Conjunctive Analyses of BSA-Seq and BSR-Seq Unveil the Msβ-GAL and MsJMT as Key Candidate Genes for Cytoplasmic Male Sterility in Alfalfa (Medicago sativa L.)
Abstract
:1. Introduction
2. Results
2.1. Sterility Survey of Parental Line and F2 Segregation Population
2.2. Bulked-Segregant Analysis
2.2.1. Sequencing Data Analysis of Sterile and Fertile Bulks
2.2.2. Association Analysis of BSA-Seq Data
2.3. Bulked Segregant RNA-Seq Analysis
2.3.1. Sequencing Data Analysis of Four Bulks
2.3.2. Comparison of Gene Expression Pattern between Two Set Pairs of Sequencing Bulks
2.3.3. Association Analysis of BSR-Seq Data
2.4. The DEGs Involved in Cytoplasmic Male Sterility
2.5. The Candidate Genes Involved in Cytoplasmic Male Sterility
2.6. Validation of Quantitative Real Time PCR for Key Genes Associated with Male Sterility in Alfalfa
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. Fertility Identification of Alfalfa
4.3. Construction of Illumina Library of BSA-Seq
4.4. Analysis of BSA-Seq Data
4.5. cDNA Library Preparation of BSR-Seq
4.6. Analysis of BSR-Seq Data
4.7. qRT-PCR Validation
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Bulk | Clean Reads | Data Generated | Q30 (%) | Genome Coverage (%) | Average Depth (×) | Alignment Efficiency (%) |
---|---|---|---|---|---|---|
R03 (sterile) | 238,377,744 | 67,766,104,095 | 94.29 | 83.69 | 45.92 | 90.01 |
R04 (fertile) | 271,300,098 | 77,009,673,959 | 93.82 | 84.53 | 52.12 | 91.03 |
Method. | Chromosome | Start (Mb) | End (Mb) | Length (Mb) | Genes Number |
---|---|---|---|---|---|
SNP-index | Chromosome 5 | 8.54 | 17.02 | 8.47 | 626 |
Bulk | Clean Reads | Data Generated (Gb) | Q30 (%) | Mapped Reads | Mapped Efficiency |
---|---|---|---|---|---|
T01 (male parent (♂)) | 24,944,663 | 7.43 | 94.14% | 17,230,648 | 85.05% |
T02 (female parent (♀)) | 27,026,088 | 8.06 | 94.01% | 19,151,540 | 84.11% |
T03 (sterile) | 39,503,495 | 11.76 | 93.76% | 27,499,773 | 85.60% |
T04 (fertile) | 38,797,934 | 11.54 | 94.03% | 27,389,445 | 85.18% |
Pathways | Transcript_ID | FPKM (T03 vs. T04) | Log2FC (T04/T03) | NR_Annotation | Gene Name | |
---|---|---|---|---|---|---|
T03 | T04 | |||||
ko 04075 (Plant hormone signal transduction) | c101056.graph_c0 | 22.93 | 77.45 | 1.90 | SAUR-like auxin-responsive family protein (Medicago truncatula) | SAUR |
c102778.graph_c0 | 0.05 | 1.21 | 3.84 | Indole-3-acetic acid-amido synthetase (Medicago truncatula) | GH3 | |
c116678.graph_c0 | 16.29 | 39.07 | 1.35 | indole-3-acetic acid-amido synthetase (Medicago truncatula) | GH3 | |
c90443.graph_c0 | 0.21 | 1.25 | 2.47 | BZIP transcription factor (Medicago truncatula) | ABF | |
c108656.graph_c0 | 10.01 | 3.89 | −1.18 | brassinosteroid receptor(Pisum sativum) | BRI1 | |
c102842.graph_c0 | 3.9 | 0.74 | −2.15 | ethylene response factor 5 (Medicago sativa) | ERF1/2 | |
ko 00500 (Starch and sucrose metabolism) | c89515.graph_c0 | 87.9 | 326.14 | 2.03 | Pectinesterase (Medicago truncatula) | Pectinesterase |
c116661.graph_c0 | 5.48 | 16.73 | 1.74 | polygalacturonase (Medicago truncatula) | polygalacturonase | |
c118955.graph_c0 | 50.45 | 100.64 | 1.17 | UDP-glucuronate 4-epimerase (Medicago truncatula) | GAE | |
c101072.graph_c0 | 0.2 | 1.03 | 2.25 | CAZy family GT8 glycosyltransferase (Medicago truncatula) | GAUT11 | |
c100736.graph_c2 | 8.76 | 16.46 | 1.06 | UDP-glucose dehydrogenase (Medicago truncatula) | UGDH | |
c102786.graph_c0 | 0.68 | 2.28 | 1.84 | Sucrose synthase (Medicago truncatula) | SUS | |
c106305.graph_c0 | 18.08 | 57.22 | 1.80 | glycosyl hydrolase family 9 protein (Medicago truncatula) | Endoglucanase | |
c107054.graph_c0 | 54.71 | 119.48 | 1.20 | beta-fructofuranosidase, insoluble protein (Medicago truncatula) | INV | |
c111356.graph_c0 | 5.08 | 13.78 | 1.63 | Fructokinase (Medicago truncatula) | FRK | |
c99556.graph_c0 | 3.14 | 12.18 | 2.09 | Trehalose-phosphate phosphatase (Medicago truncatula) | otsB | |
c114245.graph_c0 | 29.07 | 10.13 | −1.38 | Beta-amylase (Medicago truncatula) | b-amylase | |
ko 03030 (DNA replication) | c104110.graph_c0 | 0.84 | 2.3 | 1.79 | replication factor-A carboxy-terminal domain protein (Medicago truncatula) | RFA |
c120364.graph_c1 | 2.83 | 5.28 | 1.14 | ATP-dependent DNA helicase PIF1 (Medicago truncatula) | RFA | |
c84972.graph_c0 | 0.30 | 1.8 | 5.15 | Replication factor A protein (Medicago truncatula) | RFA | |
c88743.graph_c0 | 0.20 | 2.41 | 6.54 | Replication factor A protein (Medicago truncatula) | RFA | |
c96653.graph_c0 | 10.71 | 2.52 | −1.99 | Replication factor A protein (Medicago truncatula) | RFA | |
c111708.graph_c1 | 1.91 | 0.55 | −1.61 | Replication factor A protein (Medicago truncatula) | RFA |
Transcript_ID | Regulation (T04/T03) | Gene Name | NR_Annotation |
---|---|---|---|
c123275.graph_c0 | down | Unknow | Unknow |
c111485.graph_c0 | down | Unknow | Unknow |
c102760.graph_c0 | up | EF-Tu | elongation factor Tu family protein (Medicago truncatula) |
c115887.graph_c0 | down | JMT | Jasmonate O-methyltransferase (Medicago truncatula) |
c102682.graph_c0 | up | β-GAL | Beta-galactosidase (Medicago truncatula) |
c113540.graph_c0 | down | CESA | Cellulose synthase (Medicago truncatula) |
c111062.graph_c0 | up | PHGDH | Phosphoglycerate dehydrogenase (Medicago truncatula) |
Gene_Id | Chr | Start (Kb) | End (Kb) | NR_Annotation |
---|---|---|---|---|
MsG0580024998.01 (Transcript_id: c115887.graph_c0) | Chr5 | 12668765 | 12689067 | Jasmonate O-methyltransferase (Medicago truncatula) |
MsG0580025125.01 | Chr5 | 14126518 | 14128116 | Beta-galactosidase (Medicago truncatula) |
MsG0580025201.01 | Chr5 | 15181309 | 15201352 | Beta-galactosidase (Medicago truncatula) |
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Zhou, L.; Wang, Y.; Xu, X.; Yan, D.; Yu, W.; Miao, Y.; Xu, B. Conjunctive Analyses of BSA-Seq and BSR-Seq Unveil the Msβ-GAL and MsJMT as Key Candidate Genes for Cytoplasmic Male Sterility in Alfalfa (Medicago sativa L.). Int. J. Mol. Sci. 2022, 23, 7172. https://doi.org/10.3390/ijms23137172
Zhou L, Wang Y, Xu X, Yan D, Yu W, Miao Y, Xu B. Conjunctive Analyses of BSA-Seq and BSR-Seq Unveil the Msβ-GAL and MsJMT as Key Candidate Genes for Cytoplasmic Male Sterility in Alfalfa (Medicago sativa L.). International Journal of Molecular Sciences. 2022; 23(13):7172. https://doi.org/10.3390/ijms23137172
Chicago/Turabian StyleZhou, Le, Yingzhe Wang, Xiaobo Xu, Dong Yan, Weijie Yu, Yifan Miao, and Bo Xu. 2022. "Conjunctive Analyses of BSA-Seq and BSR-Seq Unveil the Msβ-GAL and MsJMT as Key Candidate Genes for Cytoplasmic Male Sterility in Alfalfa (Medicago sativa L.)" International Journal of Molecular Sciences 23, no. 13: 7172. https://doi.org/10.3390/ijms23137172