An RNA Sequencing Transcriptome Analysis and Development of EST-SSR Markers in Chinese Hawthorn through Illumina Sequencing
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material and DNA Extraction
2.2. EST-SSR Mining from RNA-Seq and Primer Design
2.3. Primer Selection and PCR Amplification
2.4. Fluorescent-Labeled M13-SSR Markers and Capillary Electrophoresis
2.5. Data Analysis
2.6. Clustering Analysis
3. Results
3.1. Frequency and Distribution of EST-SSRs
3.2. Development, Screening, and Polymorphic Validation of EST-SSRs
3.3. Genetic Diversity Analysis
3.4. Cluster Analysis Using EST-SSR Markers
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Parameters Used in Screening | Data Generated by MIcroSAtellite (MISA) |
---|---|
Total number of sequences examined | 14,364 |
Total size of examined sequences (bp) | 28,888,844 |
Total number of identified SSRs | 5091 |
Number of SSR-containing sequences | 4011 |
Number of sequences containing more than one SSR | 863 |
Number of SSRs present in compound formation | 273 |
Primer Pairs | Forward Sequence (5′-3′) Reverse Sequence (5′-3′) | Motif Repeat | Product Size (bp) | Tm (°C) | Na | Ne | I | Ho | He | PIC |
---|---|---|---|---|---|---|---|---|---|---|
Pr1 | AATATTTGACCCGCTGCAAG TTCTGCAGGAAAAACCCATC | (AC)8 | 130 | 53.5 | 3 | 2.933 | 1.087 | 0.900 | 0.659 | 0.585 |
Pr19 | ATCAGTTTCCATCCCTGTCG TGGTGGACGTAACAGCACTC | (TA)6 | 206 | 56.5 | 6 | 2.632 | 1.168 | 0.797 | 0.620 | 0.554 |
Pr26 | GCTGCTGCAGTAAGCAATGA GCAACCCACAAAACTGGAAT | (CCT)6 | 103 | 54.5 | 6 | 2.271 | 0.986 | 0.913 | 0.560 | 0.469 |
Pr29 | ATGAGGCTGACGAGAGAGGA TTTGCAGAACCCAAAAGTCC | (AG)7 | 235 | 55.5 | 4 | 1.407 | 0.585 | 0.214 | 0.289 | 0.272 |
Pr31 | TTACACTCGCCAGAACCCTC CCGTCATGTTGAATCCTGTG | (CCG)5 | 182 | 56.5 | 3 | 2.010 | 0.790 | 0.729 | 0.503 | 0.407 |
Pr56 | GACGAGAATAACTCGCCTCG TGCACGACTTGCAGTACCTC | (TCG)5 | 199 | 57.5 | 6 | 2.454 | 1.117 | 0.824 | 0.592 | 0.522 |
Pr59 | CTGCAAGATTGGAGGAGGAG TCAAGGGGAGTGCTCTCAGT | (TGT)5 | 166 | 57.5 | 2 | 2.000 | 0.693 | 0.829 | 0.500 | 0.375 |
Pr81 | AGGAAGAGGAGGAATAGCCG GGATTCGCAGAGGATGTTGT | (GA)7 | 157 | 56.5 | 4 | 2.181 | 0.873 | 0.754 | 0.542 | 0.441 |
Pr83 | TTCCTTCTCACGCAAAATCC TGGGTTTTGGAAGCTTTGAG | (CT)6 | 116 | 53.5 | 4 | 1.280 | 0.457 | 0.194 | 0.219 | 0.206 |
Pr100 | GGACACCTTCTTTGGCACTC TGTGGGTTGTGTGTTTTGCT | (CT)9 | 173 | 55.5 | 4 | 1.479 | 0.561 | 0.250 | 0.324 | 0.280 |
Pr110 | CGCCGTAAACAGAGAGAGGA GTCGGAGAAAATGGTGTCGT | (CT)8 | 144 | 56.0 | 4 | 2.537 | 1.071 | 0.841 | 0.606 | 0.533 |
Pr114 | TTCAACCTCCATCCATCCAT CAAGCCTCATCAGAACACGA | (GCC)6 | 123 | 54.0 | 5 | 2.340 | 0.976 | 0.855 | 0.573 | 0.480 |
Pr117 | CTCAATGCAGTGGGAACTCA AGAGCTTGAGCAAGCAGAGG | (AG)6 | 262 | 56.0 | 2 | 1.258 | 0.359 | 0.232 | 0.205 | 0.184 |
Pr125 | GATGTGGACGATTGAGTTGC GCATGTAGCCCACAAGACAA | (TG)6 | 266 | 54.5 | 3 | 1.540 | 0.582 | 0.414 | 0.351 | 0.298 |
Pr134 | GGGTTGGTGAAAGCCCTAAT ATGCATACGCAGCAGTCTTG | (ATC)6 | 130 | 56.0 | 4 | 2.654 | 1.054 | 0.814 | 0.623 | 0.548 |
Pr146 | AGAAGATGACGACCACGACC TGCGATTCGAAACCCTAATC | (TTC)6 | 269 | 54.5 | 4 | 2.373 | 1.008 | 0.786 | 0.579 | 0.518 |
Pr152 | CGCTTGGTTAACTGTGGAGC TTGGACTCCACACATGCAAT | (AC)6 | 127 | 56.0 | 4 | 2.133 | 0.880 | 0.714 | 0.531 | 0.444 |
Pr167 | AACACTTCCCTCACCGTCAC TAGATTGCCAGGACCAGACC | (GTC)5 | 209 | 57.0 | 6 | 2.090 | 0.878 | 0.754 | 0.521 | 0.425 |
Pr171 | CATACCCACTCAAGGGCTGT GGCCTTTGACTCCAAATGAA | (CA)8 | 226 | 56.0 | 14 | 3.214 | 1.571 | 0.857 | 0.689 | 0.642 |
Pr174 | TCTTTCTCACCGAACCCATC TCGTAATCGGCGAGAGAGTT | (CT)8 | 227 | 54.5 | 4 | 1.773 | 0.785 | 0.132 | 0.436 | 0.389 |
Pr175 | TTGCACTTGTGGCTAGATCG CAATTCATTGCACTTCGGTG | (AT)7 | 133 | 53.5 | 9 | 3.010 | 1.383 | 0.870 | 0.668 | 0.616 |
Pr205 | ACCGTAGGAAGTCCAACACG CCCTCAGTTCAAGGAATACTCG | (GAG)5 | 125 | 57.5 | 4 | 2.510 | 1.016 | 0.899 | 0.602 | 0.523 |
Pr211 | TGTTGCTGCAATCTGCTTTC CCACTTGGAGGCAGTAGTGA | (AAT)6 | 183 | 56.0 | 4 | 3.082 | 1.228 | 0.900 | 0.676 | 0.615 |
Pr224 | AAACTTCATCGGTCCGTCAG CTCTTCCTTGCTGCATCCTC | (AG)6 | 242 | 56.0 | 10 | 2.456 | 1.207 | 0.836 | 0.593 | 0.527 |
Pr226 | ACGAGAGCATGCAGGAGAAT GCTTTCCGAACCTTCAACAG | (GGC)5 | 242 | 56.0 | 4 | 2.007 | 0.755 | 0.829 | 0.502 | 0.390 |
Pr235 | GCAAACAATGGGCACACATA GCTGTGCATGAGATGGAAGA | (GAG)5 | 163 | 54.5 | 9 | 2.563 | 1.191 | 1.000 | 0.610 | 0.533 |
Pr237 | CGGGATCAAGCAATGAAGAT CAGAGCCATGGTTCAGGAAT | (CCT)5 | 242 | 54.5 | 2 | 2.000 | 0.693 | 1.000 | 0.500 | 0.375 |
Pr239 | ACCATGGAAGATTCGTGGAG CCAATGAGCAAAAGCGTGTA | (CTT)5 | 196 | 54.5 | 4 | 2.196 | 0.902 | 0.729 | 0.545 | 0.456 |
Pr244 | GCCGACTCTACTTGAAGGGA TACGGATAAACCCAGCTTCG | (GTT)6 | 173 | 56.0 | 2 | 1.998 | 0.693 | 0.657 | 0.500 | 0.375 |
Pr246 | GCAAGGGACTTGAAATTGGA ATCCGATCGGTTCTGTGAAG | (GA)9 | 174 | 54.5 | 13 | 2.259 | 1.397 | 0.609 | 0.557 | 0.542 |
Pr251 | TCTTCCTTCTCCGCTCTCTG CCCATTTCTGTCTGCTCCAT | (AG)9 | 227 | 56.0 | 5 | 2.771 | 1.112 | 0.838 | 0.639 | 0.569 |
Pr255 | GCCGTATGGAGGGTCCTTAG GCGGAGGTGATTGGAGACTA | (TC)6 | 177 | 58.5 | 2 | 1.845 | 0.651 | 0.710 | 0.458 | 0.353 |
Pr295 | GCAATCGATGCATACAGCTC ACAAGAGTGTCAAGGCCCAG | (AG)9 | 225 | 56.0 | 3 | 2.047 | 0.772 | 0.729 | 0.512 | 0.401 |
Mean | / | / | / | / | 4.939 | 2.221 | 0.924 | 0.709 | 0.524 | 0.450 |
Standard Error | / | / | / | / | 0.515 | 0.088 | 0.049 | 0.042 | 0.022 | 0.118 |
Source | Dai et al., 2013 [11] | Yang et al., 2015 [42] | Xu et al., 2016 [43] | |
---|---|---|---|---|
Materials | C. pinnatifida H8 | C. pinnatifida S7 | C. pinnatifida “Zezhouhong” | C. pinnatifida “Qiujinxing” and “Ruanroushanlihong3Hao” |
Clean reads | 11,538,395 | 14,659,624 | 32,332,207 | 23,183,612 * |
Total bases (bp) | 2,140,589,552 | 2,743,145,672 | 6,530,452,306 | 5,839,454,759 * |
Number of >200 bp contigs | 460,119 | 515,118 | / | 2,182,914 |
Mean length of contigs (bp) | 130 | 131 | / | 77 |
Number of >200 bp transcripts | 54,662 | 62,653 | 83,817 | 199,204 |
Mean length of transcripts (bp) | 756 | 846 | 938 | 1056 |
N50 length of transcripts (bp) | 1237 | 1421 | 1615 | 1656 |
Number of unigenes | 39,663 | 41,723 | / | 72,837 |
Mean length of unigenes (bp) | 656 | 703 | / | 696 |
Sequencer | Illumina HiSeq 2000 | Illumina HiSeq 2000 | Illumina HiSeq 2500 |
Source | Dai et al., 2013 [11] | Yang et al., 2015 [42] | Our Study | |||
---|---|---|---|---|---|---|
Mono-nucleotide | 992 | 31.25% | / | / | 1989 | 39.07% |
Di-nucleotide | 1254 | 39.51% | 6707 | 64.05% | 2012 | 39.52% |
Tri-nucleotide | 677 | 21.33% | 3118 | 29.77% | 1024 | 20.11% |
Tetra-nucleotide | 29 | 0.91% | 159 | 1.52% | 46 | 0.90% |
Penta-nucleotide | 4 | 0.13% | 156 | 1.49% | 9 | 0.18% |
Hexa-nucleotide | 4 | 0.13% | 332 | 3.17% | 11 | 0.22% |
Number of compound SSRs | 214 | 6.74% | / | / | 273 | 5.36% |
Total number of identified SSRs | 3174 * | / | 10,472 | / | 5091 ** | / |
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Ma, S.; Dong, W.; Lyu, T.; Lyu, Y. An RNA Sequencing Transcriptome Analysis and Development of EST-SSR Markers in Chinese Hawthorn through Illumina Sequencing. Forests 2019, 10, 82. https://doi.org/10.3390/f10020082
Ma S, Dong W, Lyu T, Lyu Y. An RNA Sequencing Transcriptome Analysis and Development of EST-SSR Markers in Chinese Hawthorn through Illumina Sequencing. Forests. 2019; 10(2):82. https://doi.org/10.3390/f10020082
Chicago/Turabian StyleMa, Suliya, Wenxuan Dong, Tong Lyu, and Yingmin Lyu. 2019. "An RNA Sequencing Transcriptome Analysis and Development of EST-SSR Markers in Chinese Hawthorn through Illumina Sequencing" Forests 10, no. 2: 82. https://doi.org/10.3390/f10020082
APA StyleMa, S., Dong, W., Lyu, T., & Lyu, Y. (2019). An RNA Sequencing Transcriptome Analysis and Development of EST-SSR Markers in Chinese Hawthorn through Illumina Sequencing. Forests, 10(2), 82. https://doi.org/10.3390/f10020082