Isolation and identification of EST-SSR markers in Ixonanthes chinensis (Ixonanthaceae).

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.

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.

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.

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.