Reference Genes Selection of <i>Gymnosporangium yamadae</i> during the Interaction with Apple Leaves
Apple rust disease caused by <i>Gymnosporangium yamadae</i> is the one of the major threats to the development of the apple industry in China, but the pathogenic molecular mechanism of the disease remains unclear. It is imperative to screen out appropriate reference genes during the inte...
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MDPI AG
2022-08-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/8/8/830 |
| _version_ | 1797432020634173440 |
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| author | Chenxi Shao Wenhao Lao Yingmei Liang |
| author_facet | Chenxi Shao Wenhao Lao Yingmei Liang |
| author_sort | Chenxi Shao |
| collection | DOAJ |
| description | Apple rust disease caused by <i>Gymnosporangium yamadae</i> is the one of the major threats to the development of the apple industry in China, but the pathogenic molecular mechanism of the disease remains unclear. It is imperative to screen out appropriate reference genes during the interaction between <i>G. yamadae</i> and apple leaves to analyze the gene expression patterns during the pathogenesis of <i>G. yamadae</i>. <i>ACT</i>, <i>EF1</i>, <i>EF2</i>, <i>GAPDH</i>, <i>40S</i>, <i>60S</i>, <i>α-TUB</i>, <i>β-TUB</i> and <i>UBCE3</i> were selected as candidate reference genes based on the transcriptomic dataset of <i>G. yamadae</i>. The expression levels were tested by real-time quantitative PCR during time-course infection of apple leaves and the expression stabilities were evaluated by <i>△</i>Ct method as well as by three software (NormFinder, geNorm and BestKeeper) and one web-based analysis software (RefFinder). The expression stability of the candidate reference genes was further validated by using the effector candidate gene <i>Cluster-3395.48660</i> as the target gene in RT-qPCR. According to the results by <i>△</i>Ct and BestKeeper, <i>40S</i>, <i>EF2</i> and <i>EF1</i> were the most stable reference genes, while <i>EF1</i>, <i>EF2</i> and <i>GAPDH</i> were the most stable reference genes based on the NormFinder analysis result. The geNorm recommended the most stable genes <i>EF1</i>, <i>EF2</i> and <i>α-TUB</i> as reference genes. Comprehensive analysis results of the RefFinder indicated <i>EF1</i>, <i>EF2</i> and <i>α-TUB</i> were the most suitable genes. Based on these results, <i>EF1</i>, <i>EF2</i> and <i>α-TUB</i> were considered as reference genes for analyzing the gene expression profiles of <i>Cluster-3395.48660</i> in different infection stages, and the results were consistent with the transcriptome data. All the results suggest that the combination of <i>EF1</i>, <i>EF2</i> and <i>α-TUB</i> proved to be acceptable reference genes during the interaction between <i>G. yamadae</i> and apple leaves. |
| first_indexed | 2024-03-09T09:53:44Z |
| format | Article |
| id | doaj.art-4819e17154c4465991cec971aed74012 |
| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-09T09:53:44Z |
| publishDate | 2022-08-01 |
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| spelling | doaj.art-4819e17154c4465991cec971aed740122023-12-01T23:52:16ZengMDPI AGJournal of Fungi2309-608X2022-08-018883010.3390/jof8080830Reference Genes Selection of <i>Gymnosporangium yamadae</i> during the Interaction with Apple LeavesChenxi Shao0Wenhao Lao1Yingmei Liang2The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, ChinaThe Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, ChinaMuseum of Beijing Forestry University, Beijing Forestry University, Beijing 100083, ChinaApple rust disease caused by <i>Gymnosporangium yamadae</i> is the one of the major threats to the development of the apple industry in China, but the pathogenic molecular mechanism of the disease remains unclear. It is imperative to screen out appropriate reference genes during the interaction between <i>G. yamadae</i> and apple leaves to analyze the gene expression patterns during the pathogenesis of <i>G. yamadae</i>. <i>ACT</i>, <i>EF1</i>, <i>EF2</i>, <i>GAPDH</i>, <i>40S</i>, <i>60S</i>, <i>α-TUB</i>, <i>β-TUB</i> and <i>UBCE3</i> were selected as candidate reference genes based on the transcriptomic dataset of <i>G. yamadae</i>. The expression levels were tested by real-time quantitative PCR during time-course infection of apple leaves and the expression stabilities were evaluated by <i>△</i>Ct method as well as by three software (NormFinder, geNorm and BestKeeper) and one web-based analysis software (RefFinder). The expression stability of the candidate reference genes was further validated by using the effector candidate gene <i>Cluster-3395.48660</i> as the target gene in RT-qPCR. According to the results by <i>△</i>Ct and BestKeeper, <i>40S</i>, <i>EF2</i> and <i>EF1</i> were the most stable reference genes, while <i>EF1</i>, <i>EF2</i> and <i>GAPDH</i> were the most stable reference genes based on the NormFinder analysis result. The geNorm recommended the most stable genes <i>EF1</i>, <i>EF2</i> and <i>α-TUB</i> as reference genes. Comprehensive analysis results of the RefFinder indicated <i>EF1</i>, <i>EF2</i> and <i>α-TUB</i> were the most suitable genes. Based on these results, <i>EF1</i>, <i>EF2</i> and <i>α-TUB</i> were considered as reference genes for analyzing the gene expression profiles of <i>Cluster-3395.48660</i> in different infection stages, and the results were consistent with the transcriptome data. All the results suggest that the combination of <i>EF1</i>, <i>EF2</i> and <i>α-TUB</i> proved to be acceptable reference genes during the interaction between <i>G. yamadae</i> and apple leaves.https://www.mdpi.com/2309-608X/8/8/830<i>Gymnosporangium yamadae</i>reference genespathogenic mechanismapple |
| spellingShingle | Chenxi Shao Wenhao Lao Yingmei Liang Reference Genes Selection of <i>Gymnosporangium yamadae</i> during the Interaction with Apple Leaves Journal of Fungi <i>Gymnosporangium yamadae</i> reference genes pathogenic mechanism apple |
| title | Reference Genes Selection of <i>Gymnosporangium yamadae</i> during the Interaction with Apple Leaves |
| title_full | Reference Genes Selection of <i>Gymnosporangium yamadae</i> during the Interaction with Apple Leaves |
| title_fullStr | Reference Genes Selection of <i>Gymnosporangium yamadae</i> during the Interaction with Apple Leaves |
| title_full_unstemmed | Reference Genes Selection of <i>Gymnosporangium yamadae</i> during the Interaction with Apple Leaves |
| title_short | Reference Genes Selection of <i>Gymnosporangium yamadae</i> during the Interaction with Apple Leaves |
| title_sort | reference genes selection of i gymnosporangium yamadae i during the interaction with apple leaves |
| topic | <i>Gymnosporangium yamadae</i> reference genes pathogenic mechanism apple |
| url | https://www.mdpi.com/2309-608X/8/8/830 |
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