Study on differentially expressed genes related to defoliation traits in two alfalfa varieties based on RNA-Seq

- Study on differentially expressed genes related to defoliation traits in two alfalfa varieties based on RNA-Seq.
- Thus far, the understanding of the molecular mechanism of alfalfa defoliation traits remains unclear.
- The transcriptome database created by RNA-Seq is used to identify critical genes related to defoliation traits..
- Results: In this study, we sequenced the transcriptomes of the Zhungeer variety (with easy leaf abscission) and WL319HQ variety (without easy leaf abscission).
- Among the identified 66,734 unigenes, 706 differentially expressed genes (DEGs) upregulated, and 392 unigenes downregulated in the Zhungeer vs WL319HQ leaf.
- Six DEGs belonging to the “ Carotenoid biosynthesis.
- Plant hormone signal transduction ” and “ Circadian rhythm-plant ” pathways involved in defoliation traits were identified and validated by RT-qPCR analyses..
- Conclusions: This study used RNA-Seq to discover genes associated with defoliation traits between two alfalfa varieties.
- Alfalfa (Medicago sativa L.) is a member of the plant fam- ily Fabaceae.
- However, the leaves fall off very easily due to external pressure dur- ing harvest [4], and the resulting low-quality hay cannot satisfy livestock nutritional requirements..
- The main factors in leaf senescence are endogenous abscisic acid (ABA) and ethylene (ETH) [5, 6].
- ABA is a natural plant hormone identified in the 1960s, with roles in promoting plant organ shedding and seed germin- ation [7, 8].
- ETH is another early-discovered plant hormone with function in the regulation of plant growth, such as the promotion of fruit ripening, acceler- ation of organ aging and shedding [13].
- ETH synthesis is regulated by a variety of growth signals with tissue specificity at different development stages, and the accumulation of ETH can also stimulate further ETH production [15].
- 1 College of Grassland Resources and Environment, Key Laboratory of Forage Cultivation, Processing and High Efficient Utilization of the Ministry of Agriculture and Key Laboratory of Grassland Resources of the Ministry of Education, Inner Mongolia Agricultural University, Hohhot 010011, China Full list of author information is available at the end of the article.
- 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated..
- significantly higher in the processes of fruit ripening, seed germination, and leaf and flower aging off [16, 17]..
- Transcriptome sequencing re- search is essential for novel gene discovery, functional gene annotation, gene differential expression and the de- velopment of molecular markers [23–25].
- This study aimed to construct a tran- scriptome database of two alfalfa leaf varieties in the early blooming stage.
- Based on DEGs, we aimed to identify the critical genes related to defoliation traits in the Zhungeer variety (characterized by easy leaf abscis- sion) and the WL319HQ variety (without easy leaf ab- scission).
- The results of this study provide a theoretical basis for future alfalfa molecular breeding and provide a reference for subsequent study of the transcription of alfalfa leaves..
- Six libraries of the total RNA extracted from the leaves of Zhungeer and WL319HQ varieties at the early flow- ering stage were constructed for high-throughput se- quencing.
- and unigenes were annotated to the Nr, Swiss-Prot, KOG and KEGG databases, respectively (Fig.
- According to the Nr database, 19.26% of unigenes showed homology (1 x E − 20 <.
- and the remaining of 38.26% of unigenes had very strong homology (E-value ≤1E − 100 ) (Fig.
- For the spe- cies distribution of the top BLAST hits, 3,441 unigenes matched to the homologous sequences of Medicago trun- catula, while and 551 unigenes matched to the homologous sequences of Cicer arietinum, Cajanus cajan and Glycine max, respectively (Fig.
- 3), 706 uni- genes were upregulated, and the other 392 unigenes were downregulated (Zhungeer vs WL319HQ).
- The abscissa is the length of the assembled unigenes from 200 nt to ≥ 3000 nt, and the ordinate is the number of unigenes of the corresponding length.
- The numbers in the circles indicate the number of unigenes annotated by single or multiple databases.
- b E-value distribution of the top BLASTx hits against the Nr database.
- unigenes and 64 DEGs accounting for 22.78% of 281 DEGs were annotated to the “Ribosome”.
- In the 87 KEGG pathways, the DEGs related to direct or indirect effects on the content of ABA and ETH were predicted.
- In the “Plant hormone signal transduction”.
- In the “Circadian rhythm-plant” pathway (ko04712), 61 unigenes with 4 DEGs were annotated.
- To test the reliability of transcriptome sequencing data in the three pathways, Unigene0044746 (Auxin response fac- tor, ARF), Unigene0002039 (Phytochrome interacting factor 3, PIF3), Unigene0027311 (Ethylene receptor protein, ETR), Unigene0053251 (Phytochrome B, PHYB), Unigene0053032 (Cryptochrome, CRY) and Unigene0014585 (9-cis-epoxy- carotenoid dioxygenase 3, NCED3) (Additional file 4: Table.
- S4) were selected for One-Step RT-qPCR analysis.
- In addition to DEGs, Unigene0030464 (Tubulin alpha, TUBA) and Unigene0032887 (indole-3-acetic acid-amido synthe- tase, GH) were selected in the RT-qPCR analysis as un- affected genes.
- The significant difference of RT- qPCR data between two varieties was analyzed by t-test, and the expression patterns revealed by RT-qPCR analysis were similar to those revealed by transcriptome sequencing for the same genes (Fig.
- When plants are under environmental stress, such as drought, salinity or low temperature, the leaves will accelerate senescence and abscission due to the elevation of ABA concentration [11, 30].
- The RNA-Seq data revealed that the alfalfa 9-cis-epoxycarotenoid dioxygenase 3 (NCED3) gene differentially expressed between WL319HQ and Zhungeer varieties.
- The RT-qPCR analysis showed that the transcription of the Zhungeer variety was approxi- mately 2.3 times higher than the WL319HQ variety..
- thaliana NCED3 resulted in a significantly higher level of endogen- ous ABA in the plant [40], and similar results were ob- tained in transgenic tobacco to overexpress the tomato NCED1 gene [38]..
- The “Plant hormone signal transduction” (ko04075) pathway plays a vital role in the metabolism of phyto- hormones in higher plants.
- In the RNA-Seq data, the ex- pression level of the ethylene receptor protein (ETR) gene in Zhungeer variety was approximately 1.4 times higher than the WL319HQ variety.
- In the present study, the ETR gene was downregulated in WL319HQ compared to the Zhungeer variety, indicating that Zhungeer is more likely to accumulate ETH than the WL319HQ variety..
- Auxin response factors (ARF) are transcription factors that bind specifically to the DNA sequence 5’-TGTCTC-3′.
- Study of the A.
- The ARF gene was significantly downregulated in alfalfa leaves in the WL319HQ variety..
- Plant circadian rhythm is a hereditary physiological regulatory mechanism that synchronizes with circadian alternation in the plant.
- Circadian rhythm plays an essential role in the plant.
- At the same time, the plant hormone signaling network also acts on circa- dian rhythm to transmit different metabolic signals and environmental signals to the endogenous circadian rhythm system, thus forming a complex regulatory net- work [51, 52].
- In this study, three DEGs involved in the plant circadian rhythm pathway were identified that may affect the regulation of ABA and ETH.
- PIF3 plays a critical role not only in light and temperature-mediated environmental signaling but also in the signaling of ABA and ETH.
- This study is the first to use RNA-Seq to identify the de- foliation traits between alfalfa varieties.
- 4 The relative expression level of DEGs involved in “ Plant hormone signal transduction.
- Circadian rhythm-plant ” and “ Carotenoid biosynthesis ” pathways revealed by RT-qPCR analysis.
- We selected two varieties of the tetraploid M.
- sativa: the Zhungeer variety that has easy leaf abscission and the WL319HQ variety that is not characterized by easy leaf abscission.
- Leaves of the Zhungeer and WL319HQ var- ieties were collected from two-year-old plants at the early flowering stage in June.
- RNA extraction and RNA-Seq library construction.
- Total RNA was extracted from three biological replicates of the leaves of each M.
- Finally, Butterfly analyzed the paths from the de Bruijn graphs that were taken by reads and pairs of reads, and the complete transcripts and unigenes sets were ob- tained from the outputs [54]..
- The unigenes were annotated using the BLASTx program (http://www.ncbi.nlm.nih.gov/BLAST/) with an E-value threshold of to Nr (NCBI nonredundant protein) database (http://www.ncbi.nlm.nih.gov), the Swiss-Prot (A manually annotated and reviewed protein sequence data- base) protein database (http://www.expasy.ch/sprot), the KEGG (Kyoto Encyclopedia of Genes and Genomes) data- base (http://www.genome.jp/kegg) and the KOG (Clusters of Orthologous Groups of proteins) database (http://.
- Protein functional annota- tions were obtained according to the best alignment results..
- Analysis of DEGs.
- One-Step Real-time quantitative PCR (RT-qPCR) was carried out to confirm the RNA-Seq data from the ex- tracted M.
- Primers for One-Step RT- qPCR were designed using Primer Premier 5.0 software (Premier, Canada).
- The One-Step RT-qPCR analysis was performed on the extracted total RNA in three biological replicates of the leaves of each M.
- The relative expression changes of the endogenous reference and tested genes were analyzed by the 2.
- GO functional annotations and the number of DEGs statistics.
- Genes for RT-qPCR analysis and the list of GO ID.
- Primers used for RT-qPCR analysis.
- ABA: Abscisic acid.
- RT-qPCR: Real-time quantitative PCR.
- The funding body had no role in the design of the study, collection, analysis, or interpretation of data or in the writing of the manuscript..
- No specific permit was required for the field collection of alfalfa cultivars and the location is not privately-owned or protected in any way..
- 1 College of Grassland Resources and Environment, Key Laboratory of Forage Cultivation, Processing and High Efficient Utilization of the Ministry of Agriculture and Key Laboratory of Grassland Resources of the Ministry of Education, Inner Mongolia Agricultural University, Hohhot 010011, China..
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