De Novo Transcriptomic Analysis and Development of EST–SSRs for Styrax japonicus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. DNA and RNA Isolation
2.3. cDNA Library Construction and Sequencing
2.4. Sequence Assembly and Annotation
2.5. Identification of EST–SSR Loci and Primer Design
2.6. Validation and Application of SSR Markers
3. Results
3.1. Sequencing and De Novo Assembly
3.2. Functional Annotation of Unigenes
3.3. Frequency and Distribution of SSRs
3.4. Development and Validation of Novel EST–SSRs
4. Discussion
4.1. Characterization of the S. japonicus Transcriptome
4.2. SSR Markers in the Transcriptome of S. japonicus
4.3. Validation of EST-SSR Markers
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Category | Items | Number |
---|---|---|
Raw reads | Total raw reads | 364,794,740 |
Clean reads | Total clean reads | 338,570,222 |
Total clean nucleotide (nt) | 49,540,935,462 | |
Q20 percentage (average) | 98.01% | |
Q30 percentage (average) | 94.67% | |
GC percentage (average) | 41.58% | |
Unigenes | Total sequence number | 136,071 |
Total sequence Length | 83,181,839 | |
Largest (bp) | 10,742 | |
Smallest (bp) | 224 | |
Average (bp) | 611 | |
N50 (bp) | 846 | |
EST–SSRs | Sequences examined | 136,071 |
Size of examined sequences (bp) | 83,181,839 | |
Identified SSRs | 55,977 | |
SSR-containing sequences | 38,611 | |
Sequences containing more than one SSR | 11,652 | |
SSRs present in compound formation | 8121 |
Repeats | Di- | Tri- | Tetra- | Penta- | Hexa- | Total | Percentage (%) |
---|---|---|---|---|---|---|---|
3 | 5978 | 2170 | 2291 | 10,439 | 25.14 | ||
4 | 7072 | 974 | 390 | 624 | 9,060 | 21.82 | |
5 | 7064 | 2256 | 262 | 91 | 66 | 9,739 | 23.45 |
6 | 2826 | 909 | 80 | 6 | 51 | 3,872 | 9.32 |
7 | 1747 | 438 | 11 | 4 | 27 | 2,227 | 5.36 |
8 | 1486 | 308 | 8 | 3 | 12 | 1,817 | 4.38 |
9 | 1076 | 51 | 4 | 9 | 1,140 | 2.75 | |
10 | 824 | 63 | 4 | 3 | 894 | 2.15 | |
11 | 687 | 44 | 1 | 3 | 735 | 1.77 | |
12 | 453 | 32 | 1 | 486 | 1.17 | ||
13 | 130 | 31 | 1 | 162 | 0.39 | ||
14 | 139 | 9 | 1 | 149 | 0.36 | ||
15 | 147 | 12 | 159 | 0.38 | |||
16 | 102 | 6 | 108 | 0.26 | |||
17 | 93 | 7 | 100 | 0.24 | |||
18 | 87 | 3 | 90 | 0.22 | |||
19 | 68 | 7 | 75 | 0.18 | |||
20 | 55 | 3 | 58 | 0.14 | |||
21 | 41 | 3 | 44 | 0.11 | |||
22 | 31 | 2 | 33 | 0.08 | |||
23 | 16 | 1 | 17 | 0.04 | |||
≥24 | 121 | 3 | 2 | 126 | 0.30 | ||
Total | 17,193 | 11,260 | 7323 | 2664 | 3090 | 41,530 | |
Percentage (%) | 41.40 | 27.11 | 17.64 | 6.41 | 7.44 |
Primer | Forward Primer (5′–3′) | Reverse Primer (5′–3′) | Tm (°C) | SSRs | Alleles Number | Ho | He | PIC | Fst | r |
---|---|---|---|---|---|---|---|---|---|---|
Sty-3 | GGATCGATCCCAATGGCGTA | TCCTTAGCTCCATCAAGTGCA | 59.091 | (GTTTG) 5 | 7 | 0.800 | 0.625 | 0.582 | 0.023 | 0.143 |
Sty-7 | ACAAGCCATGAGCCGATGAA | TGTTGTCCTTTGTTAATTCCCCT | 58.19 | (AGC) 4 | 3 | 0.325 | 0.308 | 0.282 | 0.049 | 0.333 |
Sty-16 | CTCTCCACCCCAATTGCCAT | CCAAGCTTGCCAAATCCACC | 60.037 | (CCG) 8 | 7 | 0.475 | 0.464 | 0.441 | 0.037 | 0.143 |
Sty-20 | GGGTTGTATAAGGGTTGCCA | GACGGTGGTACTTCACAGTGT | 59.932 | (AT) 10 | 7 | 0.692 | 0.767 | 0.732 | 0.019 | 0.143 |
Sty-39 | TTCATCGGCTCTTGGCTTGT | CAGCGTCAAGTTGGTTTCGG | 60.041 | (GTG) 4 | 9 | 0.950 | 0.811 | 0.776 | 0.027 | 0.111 |
Sty-43 | GGACGCTCAAGGTCAACTCA | AGGCTGCCATATCTAGGGGT | 59.805 | (TCGGT) 3 | 5 | 0.875 | 0.603 | 0.523 | 0.005 | 0.200 |
Sty-44 | CCTTCCCCACAACCCACTAC | ATGCTTCCTCATTGTGCAGC | 59.184 | (TC) 12 | 12 | 0.939 | 0.882 | 0.855 | 0.039 | 0.083 |
Sty-47 | GGGTTGGCCACCTTGATCTT | TCAGAGGCTCCAATACGTAGC | 59.318 | (TC) 11 (TA) 8 | 14 | 1.000 | 0.873 | 0.848 | 0.073 | 0.071 |
Sty-49 | TCACCAGTTCGCGTGTTAGT | GCATGGGGTTTCTTGCGAAG | 60.109 | (TCAT) 3 | 6 | 0.846 | 0.725 | 0.678 | 0.027 | 0.167 |
Sty-55 | TGTTGCTTGAAAGTGCGGTC | GTGTGGACACATCACCGGAT | 60.036 | (AATA) 4 | 8 | 0.256 | 0.491 | 0.450 | 0.043 | 0.125 |
Sty-57 | TCGGGATCTTTTAAGTTTTGGAGC | GAATCCGTTTGGTTGCCGAG | 59.832 | (AAAT) 3 | 3 | 0.025 | 0.074 | 0.071 | 0.030 | 0.333 |
Sty-58 | TCCTTGTGCACTCAGTCCAC | TGTAGCACACGGTCCTGTTC | 59.967 | (GGTTC) 3 | 2 | 0.077 | 0.266 | 0.226 | 0.120 | 0.500 |
Sty-59 | TGCTTCAAAATGCATTGCCA | GGATCCACAGTTGCATCAGC | 59.263 | (GAAT) 3 | 3 | 0.900 | 0.553 | 0.442 | 0.012 | 0.333 |
Sty-61 | ACTTGGCTAGAGAGGGGGAG | AGGTGAGTGAGAATTGGGCG | 60.036 | (CGC) 4 | 2 | 0.550 | 0.404 | 0.319 | 0.154 | 0.500 |
Sty-66 | CGGTGTTGAGAGGAGTCGTC | CCAGCATCTCCTCCTTCCAG | 59.531 | (AGT) 5 | 4 | 0.316 | 0.413 | 0.374 | 0.041 | 0.250 |
Sty-69 | CCACTCGAGCCAACCAACAT | AGACGAGACTGAGTGGAGTT | 57.72 | (TC) 5 | 2 | 0.400 | 0.324 | 0.269 | 0.004 | 0.500 |
Sty-72 | GTGACAGAGGGGAGCACTTC | CCGCCCAATGTAGTGAAGGT | 60.035 | (GTAGGA) 3 | 5 | 0.950 | 0.574 | 0.474 | 0.004 | 0.200 |
Sty-77 | AAGAGAGGAACAGTGGCAGC | TCGTGTCTACCTCCAACAAAGT | 59.303 | (AG) 11 | 5 | 0.450 | 0.704 | 0.636 | 0.026 | 0.200 |
Sty-86 | GCAATCGAGAGGGAGTGAGG | CGGTTGGCCAGTTTCTTTGG | 59.968 | (AG) 5 | 2 | 0.375 | 0.309 | 0.258 | 0.173 | 0.500 |
Sty-87 | GGGCTGGGTCAGAATGTGAA | CGACAACAGTTGCCACCTTG | 59.971 | (GTT) 5 | 5 | 0.725 | 0.565 | 0.517 | 0.017 | 0.200 |
Sty-88 | GTCGAATTCCGCAAGCCATC | GGTTCACCGTGATTCACCGA | 60.32 | (AT) 6 | 15 | 0.975 | 0.854 | 0.825 | 0.046 | 0.067 |
Mean | 59.6042 | 6 | 0.614 | 0.552 | 0.504 | 0.046 | 0.243 | |||
min | 57.72 | 2 | 0.025 | 0.074 | 0.071 | 0.004 | 0.067 | |||
max | 60.32 | 15 | 1.000 | 0.882 | 0.855 | 0.173 | 0.500 |
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Li, W.; Zhang, C.; Jiang, X.; Liu, Q.; Liu, Q.; Wang, K. De Novo Transcriptomic Analysis and Development of EST–SSRs for Styrax japonicus. Forests 2018, 9, 748. https://doi.org/10.3390/f9120748
Li W, Zhang C, Jiang X, Liu Q, Liu Q, Wang K. De Novo Transcriptomic Analysis and Development of EST–SSRs for Styrax japonicus. Forests. 2018; 9(12):748. https://doi.org/10.3390/f9120748
Chicago/Turabian StyleLi, Wei, Cuiping Zhang, Xinqiang Jiang, Qingchao Liu, Qinghua Liu, and Kuiling Wang. 2018. "De Novo Transcriptomic Analysis and Development of EST–SSRs for Styrax japonicus" Forests 9, no. 12: 748. https://doi.org/10.3390/f9120748