DNA Fingerprint Profiles Research Articles

Identification of New Cultivar and Different Provenances of Dendrocalamus brandisii (Poaceae: Bambusoideae) Using Simple Sequence Repeats Developed from the Whole Genome.

Dendrocalamus brandisii is a high-quality bamboo species that can be used for both bamboo shoots and wood. The nutritional components and flavors of D. brandisii vary from different geographical provenances. However, the unique biological characteristics of bamboo make morphological classification methods unsuitable for distinguishing them. Although the new cultivar 'Manxie No.1' has significant differences in the branch characteristics and the color of shoot sheaths compared to the D. brandisii, it still lacks precise genetic information at the molecular level. This study identified 231,789 microsatellite markers based on the whole genome of D. brandisii and analyzed their type composition and distribution on chromosomes in detail. Then, using TP-M13-SSR fluorescence-labeling technology, 34 pairs of polymorphic primers were screened to identify the new cultivar 'Manxie No.1' and 11 different geographical provenances of D. brandisii. We also constructed DNA fingerprinting profiles for them. At the same time, we mapped six polymorphic SSRs to the gene of D. brandisii, among which SSR673 was mapped to DhB10G011540, which is related to plant immunity. The specific markers selected in this study can rapidly identify the provenances and the new cultivar of D. brandisii and help explore candidate genes related to some important traits.

Assessment of genetic homogeneity of in-vitro propagated apple root stock MM 104 using ISSR and SCoT primers

Apple is an important fruit crop that is always in demand due to its commercial and nutraceutical value. Also, the requirement for quality planting material for this fruit crop for new plantations is increasing continuously. In-vitro propagation is an alternative approach, which may help to produce genetically identical high grade planting material. In this study, for the first time, an efficient and reproducible propagation protocol has been established for apple root stock MM 104 via axillary bud. Culturing axillary buds on Murashige and Skoog apple rootstock (MM 104) resulted in better in-vitro propagation. (MS) basal medium supplemented with 3.0% (w/v) sucrose and 0.8% (w/v) agar. The axillary buds were established in MS basal medium with BA (5.0 µM), NAA (1.0 µM) and further used to establish invitro propagation protocol. Plant Growth Regulators (PGRs), BA (1.0 µM) in combination with NAA (1.0 µM) was found most efficient for shoot multiplication (100%) and produced 9.8 shoots/explants with an average shoot length of (2.4 ± cm). All the shoots produced roots in 0.1 µM IBA with a 5-day dark period. Acclimatization of in-vitro raised plantlets was obtained with vermiculite: perlite: sand: soil (2:2:1:1) resulting in 76% survival under field conditions. The study showed that the use of axillary bud is efficient for multiple-shoot production of apple rootstock (MM 104). This is the first comprehensive report on in-vitro growth of apple root stock MM 104 with an assessment of genetic stability using DNA fingerprinting profiles based on Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT). The genetic stability of in-vitro-produced plants, as determined by SCoT and ISSR primers, demonstrated genetic closeness to the mother plant.

Genomic structure analysis and construction of DNA fingerprint for four sheep populations

The Yongdeng Qishan sheep (QS) is a sheep population found locally in China. To gain in-depth knowledge of its population characteristics, three control groups were chosen, comprising the Lanzhou fat-tailed sheep (LFT), TAN sheep (TAN), and Minxian black fur sheep (MBF), inhabiting the nearby environments. This study genotyped a total of 120 individuals from four sheep populations: QS, LFT, TAN, and MBF. Using Specific-Locus Amplified Fragment Sequencing, we conducted genetic diversity, population structure, and selective sweep analysis, and constructed the fingerprint of each population. In total, there were 782 535 single nucleotide polymorphism (SNP) variations identified, with most being situated within regions that are intergenic or intronic. The genetic diversity analysis revealed that the QS population exhibited lower genetic diversity compared to the other three populations. Consistent results were obtained from the principal component, phylogenetic tree, and population structure analysis, indicating significant genetic differences between QS and the other three populations. However, a certain degree of differentiation was observed within the QS population. The linkage disequilibrium (LD) patterns among the four populations showed clear distinctions, with the QS group demonstrating the most rapid LD decline. Kinship analysis supported the findings of population structure, dividing the 90 QS individuals into two subgroups consisting of 23 and 67 individuals. Selective sweep analysis identified a range of genes associated with reproduction, immunity, and adaptation to high-altitude hypoxia. These genes hold potential as candidate genes for marker-assisted selection breeding. Additionally, a total of 86 523 runs of homozygosity (ROHs) were detected, showing non-uniform distribution across chromosomes, with chromosome 1 having the highest coverage percentage and chromosome 26 the lowest. In the high-frequency ROH islands, 79 candidate genes were associated with biological processes such as reproduction and fat digestion and absorption. Furthermore, a DNA fingerprint was constructed for the four populations using 349 highly polymorphic SNPs. In summary, our research delves into the genetic diversity and population structure of QS population. The construction of DNA fingerprint profiles for each population can provide valuable references for the identification of sheep breeds both domestically and internationally.

A rare cultivar of horse chestnut (Aesculus hippocastanum L. ˈBaumaniiˈ) in Slovakia: morphological, molecular and antioxidant analysis

Aesculus hippocastanum L. ˈBaumaniiˈ (syn.: A. hippocastanum L. ˈFlore Plenoˈ; A. hippocastanum ˈPlenaˈ) is a rarely cultivated cultivar characterized by fully white and sterile flowers, and as a result it does not produce fruit. For identification and characterization of the morphological, molecular, and biochemical parameters of this cultivar, we performed the pollen viability test, morphological analysis of flowers, assay of miRNA-based markers and biochemical analyses. The results show that the germination of pollen grains in artificial media reached 0 % and the pistil in flowers was undeveloped. The antioxidant activity, total polyphenol and flavonoid content exhibited the highest values of all three tested parameters (1.5540 mg TEAC/g FM; 3.7278 mg GAE/g FM and 1.328 mg CAE/g FM) in the extracts from A. hippocastanum ˈBaumaniiˈ. The applied miRNA-based markers showed a sample-specific and tissue-specific genomic polymorphism. Various DNA fingerprinting profiles between the flowers and leaf samples of fruiting and barren trees were noted. URL: https://www.upjs.sk/pracoviska/botanicka-zahrada/odborne-aktivity/contents-abstracts/

Elevated temperature may reduce functional but not taxonomic diversity of fungal assemblages on decomposing leaf litter in streams.

Mounting evidence points to a linkage between biodiversity and ecosystem functioning (B-EF). Global drivers, such as warming and nutrient enrichment, can alter species richness and composition of aquatic fungal assemblages associated with leaf-litter decomposition, a key ecosystem process in headwater streams. However, effects of biodiversity changes on ecosystem functions might be countered by the presumed high functional redundancy of fungal species. Here, we examined how environmental variables and leaf-litter traits (based on leaf chemistry) affect taxonomic and functional α- and β-diversity of fungal decomposers. We analysed taxonomic diversity (DNA-fingerprinting profiles) and functional diversity (community-level physiological profiles) of fungal communities in four leaf-litter species from four subregions differing in stream-water characteristics and riparian vegetation. We hypothesized that increasing stream-water temperature and nutrients would alter taxonomic diversity more than functional diversity due to the functional redundancy among aquatic fungi. Contrary to our expectations, fungal taxonomic diversity varied little with stream-water characteristics across subregions, and instead taxon replacement occurred. Overall taxonomic β-diversity was fourfold higher than functional diversity, suggesting a high degree of functional redundancy among aquatic fungi. Elevated temperature appeared to boost assemblage uniqueness by increasing β-diversity while the increase in nutrient concentrations appeared to homogenize fungal assemblages. Functional richness showed a negative relationship with temperature. Nonetheless, a positive relationship between leaf-litter decomposition and functional richness suggests higher carbon use efficiency of fungal communities in cold waters.

Determining the DNA Fingerprinting Profiles of Salmonella Isolates from Raw Poultry Meats and Human Clinical Samples from the Same Geographic Area Using Pulsed-Field Gel Electrophoresis

Foods of animal origin, such as poultry, eggs, and pork, are recognized sources of Salmonella infections, but determination of the proportion of foodborne infections associated with various food sources has been challenging. In the present study, 141 Salmonella isolates recovered from 1,322 poultry product samples purchased over a 1-year period from retail stores across Seattle, WA were subtyped by pulsed-field gel electrophoresis (PFGE) using restriction enzyme XbaI. The objectives of the study were (i) to analyze the longitudinal distribution of Salmonella PFGE profiles throughout the sampling period and their clonality within and between poultry processing establishments, (ii) to determine the association between PFGE profiles of Salmonella isolates from locally distributed poultry products and those of clinical isolates submitted to the Washington State Department of Health (WA-DOH) laboratories, and (iii) to compare the PFGE profiles of Salmonella isolates from the National Antimicrobial Resistance Monitoring System (NARMS) retail meats program. During the 1-year sampling period, multiple indistinguishable PFGE patterns were found across multiple poultry processing establishments. Twelve of the 30 unique PFGE profiles of Salmonella isolates from locally purchased poultry products were indistinguishable from the PFGE profiles of clinical Salmonella isolates submitted to the WA-DOH. When the PFGE profiles from the poultry samples were compared with those found in the NARMS database, eight indistinguishable PFGE matches were found with isolates recovered from chicken breasts, ground turkey, and ground beef from multiple states. Although this study revealed some association between PFGE profiles from raw poultry products and those of clinical isolates from the same geographical area, these results do not prove that all of those clinical isolates were from infections acquired through consumption or handling of poultry.

Characterisation of Salmonella enterica clones carrying mcr-1 plasmids in meat products and patients in Northern Thailand using long read sequencing

Salmonella spp. is an important foodborne pathogen associated with consumption of contaminated food, especially food of livestock origin. Antimicrobial resistance (AMR) in Salmonella has been reported globally and increasing AMR in food production is a major public health issue worldwide. The objective of this study was to describe the genetic relatedness among Salmonella enterica isolates, which displayed identical DNA fingerprint profiles. Ten S. enterica isolates were selected from meat and human cases with an identical rep-PCR profile of serovars Rissen (n = 4), Weltevreden (n = 4), and Stanley (n = 2). We used long-read whole genome sequencing (WGS) on the MinION sequencing platform to type isolates and investigate in silico the presence of specific AMR genes. Antimicrobial susceptibility testing was tested by disk diffusion and gradient diffusion method to corroborate the AMR phenotype. Multidrug resistance and resistance to more than one antimicrobial agent were observed in eight and nine isolates, respectively. Resistance to colistin with an accompanying mcr-1 gene was observed among the Salmonella isolates. The analysis of core genome and whole genome MLST revealed that the Salmonella from meat and human salmonellosis were genetically related. Hence, it could be concluded that meat is one of the important sources for Salmonella infection in human.

MICROSATELLITE MARKERS FOR THE MOLECULAR CHARACTERISATION OF POTENTIALLY COMMERCIAL MANGO (Mangifera indica) PROGENIES

Mango, or Mangifera indica as it is scientifically known, is a fruit crop that has significant importance to the global economy. This study characterised 10 commercially potential mango progenies using a total of 10 SSR markers. The analysis of the 10 SSR markers revealed the presence of 34 alleles. Each locus had 2–6 alleles equal to an average of 3.40 alleles per locus. The data also showed 0.115 (MiIIHR21) to 0.692 (MiIIHR28) polymorphic information content (PIC), equal to an average of
 0.431. Each locus had heterozygosity and gene diversity values ranging from 0.044 (M1) to 0.957 (MiIIHR29) and from
 0.122 (MiIIHR21) to 0.730 (MiIIHR29), respectively, averaging 0.480 and 0.476 each. The range of the pairwise genetic distance was 0.000 (ND014«ND019) to 0.611 (ND020«SB003). The dendrogram analysis showed that each mango progeny was successfully differentiated using the 10 SSR markers except for ND014 and ND019. The allele combination using these 10 SSR markers could be used to develop a DNA profile for fingerprinting purposes for each progeny except for ND014 and ND019, which can only be differentiated with the addition of an extra marker. The developed DNA fingerprinting profile could be used for cultivar identification and authentication. Additionally, breeders could also use the developed DNA profiles for plant variety protection (PVP) purposes.

SSR Marker-based DNA fingerprinting and morphological characterization for varietal identification in popular sorghum varieties of Tamil Nadu

DNA fingerprinting of varieties is mandatory for registration of germplasm and notification of newly released varieties. The present study attempted to develop a DNA fingerprinting profile of newly released varieties of sorghum using publically available SSR markers along with morphological DUS descriptors. Twenty-one SSR markers were used for the identification of unique variety-specific fingerprints in nine varieties/cultures. Of them, 14 primers (66.7%) showed clear and unambiguous amplification which is good enough to identify unique banding patterns for specific cultivars (77.8%). The SSR markers Xtxp024, Xtxp231, Xtxp075 produced unique alleles in CO 32 whereas Xtxp354 produced an unique alleles in K12. The SSR marker Xtxp003, Xtxp201 produced unique alleles in CSV 33 MF which could serve as valid genotype-specific SSR markers in varietal purity test program. The varietal-specific SSR marker will supplement the DUS test and could play a major role in varietal identification, thus resolving disputes during the seed certification process.

Genetic Diversity and Pedigree Analysis of Muscadine Grape Using SSR Markers

Muscadine grape (Vitis rotundifolia) is the first native North American grape to be domesticated. During the past century, breeding programs have created a large collection of muscadine cultivars. Muscadine cultivars are usually identified by evaluating morphological traits and checking breeding records, which can be ambiguous and unauthentic. During this study, simple sequence repeat (SSR) markers were used to generate DNA fingerprinting profiles to identify muscadine cultivars and verify their reported pedigrees. Eighty-nine Vitis accessions were genotyped using 20 SSRs from 13 linkage groups. From these, 81 unique subgenus Muscadinia accessions were identified, and a core set of five SSR markers was able to distinguish all of them. Eighteen misidentifications were found, and five previously unknown accessions were matched with cultivars in the dataset. The profiles of 12 cultivars were not consistent with their reported parentage–progeny relationships. Genetic diversity was analyzed at four levels: all V. rotundifolia cultivars (N = 67); current cultivars (N = 39); historical cultivars (N = 28); and wild V. rotundifolia accessions (N = 9). There was substantial genetic diversity in both wild and historically cultivated muscadines. The principle coordinate analysis (PCoA) showed clear separation among subgenus Vitis cultivars, wild muscadine accessions, and cultivated muscadines, with PCoA1 and PCoA2 explaining 11.0% and 9.3% of the total variation, respectively.

Construction of DNA fingerprint for chili pepper varieties using SNAP markers

Establishing the genetic identity of crop varieties has been considered essential for protecting plant breeder and farmer rights, particularly in developing countries like Indonesia. DNA fingerprint using molecular markers is important to give an unambiguous characteristic pattern as a valuable tool for genetic identification. In this study, eight Single Nucleotide Amplified Polymorphism (SNAP) markers were developed and applied to fingerprint 23 varieties of chili pepper. Polymorphism Information Content (PIC) detected in each primer ranged from 0.14 to 0.36 with an average of 0.17. The average of gene diversity was 0.20 among all varieties for total SNAP markers. A phylogenetic tree was subsequently constructed based on their genotypic scores for selected six markers, which separated the 23 varieties into three major groups. The cluster consisted of 2, 5 or 16 varieties. The DNA fingerprints were translated into capital letters representing presence and absence of allele, and they revealed the specific identity of five varieties. A number of varieties possessed the same DNA fingerprint profiles indicating their close genetic distance. Eventhough these SNAP markers were not able to distinguish each variety according to its unique allelic composition, this study could serve as preliminary information to establish genetic fingerprints of chili pepper varieties in Indonesia. Similar studies in the future will benefit from the SNAP found in this study.

Genetic diversity of red rice varieties originating from West Java and Banten based on SSR marker related to palatability

West Java and Banten Provinces have diverse local red rice varieties/accessions to support national food security, not only in terms of quantity but also the quality of rice. Good eating quality is closely related to palatability. Palatability is a property that is directly related to the quality of rice feeding, aroma, appearance, taste and texture. This study aimed to analyze the genetic diversity and DNA fingerprint profiles of local red rice accessions from West Java dan Banten using molecular markers related to palatability. A total of 12 red rice accessions and four local red rice accessions from Banten and West Java Provinces were estimated their genetic diversity, respectively. The SSR primers used were Ams (linked with aspartate aminotransferase), GPA (glucosamine-fructose-6-phosphate aminotransferase), GBSS1 (granule-bound starch synthase), CBG (nano cyanogenic β-glucosidase), SS1 (starch synthase), SBE1 (glucosidic linkage of a-polyglucan), RM510 (gel consistency), RM13 (protein content) and RM410 (aromatic). The dendrogram showed two main groups of red rice accessions. The first group consisted of Mayang, Tambleg, Sengkeuhan, Pare Jaketra, Jalawara Hawara, Gadok, Carogol, Beureum Batu, Waren, Segubal, Tampai Beureum and Leger Pondok (similarity reached 80.5%). The second group consists of Kapundung, Cere Beureum and Cireh Hideung with a similarity of 79.5%.

PENGEMBANGAN HANDOUT EVOLUSI BERBASIS HASIL PENELITIAN PROFIL DNA FINGERPRINTING

The purpose of this study was to develop a handout based on the results of a DNA Fingerprinting Profile in Javanese-Arabic Ethnics in Malang, East Java, Indonesia for the course of Evolution. Development (Handout) was carried out based on the 4D development model of Thiagarajan. The 4D model has 4 stages, namely define, design, develop, and disseminate, while the research to be carried out is only limited to the develop stage. The research subjects were students of the Biology Education Study Program IKIP Budi Utomo Malang who had taken as many as 40 evolutionary subjects. Data analysis techniques used to analyze quantitative data in the form of validation scores from two material experts, two media experts and readability tests. The instruments used to collect data in the study included validation sheets and questionnaires. The results of the material expert validation analysis included aspects of the completeness of the material, material, questions about evaluation and aspects of material development. The results of expert media validation analysis include aspects of Handout size, Cover Design Handout, Cover Handout Typography, Cover Handout Illustration aspects, Handout Content Design, Typography of Handout Content, Handout Illustration, Language Feasibility, and Presentation Completeness. The results of the readability test analysis include aspects of the material ease, the victory of the handout of understanding material, and renewal of material. Based on the results of the analysis data in this development research, it can be concluded that the average results of the assessment from the material expert validator was obtained with a percentage of 89.85% with the category Very Valid. The average assessment results from media expert validators with a percentage of 98.22% with the category Very Valid. The average assessment results from the readability test with a percentage of 92.73% with the category Very Valid.

Evaluation genotypes of cancer cell lines HCC1954 and SiHa by short tandem repeat (STR) analysis and DNA sequencing.

Cancer cell lines are used worldwide in biomedical researches, and data interpretation solely depends on unambiguous attribution of those respective cell lines to its original sources. Approximately one-third of all cell lines have an origin other than that assumed, leading to invalid results. It is necessary to characterize the origin of cell lines. Short-tandem-repeat (STR) fingerprinting (DNA fingerprinting) is the method for characterization of genetic identity in cultured cell lines under certain experimental conditions. We showed the fingerprinting profiles in a summed and unidentified human cancer cell line comparison to HCC1954 cell line, revealing marked alterations in DNA fingerprinting profiles up to fourteen STR loci from 16 loci. Furthermore, Sanger DNA sequencing showed no c.3140A > G heterozygous mutation in the PIK3CA gene of this suspected HCC1954 cell line. In addition, we showed the fingerprinting profiles in an unidentified cancer cell line comparison to SiHa cervical cell line, revealing same DNA fingerprinting profiles. In conclusion, we have successfully authenticated and identified both suspected HCC1954 and SiHa cell lines by STR analysis and DNA sequencing. STR analysis combined DNA sequencing may be very useful to evaluate genotypes of cancer cell lines in our cancer studies, as well as in judicial authentication and forensic sciences.

Physiological and genomic properties of Thermus tenuipuniceus sp. nov., a novel slight reddish color member isolated from a terrestrial geothermal spring

Two closely related, thermophilic bacteria, designated strains YIM 76954T and YIM 76947, were isolated from the Rehai Geothermal Field, Tengchong, Yunnan province, south-west China. Polyphasic approach and whole genome sequencing were used to determine the taxonomy status and genomic profiles of the novel strains. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the two isolates were closely related to Thermus scotoductus SE-1T (97.1% sequence similarity), and T. amyloliquefaciens YIM 77409T (96.6%). The strains could be differentiated from most recognized Thermus species by their whitish to slight reddish colony color, distinct DNA fingerprinting profiles and low ANI values. Cells stained Gram-negative, rod-shaped of diameter 0.2–0.5μm and length 1.5–5.0μm. Growth occurred at 50–75°C, pH 6.0–9.0 and in the presence of up to 1.0% (w/v) NaCl concentration. Thiosulfate was found to enhance cell growth, besides improving the intensity of its colony color. Oxygen, nitrate, sulfur, and Fe(III) could be used as terminal electron acceptors for growth. MK-8 was the major respiratory menaquinone. Major fatty acids were iso-C17:0, iso-C15:0, anteiso-C17:0, and anteiso-C15:0. The genome size was 2.26Mbp with 65.5% average GC content. A total of 2374 genes was predicted, comprising 2322 protein-coding and 52 RNA genes. On the basis of the polyphasic evidence presented, it is proposed that strain YIM 76954T represents a novel species of the genus Thermus, for which the name Thermus tenuipuniceus sp. nov. is proposed. The type strain is YIM 76954T (=JCM 30350T=KCTC 4677T).

Identification and characteristics of Testudinis Carapax et Plustrum based on fingerprint profiles of mitochondrial DNA constructed by species-specific PCR and random amplified polymorphic DNA

Traditional use of Testudinis Carapax et Plustrum (TCP) as a medicine and health food has been widely reported. We compared two DNA fingerprint profiles of mitochondrial (mtDNA) from TCP based on species-specific PCR and random amplified polymorphic DNA (RAPD) to identify their authority. A series of sequences from cytochrome b (Cyt b) of Chinemys reevesii and their counterfeits were downloaded from the Genbank, and Premier 5.0 software was used to design a set of primers. A species-specific PCR and RAPD were undertaken to obtain the different DNA fingerprints respectively. The mtDNA was successfully extracted from all samples using the modified salting-out method. A relative molecular mass of 16.6 × 103 bp was observed, and mtDNA was measured between 1.83 ± 0.02. Fragments of 78 bp were amplified from all the TCP samples tested (except adulterant animals) using species-specific PCR method. The RAPD showed different electrocardiogram between genuine and counterfeit tortoise shell goods along with stripe number and location. The salting-out method (as modified) was used to extract high-quality mtDNA from TCP. The species-specific PCR and RAPD assay proposed in this study could be used for quality control of TCP with more specificity, sensitivity, and applicability.

Population analysis of Magnaporthe oryzae by using endogenous repetitive DNA sequences and mating-type alleles in different districts of Karnataka, India.

Rice is the staple food crop of more than 60% of the population of the world. This crop suffers from blast disease caused by Magnaporthe oryzae. Information on the mating-type allele distribution and diversity of the pathogen population for the state of Karnataka, India is scanty. With this background, a total of 72 isolates of M. oryzae from rice in different districts of Karnataka were examined for identifying sexual mating alleles MAT1, MAT2 and understanding the genetic diversity based on DNA fingerprint of pot2, an inverted repeat transposon. Among 72 isolates, 44 isolates belonged to MAT1 type (male fertile) and 28 isolates were of MAT2 (female fertile) and there were no hermaphrodite isolates. In a given geographical location, only one mating type was identified. Results revealed that the isolates obtained from these regions are not sexually fertile showing predominant asexual reproduction. Hence, genetic variation observed in the pathogen may be mainly because of high copy number of transposons. A high copy number transposon, namely Pot2, was selected in our study to detect genetic diversity of the pathogen. Pot2 rep-PCR DNA fingerprinting profile showed 27 polymorphic bands with bands ranging in size from 0.65 to 4.0kb and an average of 10 to 14 bands per isolate. Five distinct clusters were formed with two major, two minor, and one outlier. Clusters 4 and 5 are further subdivided into three sub-clusters. Some of the isolates belonging to clusters 3, 4, and 5 are interlinked as these locations are close to one another sharing common geographical parameters and boundaries. This knowledge on the sexual behavior and genetic diversity of M. oryzae is important with respect to breeding for disease resistance.

HIGH YIELDING VARIETY OF TORIA (BRASSICA COMPESTRIS SYN. RAPA L.): RAJ VIJAY TORIA-1

Toria variety RVT-1 (RMT-08-7) has been developed from JT-1xT-9 and evaluated for seed yield, oil yield and reaction to insect pest and diseases under AICRP-R&M trials and on farmers’ field. Morphologically plants were erect, spreading in nature and matured in 98-105 days and had more number of primary and secondary branches and more number of seeds/siliqua than checks. It showed its superiority over checks in IVT and AVT trials by 19.20%, 23.63%; whereas, seed yield (1240 Kg/h and 1240 Kg/h) was obtained on farmers field indicated that recorded an increase of 28.50% % and 34.59 % over farmer’s own seed during two successive years respectively. In IVT and AVT, RVT-1 showed 21.33% and 23.49% increase oil yield (kg/h) over checks PT-303 and Bhawani. DNA finger printing profile indicated that primers PUT-3, PUT-181, PUT-171, PUT-154 and PUT- 173 were found useful in generating unique profile for Raj Vijay Toria-1 variety. On this basis of developed 30 bands, this variety could be identified from other variety.

Multiloci identification of Diaporthe fungi isolated from the medicinal plant Costus spiralis (Jacq.) Roscoe (Costaceae).

The purpose of this study is to identify species from genus Diaporthe associated with a medicinal plant Costus spiralis by ITS, EF 1-α, TUB and CAL gens. The 30 isolates from the genus Diaporthe associated with the medicinal plant Costus spiralis were characterized based on morphological characters and the microculture technique and grouped by DNA fingerprinting with the ISSP gene. Afterwards, a total of 12 isolates were selected for the identification of the species based on the comparative research on the blast through the sequences of the ITS gene. Phylogenetic Tree of Maximum Likelihood were generated with the ITS gene individually and with the genes ITS, TUB, CAL and EF1-α combined with the Diaporthe species recognized and with the additional sequences obtained from GenBank for these species. It was not possible to characterize the 30 isolates microscopically and macromorphologically through the microculture technique and the macromorphological characteristics. The 12 isolates selected based on the DNA fingerprinting profile identified phylogenetically, revealed five distinct species of Diaporthe which are present in C. spiralis. The molecular analyses used in this study are excellent alternatives for species-level identification of Diaporthe associated with medicinal plants.

Conservation, ex vitro direct regeneration, and genetic uniformity assessment of alginate-encapsulated nodal cuttings of Sphagneticola calendulacea (L.) Pruski

A well-organized procedure was established for the conservation and distribution of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] for the first time, using alginate-encapsulated nodal segments (NSs) as synthetic seeds. The ideal beads were obtained through a combination of 2.5% sodium alginate and 75 mM calcium chloride with 84.40 ± 2.20% rate of shoot emergence. The maximum regeneration (88.84 ± 2.24%) from synthetic seeds was achieved on liquid 1/2Murashige and Skoog (MS) medium in comparison to its other formulations. Furthermore, superior frequency (91.09 ± 2.24%) of complete plantlet (having both shoots and roots) formation was achieved when synthetic seeds were cultured on liquid 1/2MS (1.5% sucrose) fortified with 1.0 mg L−1 N6-benzyladenine plus 0.25 mg L−1 α-naphthalene acetic acid. Synthetic seeds could be effectively stored at low temperature (8 °C) up to 90 days with a survival rate of 52.38 ± 3.06%, whereas higher temperature (25 °C) did not support regeneration after 75 days of storage. The plantlets were successfully acclimatized to natural conditions with ~ 89% survival frequency. To by-pass the time-consuming in vitro culture step after encapsulation, synthetic seeds were directly regrown into complete plantlets ex vitro on sand, soil, and vermicompost (1:1:1; w/w). Regeneration rate of 42.22 ± 2.22% was attained when NSs were pretreated on 1/2MS medium containing 4.0 mg L−1 indole-3-acetic acid for 24 h in dark, prior to encapsulation. The random amplified polymorphic DNA and intersimple sequence repeat fingerprinting profiles demonstrated genetic uniformity amongst the regenerated plantlets, in vitro mother plant, as well as in vivo wild plant.