Identification and development of microsatellite markers in Hamamelis mollis (Hamamelidaceae).

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.

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).

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

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