Abstract
Premise
Ixonanthes (Ixonanthaceae) consists of between three and 19 species, among which I. chinensis and I. khasiana are considered vulnerable. Here, 58 microsatellite markers were developed for further conservation of these two Ixonanthes species.
Methods and Results
RNA transcripts of I. chinensis were sequenced and assembled into 19,545 unigenes, and 994 simple sequence repeat (SSR) loci were identified from 920 contigs. Based on these, 106 primer pairs were designed, 58 were successfully amplified, and 12 demonstrated polymorphism among five populations. The number of alleles per locus varied from three to 10, and the levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and 0.000 to 0.844, respectively. Further assessment of the transferability of the 58 amplifiable primers reported 30 being successfully cross‐amplified in I. icosandra and three in Erythroxylum sinense.
Conclusions
These novel SSR markers will be useful for future genetic conservation studies on these Ixonanthes species.
Keywords: genetic diversity, Ixonanthaceae, Ixonanthes chinensis, microsatellite marker, transcriptome
The genus Ixonanthes Jack (Ixonanthaceae) consists of 19 recorded species at present (Kool, 1980). Among them, I. chinensis Champ. and I. khasiana Hook. f. were assessed as “Vulnerable” in 1998 in the International Union for Conservation of Nature (IUCN) Red List (IUCN, 2018). Further assessment carried out in China has also listed I. chinensis in the China Species Red List as “Vulnerable” (Wang and Xie, 2004). However, the classification of Ixonanthes species is still controversial. Based on morphological characters, Kool (1980) proposed that (1) the genus Ixonanthes should contain only three species: I. reticulata Jack, I. petiolaris Blume, and I. icosandra Jack and (2) I. chinensis and I. khasiana should be considered as synonyms to I. reticulata. These opinions were confirmed by Mabberley (2008).
In China, I. chinensis is sometimes harvested for its wood as timber, which is processed for furniture and household products (Zhou and Lin, 2017). Although the survival of this species in the wild is a cause for concern among local researchers and conservationists, no studies have been carried out to assess its genetic diversity. The lack of genetic information on this vulnerable species could be due to the unavailability of useful molecular markers to carry out the work. Therefore, in this study, we have developed useful expressed sequence tag–simple sequence repeat (EST‐SSR) markers for I. chinensis. Furthermore, we also examine the cross‐transferability of these markers in the closely related species I. icosandra and Erythroxylum sinense Y. C. Wu (Erythroxylaceae).
METHODS AND RESULTS
Total RNA was extracted from fresh leaves of an I. chinensis individual (Appendix 1) using a modified cetyltrimethylammonium bromide (CTAB) method (Fu et al., 2005), and a cDNA library was constructed and sequenced using the HiSeq X Ten system (Illumina, San Diego, California, USA). Applying NGS QC Toolkit v2.3.3 (Patel and Jain, 2012), low‐quality reads containing unknown “N” bases or more than 10% bases with a Q value less than 20 were removed. Finally, applying Trinity v2.3.2 with default parameters (Grabherr et al., 2011), a total of 21 million high‐quality reads were de novo assembled into 19,545 unigenes with an average length of 517 bp and an N50 length of 621 bp. The raw data and the assembled sequences were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) and Transcriptome Shotgun Assembly (TSA) repositories (accession number: SRP127226).
SSRs containing more than six dinucleotide motifs and more than five tri‐, tetra‐, penta‐, and hexanucleotide motifs were searched from the unigenes using the online software MISA (Thiel et al., 2003). A total of 994 SSRs were identified from 920 unigenes, with 32 unigenes containing more than one SSR and 68 unigenes containing compound SSRs. The frequency of EST‐SSRs observed in the I. chinensis transcriptome was 4.7%. The most abundant repeat type was trinucleotide (53.8%), followed by dinucleotide (41.2%), tetranucleotide (3.5%), pentanucleotide (0.8%), and hexanucleotide (0.7%) repeat units. A total of 106 primer pairs were successfully designed for these SSR regions using Primer3 (Rozen and Skaletsky, 1999), specifying for an expected PCR product between 100 and 280 bp and an annealing temperature of 55°C.
Fresh leaves were collected from five populations of I. chinensis (n = 97), one population of I. icosandra (n = 5), and one population of E. sinensis (n = 14) (Appendix 1), then dried with silica gel at room temperature. In the first PCR trial, three individuals were randomly selected from each of the five I. chinensis populations and used as templates to test the 106 developed primers. DNA extractions and PCR amplifications were performed according to Fan et al. (2013). Among all tested primers, only 58 primers produced distinct bands within the expected size range (Appendix 2), and were thus included in the subsequent analysis. PCR products were then loaded onto a Fragment Analyzer Automated CE System (Advanced Analytical Technologies [AATI], Ames, Iowa, USA) using the QuantiT PicoGreen dsDNA Reagent Kit (Invitrogen, Carlsbad, California, USA). Allele sizes were determined using PROSize version 2.0 software (AATI), which resulted in only 12 primer pairs being polymorphic across the 15 individuals (Table 1).
Table 1.
Characteristics of 12 polymorphic simple sequence repeat markers isolated from Ixonanthes chinensis
| Locus | Primer sequences (5′–3′) | Repeat motif | Expected allele size (bp) | Observed size range (bp) | A | T a (°C) | Putative function [organism] | E‐value | GenBank accession no. |
|---|---|---|---|---|---|---|---|---|---|
| LC6 | F: AAGATTGCCTTCAACCAGTA | (ATGAG)5 | 333 | 313–343 | 7 | 52 | Glutamine‐fructose‐6‐phosphate aminotransferase [isomerizing] 2 [Carica papaya] | 5e‐22 | KX882016 |
| R: GGACCACGATTACATACAGT | |||||||||
| LC7 | F: GGTGAGCCAAGACAAGTG | (CCAAT)5 | 254 | 254–264 | 3 | 58 | PREDICTED: uncharacterized protein LOC18587496 [Theobroma cacao] | 1e‐97 | KX882017 |
| R: GCATTAAGCGTAAGCAACA | |||||||||
| LC12 | F: CATTCCACTCCACTCCAAT | (ATGT)6 | 124 | 120–136 | 5 | 55 | 1,4‐dihydroxy‐2‐naphthoyl‐CoA thioesterase 1 isoform X1 [Hevea brasiliensis] | 3e‐07 | KX882018 |
| R: GCTGCTGGCTAATTGAGA | |||||||||
| LC22 | F: CTCACCATCCTCGCATAC | (TGC)7 | 309 | 297–315 | 7 | 55 | PREDICTED: BRI1 kinase inhibitor 1‐like [Populus euphratica] | 5e‐81 | KX882019 |
| R: CTCTCCTCGTTCCTCCAT | |||||||||
| LC26 | F: CTCAGGAGTCAAGCCATC | (CTC)7 | 235 | 388–415 | 10 | 55 | Putative SERF‐like protein [Arachis duranensis] | 2e‐06 | KX882020 |
| R: CTGGACCGTCTCTACCTT | |||||||||
| LC33 | F: CGCCATTGTTAGAGAAGGA | (GAA)7 | 175 | 160–172 | 5 | 55 | PREDICTED: uncharacterized protein LOC8286849 [Ricinus communis] | 3e‐10 | KX882021 |
| R: TCACCACTCATCAAGAACC | |||||||||
| LC56 | F: TAGCAGCGAAGGAAGAGA | (AAGC)5 | 180 | 160–176 | 5 | 55 | — | — | KX882022 |
| R: GATAGATAGATGGTGAACAAGG | |||||||||
| LC60 | F: ACATCGGTAGCAGCATATAG | (TAT)6 | 144 | 134–155 | 6 | 55 | PREDICTED: CDPK‐related kinase 4 isoform X2 [Ricinus communis] | 3e‐28 | KX882023 |
| R: CTAATCACATCTCCTCAACAAG | |||||||||
| LC69 | F: TCTTCATGCCAACACTCAG | (TGT)6 | 126 | 120–135 | 6 | 58 | — | — | KX882024 |
| R: ATCACAGCCTCCATCTCC | |||||||||
| LC81 | F: CTTGTACTGATCGTTGTTGT | (TGA)6 | 231 | 231–243 | 5 | 55 | PREDICTED: uncharacterized protein LOC107428075 [Ziziphus jujuba] | 4e‐19 | KX882025 |
| R: GCGGAAGCATTCGTATTC | |||||||||
| LC87 | F: AGAATACCTGCCAACAATCA | (TAA)6 | 339 | 339–351 | 8 | 58 | PREDICTED: probable adenylate kinase 6, chloroplastic [Ricinus communis] | 6e‐140 | KX882026 |
| R: CGCACTGAACCTTGAAGA | |||||||||
| LC103 | F: TCAAGGAATCATCAGAGCAT | (AG)9 | 196 | 182–188 | 3 | 55 | Uncharacterized protein LOC110666353 [Hevea brasiliensis] | 3e‐28 | KX882027 |
| R: AGTGGAGGAGAAGAACAATG |
A = number of alleles; T a = annealing temperature.
For these 12 primer pairs, PCR amplifications were performed across all 97 individuals from the five natural populations of I. chinensis, and their PCR products were first electrophoresed on 10% polyacrylamide denaturing gel and then inspected with the Fragment Analyzer Automated CE System. Scoring errors and null alleles were detected using MICRO‐CHECKER (van Oosterhout et al., 2004); Hardy–Weinberg equilibrium, linkage disequilibrium, the average number of alleles per locus, and the levels of observed and expected heterozygosity were calculated using GenAlEx version 6.5 (Peakall and Smouse, 2012). Results showed that the number of alleles per locus ranged from three to 10 (Table 1), observed heterozygosity ranged from 0.000 to 1.000, and expected heterozygosity ranged from 0.000 to 0.844 (Table 2). Of the 12 polymorphic markers, eight showed significant deviation in Hardy–Weinberg equilibrium for the SZ and YC populations, seven for the HN population, six for the XG population, and five for the HSD population. No significant linkage equilibrium (P < 0.05) was detected between locus pairs (Table 2). Further cross‐species amplification was carried out using the initial 58 primer sets on two closely related species, I. icosandra (n = 5) and E. sinense (n = 14), and resulted in successful cross‐amplification of 30 primer sets in I. icosandra and three in E. sinense (Table 3). Our results showed that cross‐transferability of these EST‐SSR markers derived from I. chinensis displayed high transferability within other Ixonanthes species, but displayed rather low transferability in species of a different genus.
Table 2.
Genetic diversity of 12 polymorphic SSRs developed for Ixonanthes chinensis among five populations.a
| Locus | HN (n = 20) | HSD (n = 18) | SZ (n = 22) | XG (n = 16) | YC (n = 21) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | H o b | H e | A | H o b | H e | A | H o b | H e | A | H o b | H e | A | H o b | H e | |
| LC6 | 5 | 0.158*** | 0.738 | 5 | 0.111*** | 0.673 | 3 | 0.045*** | 0.476 | 1 | 0.000* | 0.117 | 6 | 0.095*** | 0.642 |
| LC7 | 3 | 0.500 | 0.655 | 3 | 0.333** | 0.648 | 3 | 0.682 | 0.594 | 3 | 0.533 | 0.638 | 3 | 0.524* | 0.659 |
| LC12 | 4 | 0.950 | 0.634 | 2 | 0.500 | 0.498 | 3 | 0.818 | 0.648 | 4 | 0.813 | 0.572 | 4 | 0.571 | 0.529 |
| LC22 | 6 | 0.550*** | 0.761 | 5 | 0.000 | 0.000 | 6 | 0.409*** | 0.759 | 1 | 0.438*** | 0.756 | 6 | 0.190*** | 0.630 |
| LC26 | 6 | 0.000*** | 0.770 | 6 | 0.176*** | 0.649 | 7 | 0.273*** | 0.803 | 5 | 0.063*** | 0.701 | 6 | 0.095*** | 0.785 |
| LC33 | 4 | 0.350*** | 0.666 | 4 | 0.222*** | 0.718 | 4 | 0.500 | 0.515 | 4 | 0.250*** | 0.652 | 5 | 0.143*** | 0.704 |
| LC56 | 4 | 0.450 | 0.585 | 3 | 0.556 | 0.573 | 4 | 0.364*** | 0.636 | 4 | 0.563 | 0.615 | 5 | 0.476*** | 0.685 |
| LC60 | 6 | 0.950 | 0.750 | 4 | 1.000 | 0.596 | 4 | 1.000*** | 0.758 | 5 | 0.875 | 0.844 | 6 | 1.000 | 0.693 |
| LC69 | 5 | 0.500 | 0.625 | 2 | 0.222 | 0.198 | 3 | 0.455 | 0.395 | 2 | 0.313 | 0.447 | 3 | 0.286 | 0.357 |
| LC81 | 3 | 0.050*** | 0.524 | 1 | 0.000 | 0.000 | 4 | 0.136*** | 0.520 | 3 | 0.188* | 0.439 | 4 | 0.286*** | 0.508 |
| LC87 | 4 | 0.000*** | 0.590 | 4 | 0.000*** | 0.599 | 5 | 0.273*** | 0.633 | 4 | 0.125*** | 0.609 | 5 | 0.048*** | 0.756 |
| LC103 | 3 | 0.150** | 0.476 | 1 | 0.000 | 0.000 | 2 | 0.000* | 0.087 | 2 | 0.063 | 0.061 | 1 | 0.000 | 0.000 |
— = not amplified; A = number of alleles; H e = expected heterozygosity; H o = observed heterozygosity; HWE = Hardy–Weinberg equilibrium probabilities; n = number of individuals sampled.
aLocality and voucher information are provided in Appendix 1.
bDeviations from HWE were statistically significant at *P < 0.05, **P < 0.01, and ***P ≤ 0.001.
Table 3.
Cross‐amplification of 58 microsatellite loci developed for Ixonanthes chinensis in I. icosandra and Erythroxylum sinense.a
| Locus | Ixonanthes icosandra | Erythroxylum sinense b | ||
|---|---|---|---|---|
| Length (bp) | Temperature (°C) | Length (bp) | Temperature (°C) | |
| LC3 | — | — | — | — |
| LC6 | — | — | — | — |
| LC7 | — | — | — | — |
| LC11 | 250–300 | 55 | — | — |
| LC12 | 100–150 | 55 | 100–150 | 55 |
| LC15 | 150–200 | 55 | — | — |
| LC16 | 250–300 | 61 | — | — |
| LC17 | 250–300 | 55 | — | — |
| LC18 | — | — | — | — |
| LC20 | 200–250 | 55 | — | — |
| LC22 | 250–300 | 55 | — | — |
| LC24 | — | — | — | — |
| LC25 | 200–300 | 55 | — | — |
| LC26 | 250–300 | 55 | — | — |
| LC29 | — | — | — | — |
| LC30 | — | — | — | — |
| LC33 | — | — | — | — |
| LC36 | 250–300 | 55 | — | — |
| LC39 | 200–250 | 61 | — | — |
| LC42 | 250–300 | 61 | — | — |
| LC45 | 100–150 | 61 | — | — |
| LC47 | 100–150 | 61 | — | — |
| LC48 | — | — | — | — |
| LC49 | 200–250 | 61 | — | — |
| LC51 | 200–250 | 61 | — | — |
| LC53 | — | — | — | — |
| LC56 | 200–250 | 55 | — | — |
| LC58 | 200–250 | 61 | — | — |
| LC59 | — | — | — | — |
| LC60 | 200 | 55 | — | — |
| LC61 | — | — | — | — |
| LC65 | — | — | — | — |
| LC66 | — | — | — | — |
| LC67 | — | — | — | — |
| LC69 | — | — | — | — |
| LC70 | 200–250 | 61 | — | — |
| LC71 | — | — | — | — |
| LC72 | 200–250 | 61 | — | — |
| LC76 | 250–300 | 61 | — | — |
| LC78 | — | — | — | — |
| LC79 | — | — | — | — |
| LC81 | 200–250 | 55 | — | — |
| LC83 | 250–300 | 55 | — | — |
| LC85 | — | — | — | — |
| LC87 | 250–300 | 55 | — | — |
| LC89 | 250–300 | 55 | — | — |
| LC93 | — | — | — | — |
| LC96 | 250–300 | 55 | — | — |
| LC97 | — | — | — | — |
| LC98 | — | — | — | — |
| LC99 | 200–250 | 55 | — | — |
| LC100 | 250–300 | 55 | — | — |
| LC101 | — | — | — | — |
| LC102 | — | — | — | — |
| LC103 | 200–250 | 55 | 200–250 | 58 |
| LC104 | — | — | >500 | 48 |
| LC105 | — | — | — | — |
| LC106 | — | — | — | — |
Locality and voucher information are provided in Appendix 1.
The amplifications of Erythroxylum sinense were performed in 2× Taq PCR Master Mix (KT201). The lengths were all calibrated by a 50‐bp DNA Ladder (MD108) (Tiangen Biotech Co., Beijing, China).
CONCLUSIONS
In this study, the transcriptome of I. chinensis was established using a de novo sequencing technique. By using this transcriptome library, a total of 58 EST‐SSR markers were developed, of which 12 were polymorphic across the five I. chinensis populations. Cross‐amplification of these EST‐SSR markers was also demonstrated on the closely related species I. icosandra and E. sinense. Through these efforts, our aim to provide ample population genetic information for Ixonanthes species was achieved, and the resulting markers could be useful for future studies on species delimitation, taxonomic revision, and genetic conservation of Ixonanthes species.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (31100159, 31200175, and 31570195) and by the project of Shenzhen Basic Ecological Control Line ([2012]0365).
APPENDIX 1. Voucher and locality information for populations used in this study. Specimens are deposited at the Herbarium of Sun Yat‐sen University (SYS), China.
| Species | Population code | Voucher no. | Collection locality | Geographic coordinates | n |
|---|---|---|---|---|---|
| Ixonanthes chinensis Champ. | SZ | Fan and Guo 1610 | DizhiPark, Shenzhen, Guangdong | 22°31′47.44″N, 114°32′10.61″E | 22 |
| HN | Fan and Guo 210 | Baishuiling, Hainan | 18°41′04.08″N, 109°51′11.97″E | 20 | |
| HSD | Fan and Guo 1621 | Heishiding, Guangdong | 22°24′12.62″N, 111°30′38.26″E | 18 | |
| YC | Fan and Guo 212 | Yangchun, Guangdong | 22°10′23.18″N, 111°47′11.00″E | 21 | |
| XG | Liao and Sun 002 | Hong Kong | 22°25′17.63″N, 113°56′14.36″E | 16 | |
| Ixonanthes icosandra Jack | Malaysia | Fan and Zhao HTBP‐5321 | Gunung Tahan, Pahang state | 4°38′30.84″N, 102°9′34.02″E | 5 |
| Erythroxylum sinense Y. C. Wu | JGS | Guo and Zhao JGS‐3437 | Jingangshan, Jiangxi | 26°33′47.12″N, 114°08′10.20″E | 14 |
n = number of individuals sampled.
APPENDIX 2. Characteristics of 58 microsatellite loci for Ixonanthes chinensis.
| Locus | Primer sequences (5′–3′) | Repeat motif | Expected allele size (bp) | T a (°C) |
|---|---|---|---|---|
| LC3 | F: TCTTACGAGCACGGACTT | (GCA)8 | 257 | 55 |
| R: ACCAACAGCAGCATAACAT | ||||
| LC6 | F: AAGATTGCCTTCAACCAGTA | (ATGAG)5 | 333 | 52 |
| R: GGACCACGATTACATACAGT | ||||
| LC7 | F: GGTGAGCCAAGACAAGTG | (CCAAT)5 | 254 | 58 |
| R: GCATTAAGCGTAAGCAACA | ||||
| LC11 | F: GGTGACTCCAGATATTGATTC | (GGAT)5 | 260 | 55 |
| R: AATGACACTTCCGCTATACT | ||||
| LC12 | F: CATTCCACTCCACTCCAAT | (ATGT)6 | 124 | 55 |
| R: GCTGCTGGCTAATTGAGA | ||||
| LC15 | F: ATTAGTGTAGAGCGAAGTGA | (AGG)7 | 177 | 55 |
| R: CTGAACCTGAATCATCTCCT | ||||
| LC16 | F: TCGGCAAGATAGGAATGTAT | (GCT)7 | 271 | 61 |
| R: AGCAGAGGTTCAGAAGGA | ||||
| LC17 | F: GATAGCCTGGGTAACAATGA | (AGC)7 | 262 | 55 |
| R: TTCGGTGGACACAACTCT | ||||
| LC18 | F: CGTAGACCTGGCAATGTAA | (GCC)7 | 334 | 55 |
| R: GGTGGATACTATGCTTGTTG | ||||
| LC20 | F: ACTGCTCTGGTTCTTCTTC | (GAT)7 | 218 | 55 |
| R: AGTGGCTCTATCCTATTCCT | ||||
| LC22 | F: CTCACCATCCTCGCATAC | (TGC)7 | 309 | 55 |
| R: CTCTCCTCGTTCCTCCAT | ||||
| LC24 | F: CAATGAACAGAAGCACAGAT | (GCT)6 | 298 | 55 |
| R: TAGCCAGCGAGAAGAAGA | ||||
| LC25 | F: ACACAACATCGTCCATCAT | (AGG)6 | 236 | 55 |
| R: ACAGCACAAGAAGACAGAG | ||||
| LC26 | F: CTCAGGAGTCAAGCCATC | (CTC)7 | 235 | 55 |
| R: CTGGACCGTCTCTACCTT | ||||
| LC29 | F: TGACCGATACCAGAGCAT | (GGT)6 | 309 | 55 |
| R: AGCATCTTCTTCTTCTTCCA | ||||
| LC30 | F: CACTTCTTGCTTCTGTTACC | (GGA)7 | 341 | 55 |
| R: CGTTGTTGCTGTCTTGTAG | ||||
| LC33 | F: CGCCATTGTTAGAGAAGGA | (GAA)7 | 175 | 55 |
| R: TCACCACTCATCAAGAACC | ||||
| LC36 | F: CTCCTCGTCGTCCTCTAA | (TAG)6 | 339 | 55 |
| R: GTCACCACTAGCATCCTATT | ||||
| LC39 | F: AAGTGGTGAGAATTGAAGGT | (CTG)6 | 213 | 61 |
| R: GCAGAAGTTCGTGTGGAG | ||||
| LC42 | F: TCCTCAAGCGAGAGTTCT | (GGT)7 | 259 | 61 |
| R: CTGTTACTGACTTACTGTTACC | ||||
| LC45 | F: CCTGGTCGGTCACATAGA | (GGC)7 | 155 | 61 |
| R: CGCTCCTTCTCATCATCTC | ||||
| LC47 | F: TTGCCGCTCTTTACATTTG | (GATG)6 | 157 | 61 |
| R: AACGAAGGAAGACCAACAG | ||||
| LC48 | F: CTGTTCCACCTTCACTGA | (TCTT)5 | 219 | 55 |
| R: CGTATGAATGGAGAGTAAGAG | ||||
| LC49 | F: TTCAATCCGAGTAATGATGG | (GCA)7 | 241 | 61 |
| R: CGCTCCACTTCCTAATGA | ||||
| LC51 | F: CATCCGCCGAATAATGAAC | (CTT)6 | 244 | 61 |
| R: GATTGTTGTCTCGCTTCTT | ||||
| LC53 | F: TTCTCCTCCAGTTCTCCAT | (ATAC)5 | 122 | 55 |
| R: AACACTCCAGAGCCAGAG | ||||
| LC56 | F: TAGCAGCGAAGGAAGAGA | (AAGC)5 | 180 | 55 |
| R: GATAGATAGATGGTGAACAAGG | ||||
| LC58 | F: TTCACATCACAGGTACAGAT | (AGAT)5 | 212 | 61 |
| R: GCCAGAAGAGGAGGTATTG | ||||
| LC59 | F: TGCGTTCGGTAATGACTTA | (CAT)5 | 258 | 55 |
| R: TCAGAATCAAGCCAGGATG | ||||
| LC60 | F: ACATCGGTAGCAGCATATAG | (TAT)6 | 144 | 55 |
| R: CTAATCACATCTCCTCAACAAG | ||||
| LC61 | F: GCCAACAACAACAACCATT | (GCT)6 | 216 | 55 |
| R: CGTCAGCCATAGTGTCATAA | ||||
| LC65 | F: CTCTGATACTGTCCACTTCC | (CAG)5 | 258 | 55 |
| R: TGTTCGTTCCTCCATTCTC | ||||
| LC66 | F: AGAAGAGGAAGAAGAGAAGGT | (GGT)6 | 130 | 55 |
| R: CGTCGTCGTTGCTGTTAG | ||||
| LC67 | F: ATGCGAAGGTGAGTCAAC | (TA)9 | 279 | 55 |
| R: TACAGATGAGTCGTAAGAAGG | ||||
| LC69 | F: TCTTCATGCCAACACTCAG | (TGT)6 | 126 | 58 |
| R: ATCACAGCCTCCATCTCC | ||||
| LC70 | F: GTAAGGGCTAAGACCAGAAA | (CAT)6 | 216 | 61 |
| R: ACCTCCAAGCACATCCAT | ||||
| LC71 | F: TCGTCCTTCTCCTTAACTTC | (TCT)6 | 205 | 61 |
| R: TGCTGTTGCTTCACTTCA | ||||
| LC72 | F: TCTGAACTCGCTTTCCATC | (TTC)6 | 228 | 61 |
| R: AACACGCTTATCAACAACAC | ||||
| LC76 | F: TTGTGTATGACGGCTCTG | (AAG)6 | 278 | 61 |
| R: AGGTGGAAGACAAGTATTCA | ||||
| LC78 | F: AGGTTCTGCCAATAATGTCA | (AAC)6 | 349 | 55 |
| R: GCTGTTGTTATTCTGGATGT | ||||
| LC79 | F: TGCCTCACTTGTTCTTCTC | (CCT)5 | 285 | 55 |
| R: ATCATCAGCGTCTCCAATC | ||||
| LC81 | F: CTTGTACTGATCGTTGTTGT | (TGA)6 | 231 | 55 |
| R: GCGGAAGCATTCGTATTC | ||||
| LC83 | F: AGCACAATCCTCCTCGTA | (AGC)6 | 348 | 55 |
| R: CTCCTCTTGTTCTCCTCAG | ||||
| LC85 | F: AAGAACAACAAGAGGATGC | (CAC)6 | 276 | 55 |
| R: GCGTCCGTAATCATAAGC | ||||
| LC87 | F: AGAATACCTGCCAACAATCA | (TAA)6 | 339 | 58 |
| R: CGCACTGAACCTTGAAGA | ||||
| LC89 | F: CTCAATCAAGATACGGTTGT | (GTG)6 | 252 | 55 |
| R: GAGACGGAATTGTTCATAGG | ||||
| LC93 | F: GCCAATCCAACACAATGC | (TAA)6 | 163 | 55 |
| R: CGGTGCTCATATCTCTTCC | ||||
| LC96 | F: GGATTCCAAGTGCTTAACAT | (CCA)6 | 293 | 55 |
| R: GAAGACAAGGCGGTAGAA | ||||
| LC97 | F: GGAATGCCACAGAACAAC | (CAC)6 | 303 | 55 |
| R: ATGCTCAATGTACTCTCCTC | ||||
| LC98 | F: AATGGCTGGCAATGAGAA | (TAA)5 | 342 | 55 |
| R: GCGGTATCTTCCAACACT | ||||
| LC99 | F: AGCCTTCTTCTCCTCTTCA | (CAT)5 | 149 | 55 |
| R: GGTCTGGTGTCACTGTTG | ||||
| LC100 | F: AATCGCATAGTCGCAAGG | (GAT)6 | 256 | 55 |
| R: AAGGCAAGCACATCAAGT | ||||
| LC101 | F: GTTAAGGTGGAGCAGGAG | (AGC)5 | 216 | 55 |
| R: TAGCGGATGGTTCTTCTTC | ||||
| LC102 | F: TTCGCTGGTTGTCAAGTT | (CAT)6 | 201 | 55 |
| R: CTCATATCGGTTCCAATCG | ||||
| LC103 | F: TCAAGGAATCATCAGAGCAT | (AG)9 | 196 | 55 |
| R: AGTGGAGGAGAAGAACAATG | ||||
| LC104 | F: TCTCCTCTTCTCCTCTTCTT | (CTT)6 | 252 | 55 |
| R: AACCTAGCAACACCTCCT | ||||
| LC105 | F: TGGAAGGAATCTGTCACTAC | (GAT)6 | 207 | 55 |
| R: CTGATGGATCGACCGTAAT | ||||
| LC106 | F: GTATGCTAGTGGTCACCTAC | (ACC)6 | 115 | 55 |
| R: GCTATGTTGTCGGCTTCC |
T a = annealing temperature.
Guo, W. , Fan Q., Wang J., Meng K., Chen S., Zhu L., and Liao W.. 2019. Isolation and identification of EST‐SSR markers in Ixonanthes chinensis (Ixonanthaceae). Applications in Plant Sciences 7(5): e1246.
Contributor Information
Qiang Fan, Email: fanqiang@mail.sysu.edu.cn.
Wenbo Liao, Email: lsslwb@mail.sysu.edu.cn.
DATA ACCESSIBILITY
Microsatellites and raw sequence data for the developed primers have been deposited to the National Center for Biotechnology Information (NCBI). The GenBank accession numbers for the microsatellites are provided in Table 1; the raw sequence data are available in the NCBI Sequence Read Archive (SRA) and Transcriptome Shotgun Assembly (TSA) databases (accession number: SRP127226).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Microsatellites and raw sequence data for the developed primers have been deposited to the National Center for Biotechnology Information (NCBI). The GenBank accession numbers for the microsatellites are provided in Table 1; the raw sequence data are available in the NCBI Sequence Read Archive (SRA) and Transcriptome Shotgun Assembly (TSA) databases (accession number: SRP127226).