Selection of suitable reference genes for abiotic stress-responsive gene expression studies in peanut by real-time quantitative PCR

BackgroundBecause of its strong specificity and high accuracy, real-time quantitative PCR (RT-qPCR) has been a widely used method to study the expression of genes responsive to stress. It is crucial to have a suitable set of reference genes to normalize target gene expression in peanut under different conditions using RT-qPCR. In this study, 11 candidate reference genes were selected and examined under abiotic stresses (drought, salt, heavy metal, and low temperature) and hormone (SA and ABA) conditions as well as across different organ types. Three statistical algorithms (geNorm, NormFinder and BestKeeper) were used to evaluate the expression stabilities of reference genes, and the comprehensive rankings of gene stability were generated.ResultsThe results indicated that ELF1B and YLS8 were the most stable reference genes under PEG-simulated drought treatment. For high-salt treatment using NaCl, YLS8 and GAPDH were the most stable genes. Under CdCl₂ treatment, UBI1 and YLS8 were suitable as stable reference genes. UBI1, ADH3, and ACTIN11 were sufficient for gene expression normalization in low-temperature experiment. All the 11 candidate reference genes showed relatively high stability under hormone treatments. For organs subset, UBI1, GAPDH, and ELF1B showed the maximum stability. UBI1 and ADH3 were the top two genes that could be used reliably in all the stress conditions assessed. Furthermore, the necessity of the reference genes screened was further confirmed by the expression pattern of AnnAhs.ConclusionsThe results perfect the selection of stable reference genes for future gene expression studies in peanut and provide a list of reference genes that may be used in the future.     

High temperature and UV-C treatments affect stilbenoid accumulation and related gene expression levels in Gnetum parvifolium

BackgroundGnetum parvifolium stems and roots have been used for a long time in traditional Chinese medicines. Stilbenes are bioactive compounds present in G. parvifolium plants, and they possess antioxidative and anticancer properties. However, little is known about the responses of G. parvifolium stilbene biosynthetic pathways to stress conditions. Therefore, we investigated stilbene biosynthesis, including the expression of relevant genes, in G. parvifolium exposed to high-temperature and ultraviolet-C treatments.ResultsHigh temperatures did not influence the accumulation of total stilbenes in stems but decreased stilbene concentrations in roots at 3 h, with a subsequent restoration to control levels. In contrast, ultraviolet irradiation induced the accumulation of total stilbenes in stems but not in roots. We also observed that high temperatures inhibited the production of resveratrol and piceatannol in G. parvifolium stems and roots, whereas ultraviolet treatments initially inhibited their accumulation (up to 6 h) but induced their production at later time points. Analyses of specific genes (i.e., PAL, C4H, 4CL, STS, and CYP) revealed that their expression levels generally increased in stress-treated stems and roots, although there was some variability in the expression profiles during treatments.ConclusionsOur results indicated that high temperatures and ultraviolet irradiation differentially affect the biosynthesis of specific stilbenes in G. parvifolium stems and roots. Therefore, cultivating G. parvifolium seedlings under optimal stress conditions may increase the biosynthesis of specific stilbene compounds.     

Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

BackgroundIn industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze–thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain.ResultsElite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze–thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield.ConclusionsA multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze–thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation.How to cite: Zhang Q, Jin Y, Fang Y, et al. Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high gravity bioethanol fermentation. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.06.003     

Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut

BackgroundCultivated peanut (Arachis hypogaea L.) is a major oilseed crop worldwide. Fatty acid composition of peanut oil may affect the flavor and shelf life of the resulting food products. Oleic acid and linoleic acid are the major fatty acids of peanut oil. The conversion from oleic acid to linoleic acid is controlled by the Δ12 fatty acid desaturase (FAD) encoded by AhFAD2A and AhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C → A:T) of AhFAD2A and an “A” insertion of AhFAD2B resulted in high-oleic acid phenotype. Detection of AhFAD2 mutation had been achieved by cleaved amplified polymorphic sequence (CAPS), real-time polymerase chain reaction (qRT-PCR) and allele-specific PCR (AS-PCR). However, a low cost, high throughput and high specific method is still required to detect AhFAD2 genotype of large number of seeds. Kompetitive allele specific PCR (KASP) can detect both alleles in a single reaction. The aim of this work is to develop KASP for detection AhFAD2 genotype of large number of breeding materials.ResultsHere, we developed a KASP method to detect the genotypes of progenies between high oleic acid peanut and common peanut. Validation was carried out by CAPS analysis. The results from KASP assay and CAPS analysis were consistent. The genotype of 18 out of 179 BC₄F₂ seeds was aabb.ConclusionsDue to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable for determining AhFAD2 genotype than other methods.     

Genome-wide development of polymorphic microsatellite markers and their application in peanut breeding program

BackgroundCultivated peanut (Arachis hypogaea. L) represents one of the most important oil crops in the world. Although much effort has been expended to characterize microsatellites or Simple Sequence Repeats (SSRs) in peanut, the quantity and quality of the markers in breeding applications remain limited. Here, genome-wide SSR characterization and marker development were performed using the recently assembled genome of the cultivar Tifrunner.ResultsIn total, 512,900 microsatellites were identified from 2556.9-Mb genomic sequences. Based on the flanking sequences of the identified microsatellites, 7757 primer pairs (markers) were designed, and further evaluated in the assembled genomic sequences of the tetraploid Arachis cultivars, Tifrunner and Shitouqi, and the diploid ancestral species, A. duranensis and A. ipaensis. In silico PCR analysis showed that the SSR markers had high amplification efficiency and polymorphism in four Arachis genotypes. Notably, nearly 60% of these markers were single-locus SSRs in tetraploid Arachis species, indicating they are more specific in distinguishing the alleles of the A and B sub-genomes of peanut. In addition, two markers closely related with purple testa color and 27 markers near to FAD2 genes were identified, which could be used for breeding varieties with purple testa and high-oleic acid content, respectively. Moreover, the potential application of these SSR markers in tracking introgressions from Arachis wild relatives was discussed.ConclusionsThis study reported the development of genomic SSRs from assembled genomic sequences of the tetraploid Arachis Tifrunner, which will be useful for diversity analysis, genetic mapping and functional genomics studies in peanut.How to cite: Ma J, Zhao Y, Chen H, et al. Genome-wide development of polymorphic microsatellite markers and their application in peanut breeding program. Electron J Biotechnol 2020;44. https://doi.org/10.1016/j.ejbt.2020.01.004.     

Analysis of gene expression profiles in response to Sporisorium reilianum f. sp. zeae in maize (Zea mays L.)

BackgroundHead smut of maize, which is caused by Sporisorium reilianum f. sp. zeae (Kühn), is a serious disease in maize. In order to reveal the molecular mechanism of the resistance to head smut in maize, a microarray containing ∼ 14,850 probes was used to monitor the gene expression profiles between a disease resistant near isogenic line (NIL) and a highly susceptible inbred line after S. reilianum was injected with an artificial inoculation method.ResultsLevels of expression for 3,532 genes accounting for 23.8% of the total probes changed after inoculation. Gene Ontology analysis revealed that the differentially expressed genes participated in physiological and biochemical pathways. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that plant–pathogen interaction, natural killer cell mediated cytotoxicity and benzoxazinoid biosynthesis pathways play important roles in resistance to head smut. Three head smut resistance-related candidate genes, CLAVATA1, bassinosteroid insensitive 1-associated receptor kinase 1 and LOC100217307 with leucine-rich repeat (LRR) conserved domains were identified, each of which is in maize mapping bin 2.09, a region previously shown to include a major QTL for head smut resistance. Furthermore, LOC100217307 was validated by quantitative real-time (qRT)-PCR inferring that this gene may be involved in the resistance to head smut of maize.ConclusionsThis study provided valuable information for cloning, functional analysis and marker assisted breeding of head smut resistance genes.     

Identification and characterization of profilin gene family in rice

BackgroundProfilin proteins (PRFs) are small (12–15 kD) actin-binding protein, which play a significant role in cytoskeleton dynamics and plant development via regulating actin polymerization. Profilins have been well documented in Arabidopsis, Zea mays L. as well as Phaseolus vulgaris, however no such fully characterization of rice (Oryza sativa L.) profilin gene family has been reported thus far.ResultIn the present study, a comprehensive genome-wide analysis of rice PRF genes was completed and three members were identified. OsPRF1 and OsPRF2 shared 98.5% similarity (6 nucleotide divergence), but the deduced amino acid sequences of OsPRF1 and OsPRF2 are fully identical. In contrast, the OsPRF3 presents relatively lower similarity with OsPRF1 and OsPRF2. Phylogenetic analysis also support that OsPRF1 has a closer relationship with OsPRF2. Expression pattern analysis revealed the differential expression of OsPRFs in tissues of mature plant, which suggested the potential spatial functional specificity for rice profilin genes. Subcellular localization analysis revealed the OsPRFs were localized in cytoplasm and nucleus and all of them could bind actin monomers. Furthermore, abiotic stresses and hormones treatments assay indicated that the three OsPRF genes could be differentially regulated, suggesting that OsPRF genes might participate in different stress processes in rice.ConclusionsTaken together, our study provides a comprehensive analysis of the OsPRF gene family and will provide a basis for further studies on their roles in rice development and in response to abiotic stresses.How to cite: Zhang Y, Dong G, Wu L, et al. Identification and characterization of profilin gene family in rice. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.08.004.     

Biochemical characterization of three Aspergillus niger β-galactosidases

Backgroundβ-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of β-galactosidase, but the properties of this enzyme are incompletely studied.ResultsThree new β-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative β-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal β-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4–5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All β-galactosidases performed transgalactosylation activity optimally in an acidic environment.ConclusionsThree new β-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three β-galactosidase family members with remarkably different biochemical properties.