scholarly journals Identification of stable pollen development related reference genes for accurate qRT-PCR analysis and morphological variations in autotetraploid and diploid rice

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0253244
Author(s):  
Jinwen Wu ◽  
Hao Fan ◽  
Yifan Hu ◽  
Haibin Guo ◽  
Hong Lin ◽  
...  
Keyword(s):  
Reference Genes ◽  
Pollen Development ◽  
Pollen Fertility ◽  
Expression Patterns ◽  
Cytochrome B5 ◽  
Development Stages ◽  
Qrt Pcr ◽  
Autotetraploid Rice ◽  
Fertility Genes ◽  
Diploid Rice

Autotetraploid rice exhibited hybrid vigor and greater genetic variation compared to diploid rice, but low pollen fertility is a major hindrance for its utilization. Our previous analysis revealed that large number of pollen fertility genes were exhibited down-regulation in autotetraploid rice. Hence, it is of utmost importance to reveal the expression patterns of pollen fertility genes with high accuracy. To find stable reference genes for autotetraploid rice, we compared the pollen development stages between diploid and autotetraploid rice, and 14 candidate genes were selected based on transcriptome analysis to evaluate their expression levels. Autotetraploid rice (i.e. Taichung65-4x) displayed lower seed set (40.40%) and higher percentage of abnormalities during the pollen development process than its diploid counterpart. To detect the candidate reference genes for pollen development of autotetraploid and diploid rice, we used five different algorithms, including NormFinder, BestKeeper, ΔCt method, geNorm and Re-Finder to evaluate their expression patterns stability. Consequently, we identified two genes, Cytochrome b5 and CPI, as the best candidate reference genes for qRT-PCR normalization in autotetraploid and diploid rice during pre-meiosis, meiosis, single microspore and bicellular pollen development stages. However, Cytochrome b5 was found to be the most stably expressed gene during different pollen development stages in autotetraploid rice. The results of our study provide a platform for subsequent gene expression analyses in autotetraploid rice, which could also be used in other polyploid plants.

scholarly journals Transcriptome and Gene Editing Analyses Reveal MOF1a Defect Alters the Expression of Genes Associated with Tapetum Development and Chromosome Behavior at Meiosis Stage Resulting in Low Pollen Fertility of Tetraploid Rice

2020 ◽  
Vol 21 (20) ◽  
pp. 7489
Author(s):  
Zijun Lu ◽  
Xiaotong Guo ◽  
Zhiyu Huang ◽  
Juan Xia ◽  
Xiang Li ◽  
...  
Keyword(s):  
Differential Expression ◽  
Pollen Development ◽  
Pollen Fertility ◽  
Expression Patterns ◽  
Middle Layer ◽  
Low Fertility ◽  
Myb Transcription Factor ◽  
Pcr Analysis ◽  
High Fertility ◽  
Autotetraploid Rice

Autotetraploid rice is a useful rice germplasm for polyploid rice breeding. However, low fertility limits its commercial production. A neo-tetraploid rice with high fertility was developed from the progenies of crossing between autotetraploid lines by our research group. Our previous study showed that a myeloblastosis (MYB) transcription factor, MOF1, might be associated with the pollen development in tetraploid rice. However, little information is available about its role in pollen development in tetraploid rice. Here, we identified a new haplotype of MOF1 from neo-tetraploid rice and marked it as MOF1a. Transcriptome and qRT-PCR analysis demonstrated that MOF1a highly expressed in anthers, and displayed differential expression in neo-tetraploid rice compared to tetraploid rice line with low pollen fertility. The mutant (mof1a) of MOF1a, which was generated by CRISPR/Cas9 knockout in neo-tetraploid rice, showed low pollen fertility, and also exhibited abnormal tapetum and middle layer development, and defective chromosome behaviors during meiosis. A total of 13 tapetal related genes were found to be up-regulated in meiotic anthers of MOF1a compared with wild type plants by RNA-seq analysis, including CYP703A3, PTC1, and OsABCG26, which had been demonstrated to affect tapetal development. Moreover, 335 meiosis-related genes displayed differential expression patterns at same stage, including nine important meiosis-related genes, such as metallothionein OsMT1a. These results demonstrated that MOF1a plays an important role in pollen development and provides a foundation for understanding the molecular mechanism underlying MOF1a in reproduction of tetraploid rice.

scholarly journals Selection and validation of reference genes for normalization of qRT-PCR data to study secondary metabolite related genes in industrial hemp

2021 ◽  
Author(s):  
Michihito Deguchi ◽  
Shobha Potlakayala ◽  
Zachary Spuhler ◽  
Hannah George ◽  
Vijay Sheri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heavy Metal ◽  
Expression Analysis ◽  
Reference Genes ◽  
Cannabis Sativa ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Qrt Pcr ◽  
Differential Gene ◽  
Industrial Hemp

Abstract Industrial hemp (Cannabis sativa L.) is a dioecious crop widely known for its production of phytocannabinoids, flavonoids, and terpenes. In the past two years since its legalization, there has been significant interest in researching this important crop for pharmaceutical applications. Although many scientific reports have demonstrated gene expression analysis of hemp through OMICs approaches, accurate validation of omics data cannot be performed because of lack of reliable reference genes for normalization of qRT-PCR data. The differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stress were evaluated through four software packages: geNorm, NormFinder, BestKeeper, and RefFinder. The EF-1a ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal and hormonal stress. The expression profiles of two cannabinoid pathway genes, AAE1 and THCAS, using the two most stable and single least stable reference genes confirmed that two most stables genes were apt for normalization of gene expression data. This work will contribute to the future studies on the expression analysis of hemp genes regulating the synthesis, transport and accumulation of secondary metabolites.

scholarly journals Cytological Observations and Bulked-Segregant Analysis Coupled Global Genome Sequencing Reveal Two Genes Associated with Pollen Fertility in Tetraploid Rice

2021 ◽  
Vol 22 (2) ◽  
pp. 841
Author(s):  
Nabieu Kamara ◽  
Yamin Jiao ◽  
Zijun Lu ◽  
Kelvin Dodzi Aloryi ◽  
Jinwen Wu ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Pollen Development ◽  
Pollen Fertility ◽  
Bulked Segregant Analysis ◽  
Low Fertility ◽  
High Fertility ◽  
Rna Seq ◽  
Autotetraploid Rice ◽  
Genetic Mechanisms ◽  
New Perspective

Neo-tetraploid rice with high fertility is a useful germplasm for polyploid rice breeding, which was developed from the crossing of different autotetraploid rice lines. However, little information is available on the molecular mechanism underlying the fertility of neo-tetraploid rice. Here, two contrasting populations of tetraploid rice, including one with high fertility (hereafter referred to as JG) and another with low fertility (hereafter referred to as JD), were generated by crossing Huaduo 3 (H3), a high fertility neo-tetraploid rice that was developed by crossing Jackson-4x with 96025-4x, and Huajingxian74-4x (T452), a low fertility autotetraploid rice parent. Cytological, global genome sequencing-based bulked-segregant (BSA-seq) and CRISPR/Cas9 technology were employed to study the genes associated with pollen fertility in neo-tetraploid rice. The embryo sacs of JG and JD lines were normal; however, pollen fertility was low in JD, which led to scarce fertilization and low seed setting. Cytological observations displayed low pollen fertility (25.1%) and approximately 31.3 and 27.2% chromosome lagging at metaphase I and II, and 28.8 and 24.8% chromosome straggling at anaphase I and II in JD, respectively. BSA-seq of F2–3 generations and RNA-seq of F4 generation detected a common fragment, i.e., 18,915,234–19,500,000, at chromosome 7, which was comprised of 78 genes associated with fertility. Among 78 genes, 9 genes had been known to be involved in meiosis and pollen development. Two mutants ny1 (LOC_Os07g32406) and ny2 (LOC_Os07g32040) were generated by CRISPR/Cas9 knockout in neo-tetraploid rice, and which exhibited low pollen fertility and abnormal chromosome behavior. Our study revealed that two unknown genes, LOC_Os07g32406 (NY1) and LOC_Os07g32040 (NY2) play an important role in pollen development of neo-tetraploid rice and provides a new perspective about the genetic mechanisms of fertility in polyploid rice.

scholarly journals Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Min-dong Chen ◽  
Bin Wang ◽  
Yong-ping Li ◽  
Mei-juan Zeng ◽  
Jian-ting Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Expression Patterns ◽  
Suitable Reference Gene ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Suitable Reference ◽  
Gene Expression Levels

AbstractSelecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

scholarly journals Selection and validation reference genes for qRT-PCR normalization in different cultivars during fruit ripening and softening of peach (Prunus persica)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuanghong You ◽  
Ke Cao ◽  
Changwen Chen ◽  
Yong Li ◽  
Jinlong Wu ◽  
...  
Keyword(s):  
Reference Gene ◽  
Fruit Ripening ◽  
Reference Genes ◽  
Prunus Persica ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Regulatory Mechanisms ◽  
Suitable Reference Gene ◽  
Future Research ◽  
Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been emerged as an effective method to explore the gene function and regulatory mechanisms. However, selecting appropriate reference gene (s) is a prerequisite for obtaining accurate qRT-PCR results. Peach is one of important fruit in Rosaceae and is widely cultivated worldwide. In this study, to explore reliable reference gene (s) in peach with different types during fruit ripening and softening (S1–S4), nine candidate reference genes (EF-1α, GAPDH, TBP, UBC, eIF-4α, TUB-A, TUB-B, ACTIN, and HIS) were selected from the whole-genome data. Then, the expression levels of the nine selected genes were detected using qRT-PCR in three peach types, including ‘Hakuho’ (melting type), ‘Xiacui’ (stony hard type), ‘Fantasia’ and ‘NJC108’ (non-melting type) cultivars were detected using qRT-PCR. Four software (geNorm, NormFinder, BestKeeper and RefFinder) were applied to evaluate the expression stability of these candidate reference genes. Gene expression was characterized in different peach types during fruit ripening and softening stages. The overall performance of each candidate in all samples was evaluated. The Actin gene (ACTIN) was a suitable reference gene and displayed excellent stability in ‘Total’ set, ‘Hakuho’ samples, S3 and S4 fruit developmental stages. Ubiquitin C gene (UBC) showed the best stability in most independent samples, including ‘Fantasia’, ‘NJC108’, S2 sets. Elongation factor-1α gene (EF-1α) was the most unstable gene across the set of all samples, ‘NJC108’ and S2 sets, while showed the highest stability in ‘Xiacui’ samples. The stability of candidate reference genes was further verified by analyzing the relative expression level of ethylene synthase gene of Prunus persica (PpACS1) in fruit ripening and softening periods of ‘Hakuho’. Taken together, the results from this study provide a basis for future research on the mining of important functional genes, expression patterns and regulatory mechanisms in peach.

scholarly journals Selection and Validation of Appropriate Reference Genes for qRT-PCR Analysis of Flowering Stages and Different Genotypes of Iris Germanica L.

2021 ◽  
Author(s):  
Yinjie Wang ◽  
Yongxia Zhang ◽  
Qingquan Liu ◽  
Haiying Tong ◽  
Ting Zhang ◽  
...  
Keyword(s):  
Reference Genes ◽  
Molecular Mechanisms ◽  
Gene Selection ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Herbaceous Plant ◽  
Flower Bud ◽  
Perennial Herbaceous Plant ◽  
Qrt Pcr ◽  
Iris Germanica

Abstract Iris germanica L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in I. germanica. In this study, eight candidate reference genes were examined for the normalization of qRT-PCR in three I. germanica cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that IgUBC and IgGAPDH were the most stable reference genes in ‘00246’ and ‘Elizabeth’, and IgTUB and IgUBC showed stable expression in ‘2010200’. IgUBC and IgGAPDH were the most stable in all samples, while IgUBQ showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of IgFT (Flowering Locus T gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in I. germanica.

scholarly journals Comparative cytological and transcriptome analysis reveals high pollen fertility and upregulation of related genes in neo-tetraploid rice

2019 ◽  
Author(s):  
Jinwen Wu ◽  
Yuanmou Chen ◽  
Hong Lin ◽  
Yang Chen ◽  
Hang Yu ◽  
...  
Keyword(s):  
Pollen Development ◽  
Pollen Fertility ◽  
Development Process ◽  
Yield Potential ◽  
Lower Percentage ◽  
High Fertility ◽  
Rice Breeding ◽  
Rna Seq ◽  
High Pollen ◽  
Autotetraploid Rice

Abstract Background: Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for the utilization of autotetraploid rice. Our previous study demonstrated that neo-tetraploid rice have great yield potential, which is thought to be one effective way to overcome the low fertility of autotetraploid rice. However, there is little known about the cause of high pollen fertility in neo-tetraploid rice. Here, we employed cytology and RNA-seq to study the molecular genetic mechanism of high pollen fertility in neo-tetraploid rice.Results: Cytological observations indicate that H1 displayed high pollen fertility (95.62%), lower percentage of PMC cell abnormalities, and stable chromosome configurations during the pollen development process compared with its two parents. RNA-seq analysis detected 1483 differentially expressed genes (DEGs) in neo-tetraploid rice compared with its two parents. Of these DEGs, 433 were annotated as pollen fertility-related genes, and 240 (~55.4%) exhibited significant upregulation in neo-tetraploid rice compared with its two parents, including nine cloned genes ( CSA , TMS5 etc.) that were validated by qRT-PCR and had been demonstrated to be pollen fertility-related genes. We further selected TMS5 as a candidate gene and analysed its phenotype in neo-tetraploid rice using the CRISPR/Cas9 technique. Significant variations have been detected in phenotypic charts, pollen development process and expression level in H1 and its TMS5 knockout lines.Conclusions: Our finding provides strong evidence for the regulatory mechanisms of neo-tetraploid rice, and upregulation of pollen fertility-related genes should be associated with high fertility. Moreover, the present study provides a new useful germplasm for polyploidy rice breeding.

scholarly journals Validation of Reference Genes via qRT-PCR in Multiple Conditions in Brandt’s Voles, Lasiopodomys brandtii

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 897
Author(s):  
Lin Tian ◽  
Yan Chen ◽  
Da-Wei Wang ◽  
Xiao-Hui Liu
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Developmental Stages ◽  
Development Stages ◽  
Qrt Pcr ◽  
Polymerase Chain ◽  
Experimental Treatments ◽  
Different Tissues ◽  
Lasiopodomys Brandtii ◽  
Multiple Conditions

The choice of optimal reference gene is challenging owing to the varied expression of reference genes in different organs, development stages, and experimental treatments. Brandt’s vole (Lasiopodomys brandtii) is an ideal animal to explore the regulatory mechanism of seasonal breeding, and many studies on this vole involve gene expression analysis using quantitative real-time polymerase chain reaction (qRT-PCR). In this study, we used the method of the coefficient of variation and the NormFinder algorithm to evaluate the performance of nine commonly used reference genes Gapdh, Hprt1, β-actin, PPIA, Rpl13a, Tbp, Sdha, Hmbs, and B2M using qRT-PCR in eight different tissues, five developmental stages, and three different photoperiods. We found that all nine genes were not uniformly expressed among different tissues. B2M and Rpl13a were the optimal reference genes for different postnatal development stages in the hypothalamus for males and females, respectively. Under different photoperiods in the hypothalamus, none of the selected genes were suitable as reference genes at 6 weeks postnatal; β-actin and PPIA were the optimal reference genes at 12 weeks postnatal; Hprt1, β-actin, PPIA, Hmbs, and B2M were excellent reference genes at 24 weeks postnatal. The present study provides a useful basis for selecting the appropriate reference gene in Lasiopodomys brandtii.

scholarly journals Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data

2021 ◽  
Vol 22 (5) ◽  
pp. 2569
Author(s):  
Xue Bai ◽  
Tao Chen ◽  
Yuan Wu ◽  
Mingyong Tang ◽  
Zeng-Fu Xu
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Cyperus Esculentus ◽  
Transcriptome Data ◽  
Qrt Pcr ◽  
Below Ground ◽  
The Stability

Tiger nut (Cyperus esculentus), a perennial C4 plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

scholarly journals Comparative Cytological and Transcriptome Analysis Revealed the Normal Pollen Development Process and Up-Regulation of Fertility-Related Genes in Newly Developed Tetraploid Rice

2020 ◽  
Vol 21 (19) ◽  
pp. 7046
Author(s):  
Jinwen Wu ◽  
Yuanmou Chen ◽  
Hong Lin ◽  
Yang Chen ◽  
Hang Yu ◽  
...  
Keyword(s):  
Pollen Development ◽  
Pollen Fertility ◽  
Development Process ◽  
Low Fertility ◽  
Abnormal Development ◽  
Lower Percentage ◽  
High Fertility ◽  
Rna Seq ◽  
Autotetraploid Rice ◽  
Environmentally Sensitive

Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for its utilization. Here, we reported the development of a new tetraploid rice, Huoduo1 (H1), which has the characteristic of high fertility, from crossing generations of autotetraploid rice. Cytological observations displayed the high fertility of the pollen (95.62%) in H1, a lower percentage of pollen mother cell (PMC) abnormalities, and stable chromosome configurations during the pollen development process compared with its parents. Using RNA-seq analysis, we detected 440 differentially expressed genes (DEGs) in H1 compared with its parents. Of these DEGs, 193 were annotated as pollen fertility-related genes, and 129 (~66.8%) exhibited significant up-regulation in H1 compared with the parents, including three environmentally sensitive genic male sterility genes (TMS9-1, TMS5, and CSA), one meiosis gene (RAD51D), and three tapetal-related genes (MIL2, OsAP25, and OsAP37), which were validated by qRT-PCR in this study. Two genes, TMS9-1 and TMS5, were knocked out using CRISPR/Cas9 technology, and their mutants displayed low fertility and the abnormal development of pollen. Our findings provide evidence for the regulatory mechanisms of fertility in tetraploid rice and indicated that the up-regulation of pollen fertility-related genes may contribute to the high fertility in new tetraploid rice.

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