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Molecular characterizations of somatic hybrids developed between Pleurotus florida and Lentinus squarrosulus through inter-simple sequence repeat markers and sequencing of ribosomal RNA-ITS gene.

ABSTRACT: The 12 pfls somatic hybrids and 2 parents of Pleurotus florida and Lentinus squarrosulus were characterized by ISSR and sequencing of rRNA-ITS genes. Five ISSR primers were used and amplified a total of 54 reproducible fragments with 98.14% polymorphism among all the pfls hybrid populations and parental strains. UPGMA-based cluster exhibited a dendrogram with three major groups between the parents and pfls hybrids. Parent P. florida and L. squarrosulus showed different degrees of genetic distance with all the hybrid lines and they showed closeness to hybrid pfls 1m and pfls 1h, respectively. ITS1(F) and ITS4(R) amplified the rRNA-ITS gene with 611-867 bp sequence length. The nucleotide polymorphisms were found in the ITS1, ITS2 and 5.8S rRNA region with different number of bases. Based on rRNA-ITS sequence, UPGMA cluster exhibited three distinct groups between L. squarrosulus and pfls 1p, pfls 1m and pfls 1s, and pfls 1e and P. florida.

SUBMITTER: Mallick P

PROVIDER: S-EPMC5587416 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Molecular characterizations of somatic hybrids developed between Pleurotus florida and Lentinus squarrosulus through inter-simple sequence repeat markers and sequencing of ribosomal RNA-ITS gene.

Mallick Pijush P Chattaraj Shruti S Sikdar Samir Ranjan SR

3 Biotech 20170906 5

The 12 pfls somatic hybrids and 2 parents of Pleurotus florida and Lentinus squarrosulus were characterized by ISSR and sequencing of rRNA-ITS genes. Five ISSR primers were used and amplified a total of 54 reproducible fragments with 98.14% polymorphism among all the pfls hybrid populations and parental strains. UPGMA-based cluster exhibited a dendrogram with three major groups between the parents and pfls hybrids. Parent P. florida and ...[more]

PMID: 28884065

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Project description:Edible mushrooms, including wild mushrooms, are currently being investigated as natural sources to evaluate their prebiotic potential. This study aimed to evaluate the prebiotic potential of crude polysaccharides (CPSs) extracted from wild Lentinus squarrosulus UBU_LS1 and Lentinus polychrous UBU_LP2 and their application as cryoprotectants in the freeze-drying process to formulate a novel synbiotic product. Based on fruiting body morphology and molecular identification, two wild edible mushrooms named UBU_LS1 and UBU_LP2 were identified as Lentinus squarrosulus and Lentinus polychrous, respectively. L. squarrosulus UBU_LS1 and L. polychrous UBU_LP2 contained high amounts of CPS after hot water extraction. Monosaccharide component analysis showed that CPS_UBU_LS1 and CPS_UBU_LP2 were typical heteropolysaccharides. CPS_UBU_LS1 and CPS_UBU_LP2 showed hydrolysis tolerance to the simulated human gastric acidic pH solution, indicating that these CPSs are capable of reaching the lower gastrointestinal tract. Antioxidant activity determined using the 1,1-diphenyl-2-picrylhydrazyl assay revealed that the CPS_UBU_LS1 and CPS_UBU_LP2 displayed greater antioxidant activity comparable with that of ascorbic acid. It was found that CPS_UBU_LS1 and CPS_UBU_LP2 have a high potential for stimulating growth in all probiotic strains. Moreover, both CPS compounds could possibly be used as cryoprotectants in freeze drying, since the viability of the selected probiotic L. fermentum 47-7 exhibited cell survival of greater than 70% after 90 days of storage at 4 °C. These results highlight that wild edible mushrooms L. squarrosulus UBU_LS1 and L. polychrous UBU_LP2 are potential natural sources of prebiotics and can be applied as cryoprotectants in the freeze-drying process. The crude polysaccharide derived from this study could also be considered as a potent antioxidative compound. Therefore, our study provides evidence to support the application of CPSs from wild edible mushrooms in synbiotic product development and in various functional foods. Finally, further evaluation of these prebiotics, including the determination of the potential rehabilitation of beneficial gut microbes in diseased individuals, is currently being conducted by our research group.

Project description:BackgroundThe internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), and elongation factor 1-alpha (EF1?) are often used in fungal taxonomy and phylogenetic analysis. As we know, an ideal molecular marker used in molecular identification and phylogenetic studies is homogeneous within species, and interspecific variation exceeds intraspecific variation. However, during our process of performing ITS, RPB2, and EF1? sequencing on the Pleurotus spp., we found that intra-isolate sequence polymorphism might be present in these genes because direct sequencing of PCR products failed in some isolates. Therefore, we detected intra- and inter-isolate variation of the three genes in Pleurotus by polymerase chain reaction amplification and cloning in this study.ResultsResults showed that intra-isolate variation of ITS was not uncommon but the polymorphic level in each isolate was relatively low in Pleurotus; intra-isolate variations of EF1? and RPB2 sequences were present in an unexpectedly high amount. The polymorphism level differed significantly between ITS, RPB2, and EF1? in the same individual, and the intra-isolate heterogeneity level of each gene varied between isolates within the same species. Intra-isolate and intraspecific variation of ITS in the tested isolates was less than interspecific variation, and intra-isolate and intraspecific variation of RPB2 was probably equal with interspecific divergence. Meanwhile, intra-isolate and intraspecific variation of EF1? could exceed interspecific divergence. These findings suggested that RPB2 and EF1? are not desirable barcoding candidates for Pleurotus. We also discussed the reason why rDNA and protein-coding genes showed variants within a single isolate in Pleurotus, but must be addressed in further research.ConclusionsOur study demonstrated that intra-isolate variation of ribosomal and protein-coding genes are likely widespread in fungi. This has implications for studies on fungal evolution, taxonomy, phylogenetics, and population genetics. More extensive sampling of these genes and other candidates will be required to ensure reliability as phylogenetic markers and DNA barcodes.

Dataset Information

Molecular characterizations of somatic hybrids developed between Pleurotus florida and Lentinus squarrosulus through inter-simple sequence repeat markers and sequencing of ribosomal RNA-ITS gene.

Publications

Molecular characterizations of somatic hybrids developed between <i>Pleurotus florida</i> and <i>Lentinus squarrosulus</i> through inter-simple sequence repeat markers and sequencing of ribosomal RNA-ITS gene.

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