Development and characterization of 20 novel EST-SSR markers for Pteroceltis tatarinowii, a relict tree in China.

PREMISE: Pteroceltis tatarinowii (Ulmaceae), the only species of the genus Pteroceltis, is an endangered tree in China. Here, novel expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed to illuminate its genetic diversity for conservation and assisted breeding. METHODS AND
RESULTS: Based on Illumina transcriptome data from P. tatarinowii, a total of 70 EST-SSR markers were initially designed and tested. Forty-eight of 70 loci (68.6%) were successfully amplified, of which 20 were polymorphic. The number of alleles per locus ranged from two to six, and the levels of observed and expected heterozygosity ranged from 0.018 to 0.781 and from 0.023 to 0.702, respectively. Additionally, cross-amplification was successful for 17 loci in two related species, Ulmus gaussenii and U. chenmoui.
CONCLUSIONS: These new EST-SSR markers are valuable transcriptomic resources for P. tatarinowii and will facilitate population genetics and molecular breeding of this species and its relatives in Ulmaceae.

Pteroceltis tatarinowii Maxim. (Ulmaceae), the only species of Pteroceltis Maxim., is an endangered Tertiary relict deciduous tree endemic to China (Li et al., 2013). Its bark (phloem fiber) has been famously utilized as a raw material to make the traditional Chinese Xuan paper (Cao, 1993; Li et al., 2012). Pteroceltis tatarinowii can adapt well to high‐calcium soil and barren drought conditions, which makes it a popular tree in ecological restoration (You et al., 2018; Zhang et al., 2019). However, in recent decades, the distribution and size of wild P. tatarinowii populations have dramatically decreased due to fragmentation of native habitats and overexploitation for Xuan paper. This species is categorized as a National Class III Key Protected Species in China (Zhang et al., 2019). Therefore, there is an urgent need to perform population genetic studies that will assist in the development of conservation strategies.

With the advent of high‐throughput transcriptome sequencing technologies, expressed sequence tag–simple sequence repeat (EST‐SSR) markers have been rapidly mined in recent years. Compared to anonymous genomic SSRs, EST‐SSRs have many advantages such as linking to species’ functional traits, high polymorphism, more transfer across taxonomic boundaries, and less susceptibility to null alleles and homoplasy (Varshney et al., 2005; Ellis and Burke, 2007; Yoichi et al., 2016). Previous population genetic studies of P. tatarinowii have utilized 12 genomic SSRs composed of dinucleotide repeat and imperfect SSR types (Li et al., 2015; Fan et al., 2019). However, the lack of EST‐SSR markers developed from a transcriptional data set and the insufficient SSR repeat motif types of P. tatarinowii restrict the investigation of population genetics and molecular breeding using abundant information sites. Moreover, EST‐SSR markers are relatively easy and inexpensive to develop, and are more accessible to studies based on functional traits than other types of genomic SSR markers (Hinchliffe et al., 2011; Ukoskit et al., 2018). In this study, we developed novel polymorphic EST‐SSR markers for P. tatarinowii from an Illumina transcriptome data set to elucidate its population genetic diversity and test the transferability of the studied loci in two phylogenetically related Ulmaceae species, Ulmus gaussenii W. C. Cheng and U. chenmoui W. C. Cheng.

METHODS AND RESULTS

Fresh young leaf tissue from one P. tatarinowii tree from Nanjing Botanical Garden, Memorial Sun Yat‐sen in Jiangsu Province, China (Appendix 1), was sampled for transcriptome sequencing. TRIzol reagent (Invitrogen Life Technologies, Carlsbad, California, USA) was used to extract RNA from collected leaves, and an RNA‐Seq data set containing 5.64 Gbp of paired‐end raw reads was generated by the Beijing Genomics Institute using the Illumina HiSeq 2500 platform (Illumina, San Diego, California, USA). The raw reads have been deposited into the National Center for Biotechnology Information Sequence Read Archive (SRA accession number: SRR10158849). Raw reads were initially filtered by removing ambiguous reads (N > 5%) and low‐quality reads (>20% of nucleotides with Q value ≤ 10) and trimming adapters using Trimmomatic (Bolger et al., 2014), yielding 15,264,074 clean reads. Clean reads were then assembled into 94,932 transcripts (N50 = 2212 bp) using Trinity version 2.5 (Grabherr et al., 2011) with the default parameters, and TGICL version 2.1 (Pertea et al., 2003) was used to cluster the transcripts into 42,477 unigenes (N50 = 1581 bp).

SSR screening was performed using MISA (Thiel et al., 2003) with repeat identification ranging from mono‐ to hexanucleotides within the identified unigenes. A total of 6543 EST‐SSR motifs were identified, and 3945 primer pairs were designed using Primer3 software (Rozen and Skaletsky, 1999) with default settings. Seventy primer pairs were randomly chosen for initial screening with five samples (one individual per population; Appendix 1) of P. tatarinowii to ensure the availability and optimal annealing temperature of each pair. One hundred forty‐seven individuals of P. tatarinowii from five wild populations (Appendix 1) were collected to evaluate and validate the polymorphism of the EST‐SSR markers. Two related Ulmaceae species (U. gaussenii and U. chenmoui [n = 10 for each species]; Appendix 1) were selected to test the cross‐amplification of the markers. Total genomic DNA was extracted from silica gel–dried leaves with Plant DNAzol Reagent (Invitrogen Life Technologies) following the manufacturer's protocol. DNA quality was examined on a 0.8% agarose gel stained with 1× GelRed (Biotium Inc., Fremont, California, USA), and the concentration was checked using a Nano‐Drop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). PCR amplifications were performed on a GeneAmp PCR System 9700 DNA thermal cycler (Perkin‐Elmer, Norwalk, Connecticut, USA) following the standard protocol of the 2× Taq Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) in a final volume of 15 μL, containing 1 μL (100 ng) of genomic DNA, 7.5 μL of 2× Taq Master Mix, 5.5 μL of deionized water, and 0.5 μL of forward and reverse primers (10 μM). The PCR procedure consisted of 5 min of initial denaturation at 95°C; 35 cycles of 45 s at 95°C, a temperature gradient for annealing from 48°C to 60°C for 30 s, and 30 s of synthesis at 72°C; followed by a final 15‐min extension step at 72°C and a 4°C holding temperature. The resulting 48 primer pairs (68.6%) that produced a clear band in initial screening of five individuals of P. tatarinowii were selected for further tests of polymorphism and transferability.

To screen polymorphisms of these 48 EST‐SSR loci, fluorescence‐based genotyping was performed using 147 individuals from five wild populations (Appendix 1). For all loci, the 5′ end of each forward primer was tagged with one of four fluorescent dyes (FAM, ROX, HEX, or TAMRA; Sangon Biotech, Shanghai, China). The same PCR conditions described above were used for amplification, except for 30 s of annealing at the optimal primer temperature (Table 1). After amplification, we analyzed the multiplex DNA products labeled with the four above‐mentioned fluorescent dyes on an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, California, USA) with GeneScan 500 LIZ Size Standard as an internal reference. GeneMarker version 2.2.0 (SoftGenetics, State College, Pennsylvania, USA) was then used to score the electrophoresis peaks and identify polymorphisms. Of the 48 candidate EST‐SSR markers, 20 (41.7%) exhibited polymorphism in P. tatarinowii. All 20 EST‐SSR sequences were deposited in GenBank (Table 1) and were used for cross‐amplification tests. Furthermore, the corresponding sequences of these 20 EST‐SSRs were BLASTed against the GenBank nonredundant database using BLASTX (Altschul et al., 1997) (Table 1). The characteristics of the 28 monomorphic EST‐SSR markers are provided in Appendix 2.

Table 1

Characteristics of 20 EST‐SSR markers developed for Pteroceltis tatarinowii.

Locus	Primer sequences (5′–3′)	Repeat motif	Allele size range (bp)	T a (°C)	Fluorescent dyea	BLASTX top hit description	E‐value	GenBank accession no.
E2	F: AAACGTTTTCGTTTTCGGTG	(CCAATA)6	218–248	58	FAM	Hypothetical protein L484_008773 [Morus notabilis]	2.E‐11	JZ980980
	R: TCTATTCTAGCCTTGGGCGA
E3	F: AGTTTTGTGTATGCATGCGG	(GATGAA)6	115–127	58	HEX	Serine/threonine protein kinase [Trema orientale]	4.E‐13	JZ980981
	R: CATCCTACCAAAGTCGCCAT
E4	F: CCAAGGGATCATCTGGAGAA	(AGACAA)5	154–178	58	TAMRA	Splicing factor‐like protein [Parasponia andersonii]	2.E‐69	JZ980982
	R: ATCCTTCCTCACGACAATGC
E5	F: GATTGAAGCCGGGATACTGA	(GGTTGC)5	254–272	58	ROX	Splicing factor‐like protein [Trema orientale]	3.E‐64	JZ980983
	R: ATCGAGGGGTTTTGCTCTTT
E6	F: TCTATTCCCCCAAACCACAA	(CAATTT)5	248–272	58	FAM	Zinc finger protein [Trema orientale]	7.E‐31	JZ980984
	R: GAATCCGACATGGGTAAACG
E15	F: AGGATGTTGCTATTGTGCCC	(GGTA)6	201–221	60	TAMRA	Glycosyl transferase [Trema orientale]	9.E‐14	JZ980985
	R: TCCCAGTATGTACAAAGCCGT
E16	F: AGTGGTTTGTTTTGCCCTTG	(CTTT)5	245–257	60	HEX	No significant similarity found	–	JZ980986
	R: TGGCATCTTCACACCCAATA
E19	F: ATCCCAGGCCTAATGCTTCT	(ATGT)5	110–125	60	TAMRA	MYC/MYB transcription factor [Parasponia andersonii]	6.E‐50	JZ980987
	R: CAGGAATTTGCACGATCTCA
E21	F: CACCGTATTGGAAAATAAGTTATCA	(GAAA)5	208–220	60	FAM	Mitogen‐activated protein kinase kinase kinase [Trema orientale]	1.E‐21	JZ980988
	R: GGTTTCGTCATGTTCCTGGT
E23	F: CCAGATACATCCAGGCAACA	(AGAA)5	185–191	60	HEX	No significant similarity found	–	JZ980989
	R: TTGGCAATCTCAGCTTGATG
E24	F: CCATGTCACTCCAGGGAATC	(TTGT)5	112–132	60	FAM	Golgi SNAP receptor complex, subunit [Trema orientale]	4.E‐18	JZ980990
	R: CCTCATTTTAGAGGTGCCCA
E36	F: TGTCCCAGAGAAAATGGTCC	(ATGA)5	196–202	60	HEX	Rab‐GTPase‐TBC domain‐containing protein [Trema orientale]	3.E‐03	JZ980991
	R: GTTGGCTGAGTTTCGGTCAT
E42	F: AAATCCACCACATGGCAGTT	(AGA)5	215–233	60	FAM	Chromatin structure‐remodeling complex protein BSH [Morus notabilis]	3.E‐63	JZ980992
	R: GCACTCTTATTGCCTCTGCC
E44	F: AAACGGAGACATTCGGTTTG	(ACC)6	111–114	60	HEX	Hypothetical protein Prudu_020836 [Prunus dulcis]	2.E‐30	JZ980993
	R: CTCCGAACTAGTCTGCCCTG
E48	F: AAGATGGAGGAAGAGGGCAT	(GGA)5	264–279	60	TAMRA	Chlorophyll A‐B binding protein [Parasponia andersonii]	2.E‐24	JZ980994
	R: GATTCAAAGGCATCGCAAAT
E53	F: AACTACAGCATGGGTTTGCC	(GTG)5	255–267	60	TAMRA	43‐kDa postsynaptic protein [Trema orientale]	5.E‐73	JZ980995
	R: TGATAGACCCAGGATGAGCC
E65	F: AATCGAGTCATCGGAAAACG	(TCT)5	238–256	60	ROX	Plastid division protein PDV [Parasponia andersonii]	8.E‐57	JZ980996
	R: ATGGGGAACTGTAACGCTTG
E68	F: AAATGCACGCACACAGAGAC	(AAG)5	243–279	55	ROX	DYW domain‐containing protein [Trema orientale]	2.E‐54	JZ980997
	R: AATTTGAACGATGCGTAATGG
E69	F: AAGAAGCAGCGGAAAGATCA	(CTT)5	259–279	60	ROX	Protein DA1‐related 2 isoform X1 [Sesamum indicum]	3.E‐31	JZ980998
	R: ATCCCCAACAAACACCGATA
E70	F: AACAGTAATCGTTGGCGTCC	(CTT)6	253–257	60	ROX	43‐kDa postsynaptic protein [Parasponia andersonii]	4.E‐53	JZ980999
	R: ACCATAGCCGTCGAAATCAC

T a = annealing temperature.

Fluorescent dye used to tag the 5′ of each forward primer.

Genetic diversity parameters (number of alleles and levels of expected and observed heterozygosity) were calculated in the five wild populations of P. tatarinowii using GenAlEx 6.5 (Peakall and Smouse, 2012). Significant deviation from Hardy–Weinberg equilibrium for each population and linkage disequilibrium for each primer pair were examined with GENEPOP version 4.2 (Rousset, 2008) using a Bonferroni correction. For each population of P. tatarinowii, levels of observed and expected heterozygosity varied from 0.000 to 0.917 (mean = 0.402) and from 0.000 to 0.789 (mean = 0.405), respectively (Table 2). The Hardy–Weinberg equilibrium test indicated that three primer pairs (E3 in the SD population, E4 in the JX and YZJ populations, and E5 in the LYS and TMS populations) deviated significantly from expectations after applying a Bonferroni correction (P < 0.05; Table 2), which might be caused by the Wahlund effect. No significant linkage disequilibrium was observed for any pair of loci after applying a Bonferroni correction.

Table 2

Genetic diversity statistics for five populations of Pteroceltis tatarinowii and two related taxa based on 20 newly developed EST‐SSR markers.a

Locus	Pteroceltis tatarinowii
	LYS (N=32)			JX (N =21)			TMS (N =24)			YZJ (N =34)			SD (N =36)			Ulmus gaussenii (N = 10)			Ulmus chenmoui (N = 10)
	A	H o	H e	A	H o	H e	A	H o	H e	A	H o	H e	A	H o	H e	A	H o	H e	A	H o	H e
E2	6	0.844	0.789	3	0.571	0.550	4	0.667	0.576	4	0.647	0.520	5	0.528	0.595	4	0.900	0.625	4	0.400	0.645
E3	3	0.438	0.398	3	0.571	0.543	3	0.583	0.624	2	0.382	0.344	3	0.500	0.457*	3	0.700	0.515	2	0.100	0.495
E4	5	0.469	0.511	3	0.286	0.346*	4	0.625	0.601	4	0.353	0.364*	3	0.583	0.481	4	0.700	0.625	1	0.000	0.000
E5	4	0.219	0.556*	4	0.048	0.529	4	0.208	0.605*	3	0.235	0.211	4	0.444	0.639	3	0.200	0.620	2	0.000	0.500
E6	2	0.188	0.219	3	0.143	0.135	4	0.375	0.438	2	0.412	0.327	1	0.000	0.000	2	0.100	0.095	3	0.500	0.595
E9	3	0.375	0.456	2	0.619	0.482	3	0.625	0.555	2	0.029	0.029	3	0.444	0.439	1	0.000	0.000	2	0.100	0.095
E15	4	0.719	0.667	4	0.714	0.659	3	0.750	0.625	4	0.706	0.625	4	0.917	0.674	—	—	—	3	0.300	0.555
E16	3	0.125	0.250	2	0.000	0.091	3	0.125	0.227	2	0.206	0.375	3	0.417	0.335	2	0.600	0.420	2	0.200	0.320
E19	3	0.563	0.471	3	0.571	0.490	3	0.292	0.317	3	0.471	0.514	4	0.639	0.522	1	0.000	0.000	2	0.100	0.095
E21	2	0.125	0.264	2	0.095	0.091	2	0.083	0.080	2	0.500	0.465	2	0.222	0.239	2	0.200	0.180	3	0.200	0.335
E23	2	0.281	0.242	3	0.476	0.421	2	0.542	0.457	2	0.029	0.029	1	0.000	0.000	3	0.400	0.540	2	0.300	0.255
E24	2	0.469	0.460	5	0.524	0.689	2	0.458	0.430	4	0.324	0.590	3	0.278	0.509	2	0.200	0.180	—	—	—
E36	3	0.156	0.200	3	0.238	0.217	3	0.333	0.288	2	0.364	0.397	3	0.083	0.081	3	0.200	0.185	2	0.500	0.455
E38	3	0.031	0.061	1	0.000	0.000	1	0.000	0.000	2	0.029	0.029	2	0.028	0.027	2	0.100	0.095	2	0.300	0.255
E42	3	0.563	0.510	4	0.667	0.627	3	0.375	0.555	4	0.412	0.386	5	0.500	0.482	2	0.200	0.320	1	0.000	0.000
E44	6	0.906	0.744	7	0.762	0.688	5	0.750	0.670	5	0.765	0.681	7	0.722	0.728	—	—	—	1	0.000	0.000
E48	2	0.625	0.498	3	0.429	0.441	3	0.500	0.469	3	0.706	0.501	2	0.833	0.500	2	0.400	0.420	2	0.600	0.480
E53	3	0.250	0.271	3	0.143	0.357	3	0.125	0.192	2	0.412	0.327	2	0.056	0.105	2	0.200	0.180	2	0.100	0.255
E68	2	0.531	0.488	2	0.429	0.482	2	0.333	0.278	2	0.441	0.489	2	0.500	0.500	3	0.700	0.515	2	0.500	0.375
E70	2	0.344	0.390	2	0.714	0.482	3	0.375	0.430	2	0.265	0.271	2	0.472	0.500	1	0.000	0.000	2	0.100	0.095
Average	3.15	0.411	0.422	3.10	0.400	0.416	3.00	0.406	0.421	2.80	0.384	0.374	3.05	0.408	0.391	2.55	0.405	0.373	2.50	0.320	0.395

A = number of alleles; H e = expected heterozygosity; H o = observed heterozygosity; N = number of individuals sampled.

Locality and voucher information are provided in Appendix 1.

Significant departure from Hardy–Weinberg equilibrium at P < 0.05.

Cross‐species amplification tests of the 20 loci in U. gaussenii and U. chenmoui followed the PCR procedures mentioned above. PCR products were detected using 2% agarose gels, and amplification was considered successful when one clear band was visible in the expected size range. We further analyzed the successful amplification products on an ABI 3730XL DNA Analyzer (Applied Biosystems) with GeneScan 500 LIZ Size Standard as an internal reference. GeneMarker version 2.2.0 (SoftGenetics) was then employed to score the electrophoresis peaks and identify polymorphisms. Overall, all loci were successfully amplified and exhibited polymorphisms, except for locus E36 for U. chenmoui and loci E16 and E53 for U. gaussenii (Table 2).

CONCLUSIONS

Using high‐throughput RNA sequencing data, we developed 20 polymorphic EST‐SSR markers for P. tatarinowii. Most of these markers showed high transferability in two related species, suggesting that they may contribute to the population genetics and molecular breeding of other Ulmaceae species.

Species	Voucher specimensa	Population code	N	Collection locality	Geographic coordinates
P. tatarinowii Maxim.	WGZ 20180421	LYS	32	Langya Mountain, Anhui, China	32°17′N, 118°17′E
P. tatarinowii	ZMY 20180428	JX	21	Jingxian, Anhui, China	30°39′N, 118°23′E
P. tatarinowii	ZMY 20180429	TMS	24	Tianmu Mountain, Zhejiang, China	30°19′N,119°26′E
P. tatarinowii	ZMY 20180430	YZJ	34	Swallow Promontory, Jiangsu, China	32°09′N, 118°49′E
P. tatarinowii	ZMY 20180504	SD	36	Shidu, Beijng, China	39°40′N, 115°31′E
U. gaussenii W. C. Cheng	ZX 20171012	ZWY	10	Langya Mountain, Anhui, China	32°17′N, 118°17′E
U. chenmoui W. C. Cheng	ZX 20171012	LYY	10	Langya Mountain, Anhui, China	32°17′N, 118°17′E

N = number of individuals sampled.

aVouchers were deposited in the Herbarium of Zhejiang University (HZU), Hangzhou, Zhejiang Province, China.

Locus	Primer sequences (5′–3′)	Repeat motif	T a (°C)	Allele size range (bp)
E7	F: TTCCTTGGAAGCCTCAGTTG	(AACCTG)5	58	118
	R: CGAGGTTCGGTTTGTTGTTT
E8	F: TTGATGCTGATGGTGGTGAT	(TGTTGG)5	58	117
	R: CAAGATTCAGAAAAAGGGCG
E11	F: TAACGCCCAGTAGGAATCCA	(ATATC)5	60	275
	R: GCGCAGGGTAGTGATAGAGG
E14	F: AGGAGGTTCGACTCCTGGAT	(TTCC)5	60	250
	R: AAAAAGGAAGTGTGGTATGTGTATG
E22	F: CAGTGAGCCAACAGAGTGGA	(TTTC)5	58	172
	R: AAAGGCACCATGTCCAAAAC
E25	F: CCCTTAGTTTAGCCGCAGTG	(GGTT)5	60	251
	R: ATGGGCATGGATTGCCTAAT
E27	F: CGAGGCCGAGACAAGTAAAG	(ATTA)5	55	176
	R: CCAACAAAATCTTTAAGGATATGAAA
E28	F: GGAGACGAAATTGTTGGTGG	(CTTT)5	58	219
	R: TGGTCGAATCTTTCCCACTC
E29	F: GGCTTGATGTGCTCCAAGTT	(AAAC)5	60	251
	R: CCCAAAGGAAATAAAATAGGCA
E35	F: TGATGGAGGCGACATTGATA	(TTAA)5	60	251
	R: TTCCTCAGTCCACCATGACA
E37	F: TGTGGTGCTTCAGCTCATTC	(TTTG)6	60	224
	R: CCCTGCAGATCTGTCAAACA
E39	F: AAGGAATGTACCTCGGCTCA	(CCG)5	60	280
	R: CTCCTAAACACCAGGACCCA
E41	F: AATGGCGATTTGAAAGATGG	(ATG)5	60	271
	R: CACCCTCTGCCTCCTTAACA
E43	F: AACGTTCAAATGCGCCTAGT	(CAC)6	60	251
	R: CCAGAGCTTGACCTCTTTGG
E45	F: AATGGCGAATTTGAAGATGG	(GAA)5	60	241
	R: TTTCGGTTCTCTGAATTGGG
E47	F: AACCCACACTACCGCTCATC	(GGT)7	60	219
	R: CTTTGCTTCCCATTCAGCTC
E50	F: AACAACACCCCAACCAAAAA	(CTT)5	55	208
	R: TGGGATGACTTCCATTCCAT
E51	F: AAGGACGACAAGGGTGTTTG	(GGC)5	60	206
	R: TATTTGGCTCGTAACCGAGG
E52	F: AATTAGGGCAAGGGTCGAGT	(CAA)5	60	199
	R: CCATCACCTCCCATGTTACC
E54	F: AACCCCCTCCTTTCTTGTGT	(TTA)5	60	191
	R: GACAAAAGGTTGGCTTCGTG
E56	F: AAATTCACCACCGGATGAAA	(GAA)5	60	172
	R: CTTCCGCTTTCCTCTTCCTC
E59	F: AAACCCAAAGGAAAAGTTAAAAA	(ATA)6	55	162
	R: TGTGCTTTGGGCCTTAGAGT
E60	F: AAAGGCCAGAGAATTGGGTT	(AAT)5	60	155
	R: CAAACTTTGGCTTGAACGAA
E61	F: AACTCACTCGACCCAACCAC	(CCT)5	60	152
	R: GAGGCGATGGTGAAGAAGAG
E62	F: AACAAGGCTCGTCGAATGTC	(ATC)5	60	149
	R: GGACCTATGCAGTCCTTCCA
E64	F: AAGAAGGAGGAGATGGTGGG	(GAG)6	60	134
	R: TGGCTCAGACAGTTCCAATG
E65	F: AATCGAGTCATCGGAAAACG	(TCT)5	60	132
	R: ATGGGGAACTGTAACGCTTG
E69	F: AAGAAGCAGCGGAAAGATCA	(CTT)5	60	112
	R: ATCCCCAACAAACACCGATA

T a = annealing temperature.