Methyl-Sensitive Amplification Polymorphism (MSAP) Analysis Provides Insights into the DNA Methylation Changes Underlying Adaptation to Low Temperature of Brassica rapa L.
Abstract
:1. Introduction
2. Results
2.1. Pre-Amplification Reactions and Screening of Polymorphic Primers
2.2. Effect of Low Temperature on DNA Methylation Levels in Winter Rapeseed
2.3. Effect of Low Temperature on DNA Methylation Patterns in Winter Rapeseed
2.4. Effect of Room Temperature Recovery (RG2d) on DNA Methylation Patterns in Winter Rapeseed
2.5. Sequence Analysis of Differentially Methylated DNA Fragments
2.6. Cloning and Expression Profiling of the Polymorphic Fragments
3. Discussion
4. Materials and Methods
4.1. Plant Materials and Cold Treatment
4.2. DNA Extraction, RNA Extraction, and cDNA Synthesis
4.3. MSAP Analysis
4.4. Different Bands Were Recovered and Cloned and the Sequences Were Compared
4.5. qPCR Analysis of the Differentially Methylated Genes
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Materials | Treatment | Band Type I: The Number of Unmethylated Bands | Band Type II: The Number of Semi-Methylated Bands | Band Type III: The Number of Total Methylated Bands | Total Number of Bands | Total Number of Methylated Bands | Semi- Methylation Ratio (%) | Holo-methylation Ratio (%) | Total Methylation Ratio (%) |
---|---|---|---|---|---|---|---|---|---|
CT-2360 | CK | 92 | 37 | 51 | 180 | 88 | 20.56 | 28.33 | 48.89 |
4 °C | 86 | 33 | 47 | 166 | 80 | 19.88 | 28.31 | 48.19 | |
RG2d | 87 | 35 | 48 | 170 | 83 | 20.59 | 28.24 | 48.82 | |
MXW-1 | CK | 108 | 28 | 48 | 184 | 76 | 15.22 | 26.09 | 41.30 |
4 °C | 117 | 29 | 41 | 187 | 70 | 15.51 | 21.93 | 37.43 | |
RG2d | 114 | 23 | 39 | 176 | 62 | 13.07 | 22.16 | 35.23 | |
2018-FJT | CK | 106 | 35 | 42 | 183 | 77 | 19.13 | 22.95 | 42.08 |
4 °C | 101 | 20 | 46 | 167 | 66 | 11.98 | 27.54 | 39.52 | |
RG2d | 96 | 41 | 47 | 184 | 88 | 22.28 | 25.54 | 47.83 | |
DT-7 | CK | 112 | 35 | 39 | 186 | 74 | 18.82 | 20.97 | 39.78 |
4 °C | 110 | 26 | 37 | 173 | 63 | 15.03 | 21.39 | 36.42 | |
RG2d | 92 | 27 | 59 | 178 | 86 | 15.17 | 33.15 | 48.31 | |
Total | 1221 | 369 | 544 | 2134 | 913 |
Type | CK | 4 °C | CT-2360 | MXW-1 | 2018-FJT | DT-7 | Pattern | ||
---|---|---|---|---|---|---|---|---|---|
Number of Points and Proportion | Number of Points and Proportion | Number of Points and Proportion | Number of Points and Proportion | ||||||
A (%) | EH | EM | EH | EM | 114 (62.30%) | 104 (55.62%) | 110 (62.15%) | 113 (58.55%) | no change |
A1 | 1 | 1 | 1 | 1 | 71 | 64 | 67 | 56 | |
A2 | 1 | 0 | 1 | 0 | 19 | 16 | 14 | 25 | |
A3 | 0 | 1 | 0 | 1 | 24 | 24 | 29 | 32 | |
B (%) | 27 (14.75%) | 43 (22.99%) | 35 (19.77%) | 47 (24.35%) | demethylation | ||||
B1 | 1 | 0 | 1 | 1 | 7 | 6 | 2 | 4 | |
B2 | 0 | 1 | 1 | 1 | 7 | 14 | 10 | 16 | |
B3 | 0 | 0 | 0 | 1 | 3 | 11 | 6 | 13 | |
B4 | 0 | 0 | 1 | 0 | 6 | 8 | 14 | 8 | |
B5 | 0 | 0 | 1 | 1 | 4 | 4 | 3 | 6 | |
C (%) | 42 (22.95%) | 34 (18.18%) | 27 (15.26%) | 33 (17.10%) | methylation/ hypermethylation | ||||
C1 | 1 | 0 | 0 | 0 | 14 | 7 | 5 | 9 | |
C2 | 0 | 1 | 0 | 0 | 9 | 3 | 5 | 9 | |
C3 | 1 | 1 | 0 | 1 | 7 | 11 | 6 | 5 | |
C4 | 1 | 1 | 1 | 0 | 5 | 4 | 5 | 3 | |
C5 | 1 | 1 | 0 | 0 | 7 | 9 | 6 | 7 | |
D (%) | 0 (0) | 6 (3.21%) | 5 (2.82%) | 0 (0) | other | ||||
D1 | 1 | 0 | 0 | 1 | 0 | 2 | 4 | 0 | |
D2 | 0 | 1 | 1 | 0 | 0 | 4 | 1 | 0 | |
Total | 183 | 187 | 177 | 193 |
Type | CK | RG2d | CT-2360 | MXW-1 | 2018-FJT | DT-7 | Pattern | ||
---|---|---|---|---|---|---|---|---|---|
Number of Points and Proportion | Number of Points and Proportion | Number of Points and Proportion | Number of Points and Proportion | ||||||
A (%) | EH | EM | EH | EM | 77 (49.68%) | 88 (53.99%) | 86 (50%) | 97 (48.99%) | no change |
A1 | 1 | 1 | 1 | 1 | 41 | 52 | 49 | 47 | |
A2 | 1 | 0 | 1 | 0 | 13 | 15 | 15 | 26 | |
A3 | 0 | 1 | 0 | 1 | 23 | 21 | 22 | 24 | |
B (%) | 21 (13.55%) | 31 (19.02%) | 38 (22.1%) | 43 (21.72%) | demethylation | ||||
B1 | 1 | 0 | 1 | 1 | 8 | 7 | 2 | 6 | |
B2 | 0 | 1 | 1 | 1 | 3 | 5 | 10 | 9 | |
B3 | 0 | 0 | 0 | 1 | 5 | 5 | 9 | 9 | |
B4 | 0 | 0 | 1 | 0 | 3 | 11 | 10 | 11 | |
B5 | 0 | 0 | 1 | 1 | 2 | 3 | 7 | 8 | |
C (%) | 56 (36.13%) | 37 (22.7%) | 45 (26.16%) | 55 (27.78%) | methylation/ hypermethylation | ||||
C1 | 1 | 0 | 0 | 0 | 15 | 9 | 7 | 10 | |
C2 | 0 | 1 | 0 | 0 | 10 | 11 | 13 | 13 | |
C3 | 1 | 1 | 0 | 1 | 19 | 8 | 12 | 13 | |
C4 | 1 | 1 | 1 | 0 | 6 | 4 | 9 | 11 | |
C5 | 1 | 1 | 0 | 0 | 6 | 5 | 4 | 8 | |
D (%) | 1 (0.64%) | 7 (4.29%) | 3 (1.74%) | 3 (1.51%) | other | ||||
D1 | 1 | 0 | 0 | 1 | 0 | 3 | 2 | 3 | |
D2 | 0 | 1 | 1 | 0 | 1 | 4 | 1 | 0 | |
Total | 155 | 163 | 172 | 198 |
Fragment | Methylation Pattern | Length (bp) | Gene Description | E-Value | Identities % | Accession |
---|---|---|---|---|---|---|
M-01 | Demethylation | 167 | Brassica napus protein KTI12 homolog (LOC106369112), transcript variant X2, mRNA | 2.00 × 10−46 | 91.1 | XM_013809215.2 |
M-02 | Demethylation | 143 | Brassica napus NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13-B (LOC106365400), mRNA | 1.00 × 10−53 | 100 | XM_013804832.2 |
M-03 | Demethylation | 75 | Brassica rapa probable receptor-like protein kinase At5g39020 (LOC103874456), mRNA | 3.00 × 10−16 | 100 | XM_009152882.3 |
M-04 | Methylation | 128 | Brassica rapa VQ motif-containing protein 22 (LOC103834848), mRNA | 5.00 × 10−42 | 98.08 | XM_009110941.3 |
M-05 | Demethylation | 142 | Brassica napus cysteine-rich receptor-like protein kinase 10 (LOC106398281), mRNA | 3.00 × 10−49 | 97.5 | XM_013838867.2 |
M-06 | Methylation | 93 | Brassica rapa cysteine-rich receptor-like protein kinase 10 (LOC103834757), transcript variant X4, mRNA | 9.00 × 10−28 | 100 | XM_033286881.1 |
M-07 | Demethylation | 170 | Brassica napus glucan endo-1,3-beta-glucosidase (LOC106445206), mRNA | 2.00 × 10−66 | 99.31 | XM_013886724.2 |
M-08 | Methylation | 88 | Brassica rapa glucan endo-1,3-beta-glucosidase (LOC103832330), transcript variant X2, mRNA | 5.00 × 10−20 | 96.88 | XM_009108313.3 |
M-09 | Methylation | 67 | Brassica rapa glucan endo-1,3-beta-glucosidase 11 (LOC103835240), mRNA | 1.00 × 10−8 | 95.35 | XM_009111375.3 |
M-10 | Demethylation | 129 | Brassica rapa putative glutamine amidotransferase GAT1_2.1 (LOC103850104), mRNA | 9.00 × 10−15 | 98.08 | XM_009126815.3 |
M-11 | Methylation | 133 | Brassica rapa endoglucanase 8 (LOC103831605), mRNA | 2.00 × 10−25 | 100 | XM_009107492.3 |
M-12 | Methylation | 81 | Brassica rapa O-glucosyltransferase rumi homolog (LOC103843476), mRNA | 4.00 × 10−15 | 100 | XM_009120210.3 |
M-13 | Methylation | 98 | Brassica rapa auxin-induced protein 15A (LOC103834602), mRNA | 3.00 × 10−27 | 98.65 | XM_009110705.3 |
M-14 | Demethylation | 105 | Brassica rapa transcription initiation factor IIB (LOC103863051), transcript variant X3, mRNA | 3.00 × 10−28 | 95.35 | XM_009140791.3 |
M-15 | Demethylation | 178 | Brassica rapa terpenoid synthase 17 (LOC103859580), mRNA | 4.00 × 10−59 | 94.81 | XM_009137140.2 |
M-16 | Methylation | 71 | Brassica rapa S-locus-specific glycoprotein S13 (LOC103831122), mRNA | 8.00 × 10−12 | 97.83 | XM_009106973.3 |
M-17 | Methylation | 64 | Brassica rapa monothiol glutaredoxin-S1 (LOC103844538), mRNA | 5.00 × 10−8 | 100 | XM_033282469.1 |
M-18 | Demethylation | 58 | Brassica rapa purple acid phosphatase 5 (LOC103837841), transcript variant X2, mRNA | 5.00 × 10−7 | 97.3 | XM_009114221.3 |
M-19 | Demethylation | 124 | Brassica rapa iron-sulfur assembly protein IscA-like 1, mitochondrial (LOC117127666), mRNA | 2.00 × 10−41 | 99 | XM_033278271.1 |
M-20 | Demethylation | 124 | Brassica napus homeobox-leucine zipper protein HDG2-like (LOC106398145), mRNA | 2.00 × 10−41 | 99 | XM_013838740.1 |
M-21 | Methylation | 109 | Brassica rapa elongation factor G-2, mitochondrial (LOC103866857), mRNA | 5.00 × 10−31 | 97.62 | XM_009144847.3 |
M-22 | Methylation | 97 | Brassica rapa probable protein phosphatase 2C 30 (LOC103865774), mRNA | 3.00 × 10−28 | 100 | XM_009143626.2 |
M-23 | Methylation | 184 | Brassica rapa probable clathrin assembly protein At4g32285 (LOC103851149), mRNA | 2.00 × 10−77 | 100 | XM_009127985.3 |
M-24 | Demethylation | 74 | Brassica rapa E3 ubiquitin-protein ligase ATL6-like (LOC103859080), mRNA | 1.00 × 10−5 | 100 | XM_009136564.3 |
M-25 | Demethylation | 75 | Brassica rapa testis-expressed protein 2 (LOC103831800), mRNA | 1.00 × 10−5 | 97.14 | XM_009107719.3 |
M-26 | Methylation | 89 | Brassica rapa glycine-rich RNA-binding protein 5, mitochondrial-like (LOC103860185), mRNA | 3.00 × 10−22 | 98.46 | XM_033288947.1 |
M-27 | Methylation | 113 | Brassica napus polyribonucleotide nucleotidyltransferase 2, mitochondrial-like (LOC106416529), transcript variant X3, mRNA | 3.00 × 10−14 | 98.04 | XM_013857446.2 |
M-28 | Demethylation | 91 | Brassica napus protein DETOXIFICATION 37 (LOC106419146), transcript variant X2, mRNA | 1.00 × 10−25 | 96.15 | XM_013859997.2 |
M-29 | Demethylation | 93 | Cardamine resedifolia voucher CresL-Sta1-1 haplotype 1 Ascorbate peroxidase 1 (APX1) gene, partial cds | 4.00 × 10−11 | 95.92 | KJ428054.1 |
M-30 | Demethylation | 75 | Brassica rapa subsp. pekinensis cultivar Inbred line Chiifu “clone KBrB045B23, complete sequence” | 3.00 × 10−16 | 96.49 | AC189360.2 |
M-31 | Demethylation | 164 | Brassica juncea isolate 94 mitochondrion, complete genome | 1.00 × 10−48 | 92.86 | MG872828.1 |
M-32 | Demethylation | 164 | Brassica juncea cytoplasmic male sterility-associated cytochrome oxidase subunit I (coxI-2) gene, complete cds; mitochondrial gene for mitochondrial product | 7.00 × 10−52 | 94.29 | AY300015.1 |
M-33 | Methylation | 75 | Pseudomonas fluorescens strain NCTC10038 genome assembly, chromosome: 1 | 1.00 × 10−14 | 96.3 | LS483372.1 |
M-34 | Methylation | 179 | Brassica oleracea HDEM genome, scaffold: C4 | 3.00 × 10−65 | 96.77 | LR031873.1 |
M-35 | Methylation | 102 | Tetracme recurvata isolate C236 18S ribosomal RNA gene, internal transcribed spacer 1, 5.8S ribosomal RNA gene, internal transcribed spacer 2, and 26S ribosomal RNA gene, complete sequence | 6.00 × 10−30 | 97.56 | MT819350.1 |
M-36 | Methylation | 214 | Brassica juncea chromosome og1-b mitochondrion, complete sequence | 2.00 × 10−92 | 100 | MT675106.1 |
M-37 | Demethylation | 104 | Uncultured bacterium clone Otu03067 16S ribosomal RNA gene, partial sequence | 2.00 × 10−29 | 97.53 | KX997786.1 |
M-38 | Methylation | 155 | Yosemitea repanda chloroplast, complete genome | 6.00 × 10−47 | 100 | MK637830.1 |
M-39 | Methylation | 128 | Lepidium apetalum chloroplast, complete genome | 4.00 × 10−18 | 100 | MT880914.1 |
M-40 | Methylation | 81 | Brassica rapa uncharacterized LOC103836772, transcript variant X2, mRNA | 1.00 × 10−6 | 93.18 | XM_033280897.1 |
M-41 | Demethylation | 154 | Brassica rapa uncharacterized LOC103871127, mRNA | 1.00 × 10−9 | 100 | XM_009149356.3 |
M-42 | Demethylation | 89 | Brassica rapa uncharacterized LOC103874467, mRNA | 5 × 10−20 | 96.88 | XM_009152895.3 |
M-43 | Demethylation | 109 | Brassica rapa uncharacterized LOC103833097, transcript variant X4, misc_RNA | 7.00 × 10−35 | 98.86 | XR_626125.2 |
M-44 | Methylation | 68 | Brassica rapa uncharacterized LOC117128160, ncRNA | 0.002 | 100 | XR_004451357.1 |
M-45 | Demethylation | 97 | Brassica rapa uncharacterized LOC117134590, ncRNA | 3.00 × 10−18 | 100 | XR_004458779.1 |
M-46 | Demethylation | 92 | Brassica rapa uncharacterized LOC117127533, mRNA | 4.00 × 10−26 | 100 | XM_033278112.1 |
M-47 | Demethylation | 87 | Brassica rapa uncharacterized LOC103874342, transcript variant X2, mRNA | 8.00 × 10−23 | 95.89 | XM_033292971.1 |
M-48 | Methylation | 75 | Brassica rapa uncharacterized LOC103846332, mRNA | 1.00 × 10−26 | 97.37 | XM_009123242.3 |
M-49 | Demethylation | 53 | Brassica napus uncharacterized LOC106386228, mRNA | 0.001 | 100 | XM_013826104.2 |
M-50 | Methylation | 108 | Brassica napus uncharacterized LOC106378351, mRNA | 5.00 × 10−31 | 98.77 | XM_013818495.1 |
M-51 | Methylation | 87 | Brassica napus uncharacterized LOC106388033, transcript variant X2, ncRNA | 2.00 × 10−14 | 92.06 | XR_001277777.2 |
M-52 | Methylation | 188 | Brassica napus uncharacterized LOC106451272, mRNA | 1.00 × 10−79 | 98.3 | XM_013893176.2 |
M-53 | Demethylation | 121 | Brassica napus uncharacterized mitochondrial protein LOC111207234, mRNA | 1.00 × 10−13 | 100 | XM_022705092.1 |
Materials | Place of Origin and Environmental Conditions | Root Collar Diameter (cm) | Over- Wintering Rate (%) | LT50 (°C) | Cold Resistance Ranking | Plant Height (cm) | Branch Height (cm) | Siliques per Plant | Seeds per Silique | Thousand-Seed Weight (g) | Seed Yield per Plant (g) |
---|---|---|---|---|---|---|---|---|---|---|---|
CT-2360 | Caotan, Zhangxian County, Gansu Province; Latitude: 34°38′ N; longitude: 104°28′ E; above sea level: 2360~2460 m; mean annual temperature: 4.3 °C; lowest annual temperature: −8.8 °C. | 1.1 | 89.0 | −11.32 | 4 | 112.0 | 11.2 | 145.0 | 21.5 | 3.1 | 11.0 |
MXW-1 (Longyou-7) | Shangchuan, Lanzhou City, Gansu Province; latitude: 36°03′; longitude: 103°40′; above sea level: 2150 m; mean annual temperature: 6.5 °C; lowest annual temperature: −14.6 °C. | 1.0 | 90.0 | −15.04 | 3 | 102.0 | 8.2 | 150.0 | 22.0 | 3.1 | 12.0 |
DT-7 | Datong City, Shanxi Province; Latitude: 40°04′; longitude: 113°08′; above sea level: 1000 m; mean annual temperature: 6.4 °C; lowest annual temperature: −29.2 °C. | 1.0 | 94.0 | −15.98 | 2 | 92.0 | 8.0 | 157.0 | 22.0 | 3.2 | 12.5 |
2018-FJT | Fanjiatun, Gongzhuling City, Jilin Province; Latitude: 43°43′; longitude: 124°50′; above sea level: 223 m; mean annual temperature: 5.9 °C; lowest annual temperature: −30 °C. | 1.1 | 95.0 | −16.04 | 1 | 90.0 | 6.1 | 167.0 | 23.0 | 3.3 | 13.2 |
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Liu, L.; Wang, W.; Lu, X.; Zhang, T.; Wu, J.; Fang, Y.; Ma, L.; Pu, Y.; Yang, G.; Wang, W.; et al. Methyl-Sensitive Amplification Polymorphism (MSAP) Analysis Provides Insights into the DNA Methylation Changes Underlying Adaptation to Low Temperature of Brassica rapa L. Plants 2024, 13, 1748. https://doi.org/10.3390/plants13131748
Liu L, Wang W, Lu X, Zhang T, Wu J, Fang Y, Ma L, Pu Y, Yang G, Wang W, et al. Methyl-Sensitive Amplification Polymorphism (MSAP) Analysis Provides Insights into the DNA Methylation Changes Underlying Adaptation to Low Temperature of Brassica rapa L. Plants. 2024; 13(13):1748. https://doi.org/10.3390/plants13131748
Chicago/Turabian StyleLiu, Lijun, Wanpeng Wang, Xiaoming Lu, Tianyu Zhang, Junyan Wu, Yan Fang, Li Ma, Yuanyuan Pu, Gang Yang, Wangtian Wang, and et al. 2024. "Methyl-Sensitive Amplification Polymorphism (MSAP) Analysis Provides Insights into the DNA Methylation Changes Underlying Adaptation to Low Temperature of Brassica rapa L." Plants 13, no. 13: 1748. https://doi.org/10.3390/plants13131748
APA StyleLiu, L., Wang, W., Lu, X., Zhang, T., Wu, J., Fang, Y., Ma, L., Pu, Y., Yang, G., Wang, W., & Sun, W. (2024). Methyl-Sensitive Amplification Polymorphism (MSAP) Analysis Provides Insights into the DNA Methylation Changes Underlying Adaptation to Low Temperature of Brassica rapa L. Plants, 13(13), 1748. https://doi.org/10.3390/plants13131748