Abstract
Premise of the Study
Hamamelis mollis (Hamamelidaceae) is a Tertiary relict species endemic to southern China. Polymorphic microsatellite markers were developed to reveal the genetic diversity of this species.
Methods and Results
The genome of H. mollis was sequenced and de novo assembled into 642,351 contigs. A total of 72,097 paired primers were successfully designed from 80,282 simple sequence repeat (SSR) markers identified in 63,419 contigs. PCR amplification showed that 96 of the 136 synthesized primers could be successfully amplified, and 22 demonstrated polymorphism. The mean number of alleles, levels of observed heterozygosity, and levels of expected heterozygosity were 4.602 ± 0.140, 0.632 ± 0.020, and 0.696 ± 0.010, respectively. The majority of the 96 primer pairs could be amplified in at least one other Hamamelidaceae species, including Distylium myricoides (60), Loropetalum chinense (39), Exbucklandia populnea (24), and E. tonkinensis (24).
Conclusions
These microsatellite loci provide abundant genomic SSR markers to evaluate genetic diversity of this woody ornamental plant.
Keywords: conservation genetics, Hamamelidaceae, Hamamelis mollis, microsatellite marker, shotgun genome sequencing
Hamamelidaceae comprises six subfamilies and approximately 30 genera and 140 species distributed in subtropical to temperate regions of both the Old and New World (Li et al., 1999). The fossil record of Hamamelidaceae shows that the early members of this family were present during the Late Cretaceous of the Northern Hemisphere, yet many of them disappeared at higher latitudes due to global cooling during the Late Tertiary, which was further accelerated during Quaternary glaciation. Currently the family contains many isolated and diverse genera, most of which are Tertiary relicts (Zhang and Lu, 1995).
Subfam. Hamamelidoideae contains about 19 genera and 72 species. Within the subfamily, Hamamelis Gronov. ex L. consists of four to six species distributed disjunctly between eastern Asia (two species) and eastern North America (two to four species) (Wen and Shi, 1999). Fossil leaves of Hamamelis have been found from the Paleocene in both the Old and New World (Wolfe, 1966). The modern distribution of Hamamelis is much narrower at present than the wider past distribution indicated by the fossil record (Zhang and Lu, 1995). For example, H. mollis Oliv. is present today only in central and southern China (Zhang and Lu, 1995), where it occurs at elevations of 600–1600 m. It is often planted as an ornamental tree in China.
Knowledge of the genetic characteristics of species, such as genetic diversity and population structure, is vital for the management of effective conservation strategies for relict species. For subfam. Hamamelidoideae, some simple sequence repeat (SSR) markers have been developed for Fothergilla ×intermedia Ranney & Fantz (Hatmaker et al., 2015), Chunia bucklandioides H. T. Chang (Meng et al., 2016), Distylium lepidotum Nakai (Sugai and Setsuko, 2016), and Sinowilsonia henryi Hemsl. (Li et al., 2017), yet PCR amplifications have showed low transferability of these SSR markers to H. mollis (Q. Yin, S. Chen, Q. Fan, and W. Liao, unpublished). In this study, a total of 22 polymorphic genomic SSR markers were developed for H. mollis for further use in conservation measures. These are the first SSR primers designed for this species.
METHODS AND RESULTS
Fresh leaves were collected from one seedling of H. mollis sampled from Juqiushui, Hunan Province, China (Appendix 1), and transplanted to the greenhouse of Sun Yat‐sen University, Guangzhou, Guangdong Province, China. The cetyltrimethylammonium bromide (CTAB) method of Doyle and Doyle (1987) was used to extract total genomic DNA. The DNA library was constructed with the VAHTS Universal DNA Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu Province, China) according to the manufacturer's protocol and was subsequently sequenced with the HiSeq X Ten System (Illumina, San Diego, California, USA). This yielded a total of 25.45 million high‐quality 145‐bp paired reads. Reads were filtered using NGSQCToolkit_v2.3.3 (Patel and Jain, 2012) by removing low‐quality reads (i.e., containing unknown bases [N] or more than 10% of nucleotides with Q value ≤20). Filtered reads were de novo assembled into 642,351 contigs using Edena v3.131028 with the default parameters (Hernandez et al., 2008). The average length of the contigs was 541 bp and the N50 value was 311 bp. The filtered raw data and the assembled contigs were deposited in the National Center for Biotechnology Information's (NCBI) GenBank database (BioSample: SAMN09010486, BioProject: PRJNA454742, Sequence Read Archive: SRR7110723, Transcriptome Shotgun Assembly: GGNJ00000000).
The SSR repeat motifs containing two to six nucleotides across these contigs were identified using the MISA tool (Thiel et al., 2003) with the default parameters except that mononucleotide repeats were removed from analysis. A total of 95,585 SSRs were found across 75,595 contigs. Among these SSRs, the most common motifs were dinucleotide repeats (76.75%), followed by tri‐ (17.70%), tetra‐ (3.89%), penta‐ (1.15%), and hexanucleotide (0.51%) repeats. Primer 3.0 (Rozen and Skaletsky, 1999) was used to design SSR primers with default parameters. This yielded a total of 72,097 primer pairs across 63,419 contigs. Of these primer pairs, 136 with high, medium, and low repeats were selected randomly for further characterization.
A total of 80 individuals of H. mollis were collected from four natural populations in China (Appendix 1) to test the levels of polymorphism in the target SSR loci. The transferability of these SSR primers was also tested in four other species of Hamamelidaceae (eight individuals of each species; Appendix 1): Distylium myricoides Hemsl. (subfam. Hamamelidoideae), Loropetalum chinense (R. Br.) Oliv. (subfam. Hamamelidoideae), Exbucklandia populnea (R. Br. ex Griff.) R. W. Br. (subfam. Exbucklandioideae), and E. tonkinensis (Lecomte) H. T. Chang (subfam. Exbucklandioideae). DNA was extracted from silica‐dried leaves of all individuals using the CTAB method described above. In the first PCR trial, all 136 primer pairs were amplified for two individuals randomly selected from each population. PCR amplifications were performed according to Fan et al. (2013) and were inspected using 10% agarose gel electrophoresis. Among all primer pairs, 96 were successfully amplified across the eight test individuals with the expected product size (NCBI accessions: MH167492–MH167587). The Fragment Analyzer Automated CE System (Advanced Analytical Technologies [AATI], Ames, Iowa, USA) was used for genotyping, using the Quant‐iT PicoGreen dsDNA Reagent Kit (35–500 bp; Invitrogen, Carlsbad, California, USA). Fragment sizes were analyzed using PROSize version 2.0 (AATI). The results showed 22 polymorphic loci across all eight individuals (Table 1) and 74 monomorphic loci (Appendix 2).
Table 1.
Characteristics of 22 polymorphic EST‐SSR loci developed for Hamamelis mollis
| Locus | Primer sequences (5′–3′) | Repeat motif | Product size (bp) | Allele size range (bp) | T a (°C) | GenBank accession no. | Putative function |
|---|---|---|---|---|---|---|---|
| H4 | F: ACACCTAATTCGCAGGCATC | (AAAAAT)6 | 215 | 191–227 | 60 | MH167495 | Swertia tetraptera microsatellite ST40 sequence |
| R: ATGAAGTGGCATTCGGAAAC | |||||||
| H7 | F: TTGATGGGTTTTGTGGGAAT | (GAAAA)8 | 238 | 218–238 | 62 | MH167497 | PREDICTED: Glycine max homeobox‐leucine zipper protein HOX11‐like (LOC100805312), mRNA |
| R: TGAACCACGGAACAAAACAA | |||||||
| H11 | F: TCAACCATGAGTGTGTACCTAGC | (ATAC)11 | 243 | 231–247 | 60 | MH167500 | Chrysanthemum ×morifolium microsatellite JH‐1484 sequence |
| R: CCTCTAATCACAGGCAACCAA | |||||||
| H22 | F: CATGGGTTACGGCTGTCTTT | (ATC)15 | 237 | 217–250 | 60 | MH167508 | PREDICTED: Ziziphus jujuba uncharacterized LOC107420631 (LOC107420631), mRNA |
| R: TGCTGGTACTAACCTTGGGG | |||||||
| H52 | F: ATGCCAAGGAGAGGGAAAAT | (AAT)9 | 184 | 172–181 | 60 | MH167529 | NOT FOUND |
| R: GCTTTTTATGCTTTAGGTTTCTGC | |||||||
| H54 | F: AAACCGAAAGAAAGCACAACA | (AAT)9 | 222 | 208–219 | 60 | MH167530 | NOT FOUND |
| R: GGGTTTTAAGCTTGCCATGT | |||||||
| H57 | F: CCTGGATAATGGAGAGCCAA | (TC)23 | 242 | 226–242 | 60 | MH167533 | PREDICTED: Durio zibethinus amino acid transporter AVT1I‐like (LOC111317757), mRNA |
| R: TTTTTGTGTTGCATTACGTGC | |||||||
| H63 | F: TATATGCGCAGTGGAGCAAA | (GAGCAA)6 | 237 | 213–243 | 60 | MH167537 | NOT FOUND |
| R: TGCCCATTAACACTGGTTCA | |||||||
| H64 | F: TCCAAGTAAAGGATCCGAACTC | (CTTTTT)6 | 251 | 233–257 | 62 | MH167538 | NOT FOUND |
| R: TTGGCTATTGATGGTGCTTT | |||||||
| H67 | F: GATTTTGTGCATGTTTCCCC | (AGCCCA)5 | 236 | 212–242 | 60 | MH167540 | NOT FOUND |
| R: AGGGGGTATCGGTGATTGTT | |||||||
| H71 | F: TGTCAACTGGAACATCAAGGA | (AAATA)6 | 255 | 240–260 | 60 | MH167543 | NOT FOUND |
| R: TGTTTCTGAGTGTCCCAACCT | |||||||
| H77 | F: CCAGCTTGGAGTACACATGG | (AC)18 | 174 | 164–178 | 60 | MH167548 | NOT FOUND |
| R: GAGGGATGCCTTTAACACCA | |||||||
| H86 | F: ATAGCAGAACCAGGCACCAG | (AAG)12 | 274 | 265–283 | 65 | MH167555 | NOT FOUND |
| R: TTCATTAGTCACCGGAAGGC | |||||||
| H94 | F: TGGAAAACGGACAGAGTGAA | (AAAT)5 | 225 | 217–229 | 60 | MH167560 | NOT FOUND |
| R: GCCATTCATTGGCTTTTTGT | |||||||
| H99 | F: ATCGCTAACCCGCTCCTAAT | (CCAGG)6 | 250 | 220–250 | 60 | MH167561 | NOT FOUND |
| R: TTCAGCTAGCAAATAAGATTGACC | |||||||
| H103 | F: GAATGCATGTGACTGATGGG | (CTTTT)6 | 220 | 210–225 | 60 | MH167563 | NOT FOUND |
| R: TTGCTTTCCTTTTCCATTGC | |||||||
| H115 | F: ATGGGCGAAAAAGATTGTTG | (CTC)12(CTT)5 | 237 | 222–240 | 62 | MH167570 | NOT FOUND |
| R: GCCTTCACGTCCTCACAAAT | |||||||
| H122 | F: GTTTGGACACGCTCGTCATA | (AAT)8…(AAT)6 | 211 | 211–232 | 60 | MH167575 | NOT FOUND |
| R: CCATCTCTGTCCTTGCATGA | |||||||
| H126 | F: TGAAAGAAACGTCACCCTCC | (TCT)7…(GAA)5… (AAG)5 | 279 | 264–282 | 60 | MH167579 | Botryotinia fuckeliana T4 SupSuperContig_200r_370_1 genomic supercontig |
| R: GATCGTCATCATCACAACCG | |||||||
| H130 | F: GGCCTTCCAACGGTCATATT | (TTGAGT)5…(TTTGAG)5 | 258 | 263–282 | 62 | MH167582 | NOT FOUND |
| R: AGGGAGGCATGTCAATTCAT | |||||||
| H131 | F: GGGAAAAAGAAGAAGGAGAAGG | (AGA)10 | 255 | 246–270 | 60 | MH167583 | NOT FOUND |
| R: GCCTTGTTTGGCATTGAACT | |||||||
| H132 | F: GCATTTGGTTGCGGTTAGAG | (TAT)10 | 272 | 266–287 | 60 | MH167584 | NOT FOUND |
| R: TCTACCAGGGGTGGAAGAGA |
T a = annealing temperature.
For these 22 polymorphic SSR loci, PCR amplification was performed for all individuals in the four populations of H. mollis. Linkage disequilibrium, departure from Hardy–Weinberg equilibrium (HWE), the average number of alleles per locus (A), the observed heterozygosity (H o), and the expected heterozygosity (H e) were calculated using GenAlEx version 6.5 (Peakall and Smouse, 2012). No pairs of loci showed linkage disequilibrium after a sequential Bonferroni correction for multiple tests, indicating that the 22 markers can be considered independent markers. Significant deviations from HWE were detected in five loci in the DLL population and four loci in the YS population (Table 2). In the DLL population, A ranged from three to eight, H e ranged from 0.515 to 0.838, and H o ranged from 0.000 to 1.000. In the TMS population, A ranged from three to eight, H e ranged from 0.445 to 0.829, and H o ranged from 0.000 to 0.858. In the WGS population, A ranged from three to seven, H e ranged from 0.445 to 0.788, and H o ranged from 0.000 to 0.850. In the YS population, A ranged from two to six, H e ranged from 0.320 to 0.788, and H o ranged from 0.000 to 0.950.
Table 2.
Polymorphism in 22 SSR loci across four populations of Hamamelis mollis.a
| Locus | DLL (n = 20) | TMS (n = 20) | WGS (n = 20) | YS (n = 20) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | H o | H e b | A | H o | H e b | A | H o | H e b | A | H o | H e b | |
| H4 | 4 | 0.65 | 0.744 | 5 | 0.50 | 0.641 | 7 | 0.55 | 0.715 | 5 | 0.60 | 0.734** |
| H7 | 3 | 0.45 | 0.540 | 5 | 0.70 | 0.759 | 5 | 0.65 | 0.731 | 5 | 0.70 | 0.773 |
| H11 | 3 | 0.40 | 0.515 | 3 | 0.50 | 0.624 | 3 | 0.55 | 0.660 | 2 | 0.45 | 0.439 |
| H22 | 8 | 0.55 | 0.853* | 8 | 0.65 | 0.784 | 6 | 0.65 | 0.788 | 5 | 0.70 | 0.788* |
| H52 | 4 | 0.50 | 0.629 | 3 | 0.65 | 0.660 | 3 | 0.55 | 0.654 | 3 | 0.55 | 0.595 |
| H54 | 5 | 0.45 | 0.665** | 4 | 0.70 | 0.726 | 5 | 0.70 | 0.781 | 4 | 0.55 | 0.716 |
| H57 | 7 | 0.60 | 0.775 | 5 | 0.86 | 0.761 | 6 | 0.70 | 0.775 | 3 | 0.65 | 0.635 |
| H63 | 5 | 0.60 | 0.665 | 3 | 0.70 | 0.636 | 5 | 0.75 | 0.736 | 4 | 0.55 | 0.648* |
| H64 | 5 | 0.85 | 0.775 | 4 | 0.70 | 0.741 | 4 | 0.85 | 0.696 | 4 | 0.60 | 0.701 |
| H67 | 6 | 0.65 | 0.771 | 6 | 0.65 | 0.749* | 5 | 0.65 | 0.739 | 5 | 0.75 | 0.709 |
| H71 | 3 | 0.55 | 0.614 | 4 | 0.55 | 0.671 | 4 | 0.70 | 0.729* | 3 | 0.50 | 0.609 |
| H77 | 7 | 0.60 | 0.838 | 7 | 0.65 | 0.759 | 5 | 0.60 | 0.726 | 5 | 0.55 | 0.741 |
| H86 | 5 | 0.65 | 0.706 | 4 | 0.70 | 0.685 | 4 | 0.60 | 0.648 | 4 | 0.65 | 0.736 |
| H94 | 4 | 0.55 | 0.644 | 3 | 0.60 | 0.651 | 4 | 0.55 | 0.643 | 3 | 0.65 | 0.629 |
| H99 | 5 | 1.00 | 0.693* | 5 | 0.90 | 0.709 | 4 | 0.80 | 0.705 | 5 | 0.65 | 0.673 |
| H103 | 3 | 0.55 | 0.609 | 4 | 0.75 | 0.738 | 4 | 0.60 | 0.729 | 4 | 0.70 | 0.748 |
| H115 | 7 | 0.55 | 0.719 | 6 | 0.75 | 0.765 | 6 | 0.65 | 0.813 | 5 | 0.80 | 0.738 |
| H122 | 6 | 0.95 | 0.779* | 6 | 0.85 | 0.829 | 7 | 0.85 | 0.808 | 6 | 0.95 | 0.765 |
| H126 | 4 | 0.60 | 0.559 | 7 | 0.80 | 0.829 | 5 | 0.70 | 0.769 | 5 | 0.75 | 0.765 |
| H130 | 3 | 0.00 | 0.645*** | 3 | 0.00 | 0.445*** | 3 | 0.00 | 0.445*** | 2 | 0.00 | 0.320*** |
| H131 | 5 | 0.55 | 0.756* | 4 | 0.70 | 0.679 | 5 | 0.75 | 0.726 | 4 | 0.65 | 0.701 |
| H132 | 4 | 0.85 | 0.686 | 5 | 0.75 | 0.711 | 5 | 0.75 | 0.735 | 4 | 0.80 | 0.636 |
A = number of alleles; H e = expected heterozygosity; H o = observed heterozygosity; n = number of individuals collected for each population.
aVoucher and locality information are available in Appendix 1.
bSignificant deviations from Hardy–Weinberg equilibrium after sequential Bonferroni corrections: *represents significance at the 5% nominal level; **represents significance at the 1% nominal level; ***represents significance at the 0.5% nominal level.
Finally, transferability tests indicated that the majority of the 96 loci could be amplified in at least one other Hamamelidaceae genus. Specifically, we found that 60, 39, 24, and 24 paired primers amplified in D. myricoides, L. chinensis, E. populnea, and E. tonkinensis, respectively, and 24 amplified in three of the four species (Table 3).
Table 3.
Cross‐amplification success and fragment size ranges (in base pairs) for 96 SSR markers in four Hamamelidaceae species.a
| Locus | Distylium myricoides | Loropetalum chinense | Exbucklandia populnea | Exbucklandia tonkinensis |
|---|---|---|---|---|
| H1 | 159 | 177–183 | 171–177 | 189 |
| H2 | — | — | — | — |
| H3 | 234–240 | — | — | — |
| H4 | 197 | 233 | — | — |
| H5 | 155 | 101 | 125–137 | 131–143 |
| H7 | — | — | — | — |
| H8 | — | — | — | — |
| H10 | 227 | — | — | — |
| H11 | — | 203 | — | — |
| H13 | — | — | — | — |
| H14 | 232–240 | 224–228 | — | — |
| H15 | 232–236 | — | — | — |
| H17 | 208 | 172–176 | 192 | 192–196 |
| H18 | 207 | — | — | — |
| H19 | 218–224 | 248 | 197 | 197 |
| H20 | — | 255 | — | — |
| H22 | 268–271 | — | 196 | 190 |
| H23 | 243 | 225–228 | 168 | 168 |
| H25 | 292–295 | 303 | 253–256 | 262 |
| H26 | 195–198 | 234 | 246 | 246 |
| H27 | 254–263 | 278–281 | 293 | 272–275 |
| H28 | 199 | — | — | — |
| H33 | 254–258 | 266 | 286–290 | 294 |
| H34 | 114 | — | — | — |
| H36 | — | — | — | — |
| H38 | 308–312 | 256–264 | — | — |
| H39 | 276–280 | 237 | 241–245 | 245 |
| H40 | 222 | 262 | — | — |
| H41 | 246–250 | 202–206 | 214–218 | 210 |
| H42 | 248 | — | — | — |
| H43 | — | — | — | — |
| H44 | 201 | — | — | — |
| H46 | — | — | 277 | 265 |
| H47 | 291–297 | 264–270 | — | — |
| H48 | — | 287 | — | — |
| H49 | 200–203 | — | — | — |
| H50 | — | — | — | — |
| H52 | — | 145 | — | 193 |
| H54 | 199 | 234 | — | — |
| H55 | — | — | — | — |
| H56 | 188 | — | — | — |
| H57 | 210 | — | — | — |
| H58 | 208 | 232 | 174 | 174 |
| H59 | 223 | — | — | — |
| H62 | 245 | 203 | — | — |
| H63 | 207–212 | — | — | — |
| H64 | — | — | — | — |
| H65 | — | — | 106–109 | 122 |
| H67 | — | — | — | — |
| H68 | 300–305 | 245 | — | — |
| H70 | 297–302 | 282 | 242 | 242 |
| H71 | 220 | — | — | — |
| H72 | 242–252 | 232 | — | — |
| H73 | 183 | — | — | — |
| H74 | — | — | — | — |
| H76 | 277–281 | 233 | — | — |
| H77 | — | — | — | — |
| H79 | — | — | — | — |
| H80 | 212–216 | 164–166 | 154 | 154 |
| H81 | 269 | 281–283 | 213 | 221–223 |
| H83 | — | 125–129 | — | — |
| H84 | — | 218 | 256 | 252–256 |
| H85 | — | — | — | — |
| H86 | — | — | — | — |
| H88 | 154 | — | — | — |
| H91 | 267–271 | — | — | — |
| H92 | 233–241 | — | 225 | 225 |
| H93 | 243–247 | — | — | — |
| H94 | 197 | — | — | — |
| H99 | — | — | — | — |
| H100 | 131 | — | — | — |
| H103 | — | — | — | — |
| H105 | 214–219 | 244 | — | — |
| H108 | — | 200 | — | — |
| H110 | — | — | — | — |
| H111 | 195–199 | 231–239 | 227 | 227–235 |
| H112 | 242–250 | 212 | — | — |
| H113 | — | — | — | — |
| H115 | 204 | — | — | — |
| H117 | 136–140 | 176 | — | — |
| H118 | 299 | — | — | — |
| H119 | 254–258 | 266–270 | — | — |
| H121 | 233 | — | — | — |
| H122 | — | 196–199 | — | — |
| H123 | — | — | — | — |
| H124 | — | — | — | — |
| H125 | 235–238 | — | — | — |
| H126 | — | — | — | — |
| H127 | 275–281 | — | — | — |
| H129 | — | 219 | — | — |
| H130 | 221–233 | — | 245 | 251 |
| H131 | — | — | — | — |
| H132 | — | — | — | — |
| H133 | — | — | — | — |
| H135 | 241–250 | — | 292–195 | 301–307 |
| H136 | 108 | — | 165 | — |
— = primers could not be amplified in any individual.
Voucher and locality information are available in Appendix 1.
CONCLUSIONS
We have developed a number of useful new primers for assessing genetic diversity in H. mollis and potentially numerous other Hamamelidaceae taxa. These loci will aid in future conservation genetics efforts across the family.
AUTHOR CONTRIBUTIONS
W.B.L. and Q.F. designed the research. Y.S.H. and H.G.Y. collected samples. C.Y.H. designed the primers. C.Y.H. and Q.Y.Y. generated the data. Q.Y.Y. and S.F.C. analyzed and interpreted the data. Q.Y.Y. wrote the manuscript, and S.F.C. modified the manuscript.
DATA ACCESSIBILITY
All genomic sequences of 136 pairs of primers were deposited in the National Center for Biotechnology Information (NCBI) GenBank database (MH167492–MH167587). The filtered raw read data and the assembled contigs were also deposited in NCBI databases (BioSample: SAMN09010486, BioProject: PRJNA454742, Sequence Read Archive [SRA]: SRR7110723, Transcriptome Shotgun Assembly [TSA]: GGNJ00000000).
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (31670189, 31800175, 31570195), the Special Program for Science and Technology Basic Research of the Ministry of Science and Technology of China (2013FY111500), the Fourth National Survey on Chinese Material Medical Resources Program for State Administration of Traditional Chinese Medicine of the People's Republic of China (2017‐152‐003), the Fundamental Research Funds for the Central Universities (161gjc38), and the Chang Hungta Science Foundation of Sun Yat‐sen University.
Appendix 1. Voucher and locality information for populations of Hamamelis mollis and four related Hamamelidaceae species used in this study.
| Species | Voucher no.a | Collection locality (Population) | Geographic coordinates | N |
|---|---|---|---|---|
| Hamamelis mollis Oliv. | W. Y. Zhao et al. 16871b | Yanling, Hunan, China | 26°33'13.4”1” N, 114°04'51.55” E | 1 |
| Q. Fan et al. 15216c | Yichang, Hubei, China (DLL) | 31°04'15.37” N, 110°55'40.56” E | 20 | |
| Q. Y. Yin et al. 17161c | Lin'an, Zhejiang, China (TMS) | 30°48'34.67” N, 120°55'55.06” E | 20 | |
| Q. Y. Yin et al. 17277c | Pingxiang, Jiangxi, China (WGS) | 27°21’46.82”N, 113°46’3.50” E | 20 | |
| Q. Y. Yin et al. 17193c | Shaoguan, Guangdong, China (YS) | 25°22’5.66”N, 114°35’32.30” E | 20 | |
| Distylium myricoides Hemsl. | X. J. Zhang et al. 17009 | Yichun, Jiangxi, China | 28°34’29.62”N, 114°35’32.30” E | 8 |
| Exbucklandia populnea (R. Br. ex Griff.) R. W. Br. | W. Y. Zhao et al. 16235 | Tongren, Guizhou, China | 27°57'55.32” N, 108°36'46.67” E | 8 |
| Exbucklandia tonkinensis (Lecomte) H. T. Chang | W. Y. Zhao et al. 16339 | Zhaoqing, Guangdong, China | 23°33'31.93” N, 111°57'52.00” E | 8 |
| Loropetalum chinense (R. Br.) Oliv. | Q. Fan et al. 17439 | Huangjiang, Guangxi, China | 25°12’9.82”N, 108°38’23.94” E | 8 |
Appendix 2. Characteristics of the 74 monomorphic SSR markers in Hamamelis mollis.
| Locus | Primer sequences (5′–3′) | Repeat motif | Expected allele size (bp) | GenBank accession no. |
|---|---|---|---|---|
| H1 | F: GTTGCTTTCGTGTTCGTCCT | (CTCGTC)9 | 171 | MH167492 |
| R: GTTTGGTAAGGCAAGGGACA | ||||
| H2 | F: CCTCCATATCGTAGTCTACCGC | (TTTTGA)8 | 255 | MH167493 |
| R: TTCTACCACACGTCACACCC | ||||
| H3 | F: CTCGACGACTTTTGGTGGAT | (TTTCTC)7 | 246 | MH167494 |
| R: CCCAATGAGGCTTTGAAAAA | ||||
| H5 | F: AGCATTGAATGTTGCGTTTG | (CTCTTC)5 | 119 | MH167496 |
| R: CTACGGGGGACAGCAGAATA | ||||
| H8 | F: TTTTGCCCTTTCTCTCCCTT | (TTCTC)7 | 261 | MH167498 |
| R: TGTTTGGATTGAAGGAATTGG | ||||
| H10 | F: TGAAAGAGAAGGGAATGGCA | (CTGCC)5 | 252 | MH167499 |
| R: TTTTTGTCCAATTCATGGCA | ||||
| H13 | F: TGTGGGGCGAGGATAAATAG | (AAAT)9 | 232 | MH167501 |
| R: GGGGAGAGGACGAGGAATAG | ||||
| H14 | F: CCGTTGCAATCCCTGTAGTT | (TTTC)8 | 248 | MH167502 |
| R: CAATTTTGGCTGCAATTCAA | ||||
| H15 | F: GTGGTAAAATTGGGTGCTGG | (TTTA)7 | 216 | MH167503 |
| R: TCGTGGTCGTCTAAGTCACG | ||||
| H17 | F: ACTCTTGTTCCCCCACCTTT | (ATTA)5 | 184 | MH167504 |
| R: GTAACCCAGGATGACCCCTT | ||||
| H18 | F: TATCCCTCGCTTGATTTTGC | (ATA)19 | 195 | MH167505 |
| R: GCAATAGAGCTCGACGGTTC | ||||
| H19 | F: AGCAAGATGGAAGGAAGCAA | (TTC)18 | 230 | MH167506 |
| R: AAACCTATCATGCATACTAACAATGAA | ||||
| H20 | F: GAAAGGCAACAGAGCTCGAC | (TAT)17 | 279 | MH167507 |
| R: CCAACAGTCGGATCAATGTG | ||||
| H23 | F: TATCCACCCCACTCCAATTC | (AAT)14 | 201 | MH167509 |
| R: CCATTTCTTGCAGGTTTGCT | ||||
| H25 | F: GCCTGGTTATTTTTGGAAACTT | (ATA)9 | 277 | MH167510 |
| R: TGTGTGCGCACTTAGGTGAT | ||||
| H26 | F: GTGTCCGCACTTCATAGGGT | (TTG)8 | 219 | MH167511 |
| R: CGCCTCCTTAACTGCATACC | ||||
| H27 | F: GCTCACTAACTCTGCCTGGG | (CAT)5 | 266 | MH167512 |
| R: TTCCGGAAAGCCAGTCATAC | ||||
| H28 | F: ATTCTGCTTTGGACCTGCAT | (ATT)5 | 172 | MH167513 |
| R: TGCTCATACAAATGTCCCAAA | ||||
| H33 | F: CCTTGGTTTCCCTCATTTCA | (TC)19 | 272 | MH167514 |
| R: TGAATCTTGTGGTTCGTCCA | ||||
| H34 | F: TTTACTTGGGGACTTGGGAA | (TA)10 | 100 | MH167515 |
| R: ACAAGAGTCCTGAAGTTTGAATGA | ||||
| H36 | F: CATAGTAAAAACACATTGAACACACTG | (TA)7 | 257 | MH167516 |
| R: TTGACAGTAAAAATACTAAAAATGGTG | ||||
| H38 | F: CAACGGAATTCAAAAATCTCG | (TTAT)9 | 276 | MH167517 |
| R: CGCTGCAATGTTCATACGAC | ||||
| H39 | F: CAACCCCTCTCCCCTCTAAA | (TACA)9 | 253 | MH167518 |
| R: GGGTCCGTTGGTTTTAGCTT | ||||
| H40 | F: ACGGGTTTAAGCGCTAAGGT | (TATG)8 | 246 | MH167519 |
| R: TTGAAGGGGAAAATGTGCTC | ||||
| H41 | F: AAGCCACATGCCAAGTTTTC | (AAAC)7 | 222 | MH167520 |
| R: TTGTTTTGAAGGTTGGGTCA | ||||
| H42 | F: AAGGCATTGCTGTCATTTCC | (TATG)7 | 274 | MH167521 |
| R: TCCTTCAAAGACCCCGTACA | ||||
| H43 | F: TCGAAGAAAAAGCTGGAAGC | (TTC)15 | 177 | MH167522 |
| R: ACTTAGGTACCCATCCCTATCAT | ||||
| H44 | F: AAAAACAACACCCAACCCAT | (TTA)15 | 183 | MH167523 |
| R: GGAGTTGGAATGCCTTTGTC | ||||
| H46 | F: TCAAAATTGATGTGGCACTAGC | (ATT)14 | 232 | MH167524 |
| R: CAAGGGAATTTTGTTGGCAT | ||||
| H47 | F: TGGCATCATTTTACTTTCTAAGCA | (TAA)14 | 279 | MH167525 |
| R: TGATGGGACTCAATCACTTTG | ||||
| H48 | F: CAAAGCCTCAATGATGACGA | (TAA)14 | 264 | MH167526 |
| R: TGAAGGGTTCAAAAAGAGATGAA | ||||
| H49 | F: TCCTTTGCATACTAGGGAAATAAAA | (AAG)13 | 188 | MH167527 |
| R: CTTGGAGTCCTTGGAGCTTG | ||||
| H50 | F: GAGGGAGCATAGCAAAGGTG | (TTA)13 | 221 | MH167528 |
| R: TCAAATGTGGACCTTAAATCACTC | ||||
| H55 | F: TCTCCCGATTTGAGGGTATG | (GA)25 | 217 | MH167531 |
| R: ACGTCATTGCGAGTCCTCTT | ||||
| H56 | F: CGAGAAAGGTCAAAGGTGGA | (GA)25 | 164 | MH167532 |
| R: ATTGCAAAACGAAGCCTCTG | ||||
| H58 | F: TCTTAAAGGGTCAATGGGCA | (TC)23 | 220 | MH167534 |
| R: AATCACACAACACCGCCTTT | ||||
| H59 | F: CGCTAATGCGCATCTGTACT | (TC)23 | 245 | MH167535 |
| R: TTGGAAAACCTGCTCGATCT | ||||
| H62 | F: TGCCTTTGCTTGTTATGTTGTC | (TTGCCT)7 | 227 | MH167536 |
| R: TCGATACCAAATGAGGGCAT | ||||
| H65 | F: TTGAAAGCAGGAAATGGACA | (ATAAAA)5 | 127 | MH167539 |
| R: AAAGTAGTGACCCCCGTCCT | ||||
| H68 | F: AACCAATTGAAAAGAAAAGAACG | (CTTTT)7 | 280 | MH167541 |
| R: GACTCCCTAATGTCGGCAAA | ||||
| H70 | F: GGTCAACAGAAATATGGCCC | (TGAGT)6 | 272 | MH167542 |
| R: GATGCCTGTGGTCTCTGGAT | ||||
| H72 | F: TATCACGACTTTGTGCCTGC | (TTTTC)5 | 222 | MH167544 |
| R: TGCTAGCAATGCTTTCCGAT | ||||
| H73 | F: GCCCGATAATCTCAACTGGA | (TTTCT)5 | 203 | MH167545 |
| R: TGGCTGCCTAGCTAACACCT | ||||
| H74 | F: CATCCTTATCCACCCACCAC | (TATTA)5 | 114 | MH167546 |
| R: AACGAAGGAGCGTAGTGTCG | ||||
| H76 | F: CATTGCCAAATTTGAGAGCA | (AAAC)6 | 245 | MH167547 |
| R: TCTAAACAATTCGTTCGGGC | ||||
| H79 | F: TCCGATTAAAAACTGCCACC | (CT)15 | 268 | MH167549 |
| R: ATATTTCCCAGCGTGTCAGG | ||||
| H80 | F: CTTTGCCTGAATGGCTGAAT | (CT)15 | 182 | MH167550 |
| R: TTCAATAGGCAAGCAATCCC | ||||
| H81 | F: GTCGAGAACACTTCCATGCC | (TG)24 | 251 | MH167551 |
| R: TACGTCACCCCGTAAGATCC | ||||
| H83 | F: TTGTGGTTCTTATGGCCCTC | (AG)21 | 147 | MH167552 |
| R: AGCTTCACTTGCCTCATCGT | ||||
| H84 | F: CACACCTGCAAAATCACCAC | (AG)21 | 240 | MH167553 |
| R: ATTTGATACATTGGCGAGGC | ||||
| H85 | F: CCTGCCGTTGCAATAACTCT | (AT)11 | 169 | MH167554 |
| R: TGGTAGACATGGCATCCGTA | ||||
| H88 | F: ATGACCTTCAACAGGCACAT | (TAT)16 | 181 | MH167556 |
| R: CATTGCAATCAAAATCGTTCA | ||||
| H91 | F: TAGTTGATTGGCTCCCTTGG | (TCTA)7 | 239 | MH167557 |
| R: CCCTCTCGCTATGTTTCTGC | ||||
| H92 | F: CGTGGGATCAAGGGAAGTAG | (CAAC)7 | 217 | MH167558 |
| R: GGATGTGACTGTGCTTCCAA | ||||
| H93 | F: GGGCAATGTCCCTTCTTGTA | (AAAT)5 | 231 | MH167559 |
| R: AGAATTGTTAGGGCCGGTTT | ||||
| H100 | F: CTGCAAATCTTGGTTTGGCT | (TTTCC)5 | 146 | MH167562 |
| R: AATTCCCCAGAAAAGGTTCG | ||||
| H105 | F: TTAGGCTCCGTTTGGTTGTC | (AGGAA)5 | 234 | MH167564 |
| R: GTGGGAAAATTGTGGACCAA | ||||
| H108 | F: TTTGGTTGAGTGGGACTTGA | (TTGG)8 | 228 | MH167565 |
| R: CAAGGGACTGGAGCTCAAAC | ||||
| H110 | F: TCAAGACCTTTTACTCCCAAAAA | (AAAG)8 | 122 | MH167566 |
| R: AAAGAGCATCGTGGCTAAAGTC | ||||
| H111 | F: GCATTGGAGGAATACGGTTG | (ACAT)7 | 219 | MH167567 |
| R: GGAGCAGAAATTCCACGAAC | ||||
| H112 | F: TGTCCTTTTGGTGTTTTCACA | (TTTA)7 | 230 | MH167568 |
| R: AATTCACTGTCACCATCCCC | ||||
| H113 | F: TTCAGTTTCTTTGTTGGGGC | (ACAT)7 | 219 | MH167569 |
| R: ATCCAACGCCTCCTAATCCT | ||||
| H117 | F: GAGTGCGTACACGGGTTCTT | (TTC)6…(TTC)5 | 152 | MH167571 |
| R: CCCATCTAGTTCCTTCTCTTTGC | ||||
| H118 | F: GGGTGAAGATTTTGGATTCG | (TA)9(CA)8 | 263 | MH167572 |
| R: AAATCACAGCCACAGAGTGAGA | ||||
| H119 | F: GCCCTATAGAGGTCACCTTCC | (AC)12(AT)12 | 272 | MH167573 |
| R: CTCACCCGAAAGCCATAAAA | ||||
| H121 | F: TTTTGAGAACCAAAATAGAGTATAGCA | (AAC)9(AAT)8 | 257 | MH167574 |
| R: TTGGAACTCATCAGTTTTTCCA | ||||
| H123 | F: ATCACTGCTAGTCCGCCACT | (CTG)6GTTCTGG(TTC)8 | 155 | MH167576 |
| R: AGGAACCGGGAAAAGAAGAA | ||||
| H124 | F: GACGAGCGTACCCTTCAAAA | (GAA)8…(AGA)7 | 156 | MH167577 |
| R: GCGACGCAGTGGTCTTCT | ||||
| H125 | F: GCCAAGTAGCCGACTTTGAA | (TTA)10TTTAT(TTA)6 | 268 | MH167578 |
| R: CTGCTCAACTCAACAAAGCCT | ||||
| H127 | F: AGCCTCAAGGCATTACACCA | (TCT)5TATTCTTA(TTC)7TTT(TTC)6 | 263 | MH167580 |
| R: GGTACCTATCCCTAGCATGGC | ||||
| H129 | F: ATGCAGAAATGCCCTTGCTA | (ATT)9…(ATT)9 | 228 | MH167581 |
| R: GCAATTACAGGTAAAGCTAATCCAA | ||||
| H133 | F: AGAGGTGGTGTTCAAAACGG | (TTA)10 | 200 | MH167585 |
| R: TGGCATACCTAAAATCCTAAATCA | ||||
| H135 | F: GGCTAAAGTGAGGTTTTGGC | (ATT)14 | 277 | MH167586 |
| R: GCTCCGAGCTAACAAAGCAC | ||||
| H136 | F: TGCAACTAATCCTAACCTTTGAA | (GAA)14 | 132 | MH167587 |
| R: AGGAGCAAAGGAGAAGGGAG |
Yin, Q. , Huang C., Huang Y., Chen S., Ye H., Fan Q., and Liao W.. 2018. Identification and development of microsatellite markers in Hamamelis mollis (Hamamelidaceae). Applications in Plant Sciences 6(10): e1189.
Notes
N = number of individuals sampled.
Vouchers are deposited in Sun Yat‐sen University (SYS), Guangzhou, Guangdong Province, China.
Sample used in genome sequencing.
Samples used in SSR trials.
Contributor Information
Qiang Fan, Email: fanqiang@mail.sysu.edu.cn.
Wenbo Liao, Email: lsslwb@mail.sysu.edu.cn.
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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
All genomic sequences of 136 pairs of primers were deposited in the National Center for Biotechnology Information (NCBI) GenBank database (MH167492–MH167587). The filtered raw read data and the assembled contigs were also deposited in NCBI databases (BioSample: SAMN09010486, BioProject: PRJNA454742, Sequence Read Archive [SRA]: SRR7110723, Transcriptome Shotgun Assembly [TSA]: GGNJ00000000).