highly polymorphic storage proteins of wheat endosperm, which together with glutenins form a gluten complex that determines the breadmaking properties of wheat. Allelic variants of gliadins are an important feature in the selection of material for breeding, but their determination by electrophoresis in acid PAGE is quite difficult. Aim. The aim of this study was to investigate the polymorphism of the Taglgap microsatellite locus and to analyze its correspondence to the polymorphism of allelic variants of gliadins that have been revealed by acid PAGE electrophoresis. Methods. 140 cultivars and lines of bread wheat of Ukrainian and foreign selection were analyzed. Electrophoresis of storage proteins was performed in an acid PAGE according to the method of F. O. Poperellia (1989), allelic variants were designated according to the international nomenclature (Metakovsky et al., 2018). DNA was isolated by CTAB method and PCR was performed with primers to the Taglgap microsatellite (Devos et al., 1995). PCR products were fractionated in 7% PAGE and stained with silver staining method. Nucleotide sequences were searched by BLAST and aligned by MAFT methods. The main results. 19 allelic variants of gliadins and 11 alleles of the Taglgap locus were identified. In the collection of Ukrainian varieties there were Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1l and Gli-B1o allelic variants and alleles of Taglgap 216 bp, 237 bp, 246 bp, 248 bp, 252 bp, 267 bp, 270 bp and null. In the foreign collection of varieties − Gli-B1a, Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1i, Gli-B1j, Gli-B1k, Gli -B1l, Gli-B1m, Gli-B1n, Gli-B1o, Gli-B1p, Gli-B1q, Gli-B1r, Gli-B1s and 213 bp, 216 bp, 237 bp, 246 bp, 248 bp, 250 bp, 252 bp, 270 bp, 285 bp and null. Nucleotide sequence analysis in the NCBI database showed the presence of a number of other alleles of the Taglgap microsatellite not only in bread wheat but also in some species of the Triticum L. and Aegilops L. genus. Conclusions. The detected polymorphism correlates with the polymorphism of allelic variants of gliadins of Gli-B1 locus and makes it possible to identify Gli-B1a, Gli-B1d, Gli-B1h and Gli-B1l allelic variants, and for Ukrainian varieties with high probability also Gli-B1b allelic variant. However, this marker does not allow identifying Gli-B1c, which is important for selection.

​SSR Based Genetic Diversity Analysis in Fenugreek (Trigonella foenum-graecum L.) Genotypes

Background: Fenugreek (Trigonella foenum-graecum L.) is an important seed spice crop widely grown all over the world. In India, the state of Rajasthan is known for fenugreek production and productivity in the world. A concerted assessment of genetic variability among the germplasm accession is essential for breeding new superior varieties. Molecular markers such as AFLP, RAPD, ISSR, SSR, SCAR, SCoT, SRAP have become for the characterization of the germplasm rapidly and accurately. The present study aimed to characterize 20 elite fenugreek genotypes using simple sequence repeat (SSR) markers to assess the existing genetic diversity of this medicinal crop. Methods: The present study was carried out at the Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan, India. Total genomic DNA was isolated from old leaves using the CTAB method (Doyle and Doyle, 1990). Further, PCR based genetic diversity was analyzed with using 50 SSR primer pairs. Dendrogram was constructed using NTSYSpc version 2.2 and clustering of the genotypes was done. Result: Twenty genotypes of fenugreek were assessed for genetic diversity analysis using SSR markers. Out of 50 markers 43 primer pairs produced 130 alleles with an average of 84.60% polymorphism. Jaccard’s similarity coefficient lied between and 0.39 to 0.82. Based on UPGMA clustering, a dendrogram consisting of five main clusters was generated with wide variability among the studied genotypes. These diverse genotypes so identified could be gainfully utilized in the fenugreek breeding programme.

Study of Parental Polymorphism and Allelic Variation for Grain Quality and Yield Traits in Rice (Oryza sativa L.) Using SSR Markers

Aim: Identification of polymorphic markers is prerequisite for conducting any QTL mapping experiment because if the parents are polymorphic for the traits of interest, then further selection of plants in the progenies becomes easy. Hence, the objective of the present study was to identify polymorphic markers for grain quality and yield traits among the parental lines Improved Samba Mahsuri and Badshabhog. Place and Duration of Study: It was carried out at Molecular Breeding Lab, Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi - 221 005, India, during 2019. Methodology: Two parents Improved Samba Mahsuri and Badshabhog were used for the present study. The DNA extraction was done as per the CTAB method suggested by Murray and Thompson. Standard PCR protocol was followed. Results: For parental polymorphism survey, a total of 576 randomly selected SSR markers including 26 gene specific markers related to aroma, cooking and eating quality, grain dimension and yield related traits distributed across the 12 chromosomes of rice were used. Overall, 96 markers including 4 gene specific markers were found to be polymorphic between the two genotypes indicating a total polymorphism percentage of 16.67%. The highest polymorphism percentage was recorded on chromosome 6 (26.67%) followed by chromosome 4 (21.43%) and the lowest polymorphism percentage was observed on chromosome 10 (8.93%). The gene specific markers nksbad2, ARO7, BADEX7_5 and SSI were found to be polymorphic. Conclusion: Based on the present study it may be concluded that the polymorphic markers identified will further be utilized in genotyping of F2:3 population, linkage analysis and mapping QTL’s for grain quality and yield traits.

RNA Extraction Protocol for Shorea v2

RNA extraction protocol using CTAB method optimized for leaf and bud samples from Shorea curtisii. Adapted from extraction protocol for Shorea beccariana (see attached publication).

Molecular and Morphological Data Confirmed First Record of Abbreviata kazakhstanica Markov and Paraskiv, 1956 (Spirurida: Physalopteridea) in Iran

Background: The genus Abbreviata (Spirurida: Physalopteridea) currently contains 47 species. Physalopteridae nematodes infect a large number of vertebrates, including mammals, birds, reptiles and amphibians. The current study is a report of the first morphological and molecular identification of A. kazakhstanica (Spirurida: Physalopteridea) in Pseudopus apodus in Iran. Methods: Eleven road-killed P. apodus, were collected from, Iran during 2016-2018. The nematodes were isolated from stomach. After morphological study, the genomic DNA of the parasites was extracted using CTAB method. The DNA was used for PCR amplification of cytochrome c oxidase subunit I (cox1). The PCR products were sequenced, the sequence data were analyzed and multiple alignments were conducted using the Clustal Omega. Results: After detailed microscopic examination, the A. kazakhstanica was identified. The cox1 sequences confirmed the species of helminth. The new sequences of A. kazakhstanica were submitted to GenBank under the accession number MK578751-2. Conclusion: Regarding the limited data on parasitological status of Iranian reptiles, more specific and comprehensive investigations are needed to identify the parasitic fauna.

The genetic potential of Taxus sumatrana medicinal plants in Kerinci Regency

Taxus -a taxol-producing medicinal plant that mostly found in highland area- is a species in genus Taxus and family Taxaceae. This study was aimed to determine the genetic diversity within and between population of T. sumatrana in Kerinci Regency, i.e. Mount Kerinci and Mount Tujuh, based on altitude. The genetic diversity was analyzed with RAPD analysis. The altitude was categorized as low (<2000 m asl) and high (>2000 m asl). The cambium extraction was carried out based on CTAB method. DNA amplification was conducted in RAPD method on machine of PCR System 9700 Applied Biosystems. Nine RAPD primers were used in this study. The results revealed that the average of polymorphic locus was 53.89%. Genetic diversity within population was fairly high with value of 0.1799 and Shannon index of 0.2746. Among the four populations, the population of High Tujuh showed the highest level of variability (He=0.2044). The Nei genetic distance between populations was ranging from 0.0567 to 0.1302. The potential of High Tujuh population is still large enough so that it can still be explored for genetic conservation and cultivated as a taxol-producing material which is useful for medicine.

Comparison of methods to extract PCR-amplifiable DNA from fruit, herbal and black teas

The success of polymerase chain reaction (PCR) assay depends on template deoxyribonucleic acid (DNA) being sufficient with respect to both quantity and quality. Some biological materials contain compounds which inhibit the functioning of DNA polymerase and thus need to be removed as part of the DNA extraction procedure. The aim of the present experiments was to optimise the process of DNA isolation from various types of black, fruit and herbal teas. A comparison was made between two cetyltrimethylammonium bromide (CTAB)-based protocols and two commercially available DNA purification kits. The yield and integrity of the extracted DNA were monitored both spectrophotometrically and using agarose gel electrophoresis. The presence/absence of inhibitors in the DNA preparations was checked by running quantitative real-time PCRs. The optimal protocol was deemed to be the CTAB method described in ISO 21571:2005, so this method is recommended for the routine sample analysis of tea products.

MODIFIKASI METODE ISOLASI DNA CETYL TRIMETHYLAMMONIUM BROMIDE (CTAB) UNTUK SAMPEL EPITEL PIPI MANUSIA

Isolation of deoxyribonucleotide (DNA) is an important step in molecular analysis. In this process, DNA must be obtained in sufficient quantities and in good quality for any further analysis. The Cetyl Trimethylammonium Bromide (CTAB) method is commonly used in DNA isolation of plant or fungal. This method is an alternative in DNA isolation since it is easy and inexpensive. This study aims to modify the CTAB method for DNA isolation from human cheek epithelium for any molecular analysis. Epithelial cells were taken from the oral cavity of the researcher. The isolation protocol included cell lysis step with CTAB buffer and proteinase-K, purification step with the addition of chloroform:isoamylalcohol (24:1), precipitation step with isopropanol. The results of the ratio analysis of DNA spectrophotometer at wavelengths of 260 and 280 nm in the range of 1.73-1.85. The quality of DNA isolation was observed by agarose gel electrophoresis and a firm band was obtained after Ethidium Bromide staining. The DNA concentration in both methods ranged from 400-480 mg/mL. The time required for both methods ranges from 2.5-3 hours. The modified CTAB method DNA isolation protocol produces DNA that has good quality and quantity for molecular analysis processes, such as Polymerase Chain Reaction (PCR).

Inter-Simple Sequence Repeat (ISSR) Markers Revealed Genetic Diversity and Population Structure Among 9 Wild Species of Clematis L.

To analyze the genetic diversity of 9 species of Clematis from 31 different populations, we extracted DNA by the improved CTAB method, used ISSR-PCR for amplification, and then selected 9 primers with clear amplified bands from amongst 220 primers. A total of 127 clear bands were amplified, of which 126 were polymorphic bands, yielding a ratio of 99.2%. The polymorphism information index (PIC) of the primers ranged from 0.9326 to 0.9649. The Nei’s genetic diversity index (H) was 0.2750, the total gene diversity (Ht) was 0.2845, and the genetic differentiation coefficient (Gst) was 0.6696, indicating high genetic differentiation among populations of Clematis. After cluster analysis, the 31 Clematis populations were divided into 3 categories. Principal coordination analysis (PCoA) of 9 Clematis species then showed that the genetic relationship between samples of the same Clematis germplasms was closer than that of samples from the same region. The mantel test revealed a significant positive correlation between genetic distance and geographical distance among the populations. The population clustering results are broadly consistent with the clustering graphs of UPGMA and PCoA. We can conclude the polymorphism of the 9 primers is good, and that the genetic diversity of 31 Clematis populations is rich. Individual Clematis germplasms are closely related and will gather together preferentially.