Development and characterization of EST-SSR markers for Camellia reticulata.

PREMISE: Camellia reticulata, which is native to southwestern China, is an economically important plant belonging to the family Theaceae. We developed expressed sequence tag-simple sequence repeat (EST-SSR) markers for C. reticulata, which can be used to investigate its genetic diversity, population structure, and evolutionary history. METHODS AND
RESULTS: We detected 4780 SSRs in C. reticulata from Camellia RNA-Seq data deposited in the National Center for Biotechnology Information's expressed sequence tags database (dbEST). Primer pairs for 70 SSR loci were designed and used for PCR amplification using 90 individuals from four populations of C. reticulata. Of these loci, 50 microsatellite markers were successfully identified, including 11 polymorphic markers. The allele number per locus ranged from two to seven (mean = 4.182), and the levels of observed and expected heterozygosity ranged from 0.044 to 0.567 and from 0.166 to 0.642, respectively. Eleven primer pairs amplified PCR products in three other species of Camellia (C. saluenensis, C. pitardii, and C. yunnanensis).
CONCLUSIONS: The set of microsatellite markers developed here can be used to study the genetic variation and population structure of C. reticulata and related species and thereby help to develop conservation strategies for this species.

Camellia reticulata Lindl. (Theaceae) is an economically important ornamental flowering shrub or small tree that grows in Yunnan Province, southwestern Sichuan Province, and western Guizhou Province of China (Ming et al., 2000). As an ornamental plant, C. reticulata has over 1000 years of history of cultivation, and many outstanding cultivars have been selected or bred from wild C. reticulata for many centuries in China (Xia et al., 1994; Gu, 1997). Camellia reticulata is notable for its large flowers, brilliant colors, numerous cultivars, and long flowering duration (Xia et al., 1994; Ming et al., 2000). Furthermore, it is valued not only as a flowering ornamental but also as a source of oils. Its seeds have a high content of oil that is rich in unsaturated fatty acids, oleic acid, vitamin E, and other physiologically active substances, making it a valuable edible oil source for daily consumption (Liu and Ma, 2010). Camellia reticulata is a perennial, outcrossing, heterogenous polypoid species (2n = 30) (Kondo et al., 1986). Its natural populations across southwestern China have different ploidy levels (i.e., 2n = 2x, 4x, 6x; x = 15), which could have resulted from natural hybridization and polyploidization (Gu, 1997). Fluorescence in situ hybridization and genomic in situ hybridization for C. reticulata indicate that C. japonica L., C. saluenensis Stapf ex Bean, C. pitardii Cohen‐Stuart, and C. yunnanensis (Pit. ex Diels) Cohen‐Stuart might have contributed to the origin and evolution of the C. reticulata polyploid complex (Gu and Xiao, 2003; Liu and Gu, 2011).

In recent years, there has been a decline in the number and size of natural populations of C. reticulata because of overharvesting and habitat destruction. This alarming situation necessitates an in‐depth understanding of the current status of the genetic diversity of the species. Studies have been conducted on the genetic diversity of C. reticulata via inter‐simple sequence repeat markers, chloroplast microsatellites, and amplified fragment length polymorphisms (Wang and Ruan, 2012; Tong et al., 2013; Yao et al., 2016; Xin et al., 2017). Yao et al. (2016) designed 20 expressed sequence tag–simple sequence repeat (EST‐SSR) primer pairs based on the transcriptome of diploid C. reticulata. Of these, 18 were successfully amplified, detecting seven polymorphic loci in 24 C. reticulata individuals. We further tested these 20 markers in four natural populations, showing four loci with polymorphisms. These are not sufficient for inclusive studies on C. reticulata. Therefore, in this study, we aimed to develop new microsatellite markers that will help to investigate the reproductive characteristics of C. reticulata, evaluate its evolutionary potential, and develop effective strategies for the conservation, development, and utilization of wild natural populations. In addition, we tested the cross‐species transferability of these markers in three other species of Camellia: C. saluenensis, C. pitardii, and C. yunnanensis, which are thought to be involved in the polyploidy of C. reticulata.

METHODS AND RESULTS

Fresh healthy leaves collected from 90 individuals of C. reticulata sampled from four wild populations from Yunnan Province, China, were freeze‐dried or silica‐dried. Forty samples from three populations of C. saluenensis, C. pitardii, and C. yunnanensis were also collected to test the cross‐amplification of the markers. Voucher specimens were deposited at the Kunming Institute of Botany, Chinese Academy of Sciences (KUN) (Appendix 1). Genomic DNA was extracted from 20–30 mg of dried leaf tissue using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987).

We obtained 50,287 EST sequences from the National Center for Biotechnology Information (NCBI) expressed sequence tags database (dbEST) (accessed on June 2019) (Boguski et al., 1993). To obtain a nonredundant EST data set for SSR identification and primer design, vectors were removed from EST sequences using SeqTrim (Falgueras et al., 2010) and poly(A) tails were trimmed using est‐trimmer.pl. Clean EST sequences were then clustered and assembled into contigs and singletons using CAP3 (Huang and Madan, 1999), generating 17,989 unigenes consisting of 5099 contigs and 12,890 singletons. These unique sequences were further used to screen for the presence of microsatellites using the MISA Perl program (Thiel et al., 2003). The criteria for SSRs were set as sequences having at least 10, six, five, five, five, and five repeat units for mono‐, di‐, tri‐, tetra‐, penta‐, and hexanucleotide motifs, respectively. In total, 4780 SSRs were identified, with an average frequency of 1 SSR/1.57 kbp. Primers were designed using Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, California, USA) with the following criteria: primer lengths of 16–22 bp, GC content of 40–65%, annealing temperature ranging from 40°C to 60°C, and a predicted PCR product size ranging from 100 to 300 bp.

We randomly selected 70 primer pairs and tested them for PCR amplification in 12 accessions of C. reticulata (three individuals in each population, Appendix 1) in an initial screening test. PCR amplification was performed in an 18‐μL reaction mixture containing 20–30 ng of genomic DNA, 9 μL of 2× Easy Taq PCR Super Mix (TransGen Biotech, Beijing, China), and 1 μL of each primer (10 μM), adding ddH2O to a final volume of 18 μL. Cycling consisted of 30 s of denaturation at 94°C, 30 s at the optimized annealing temperature (Table 1), and a 1‐min extension at 72°C for 32 cycles, followed by a final extension at 72°C for 5 min. The amplified products were separated on 8% polyacrylamide denaturing gels, and the bands were developed with silver staining with a 2‐kbp DNA Ladder Marker (Hangzhou Bioer Technology Co. Ltd., Hangzhou, China) as a reference. The ploidy level of the sampled populations was unknown, but multiple copy bands per locus due to polyploidy were not observed. Of the 70 primer pairs tested, 50 yielded clear and reproducible amplicons in C. reticulata; the others were unstable or gave no product. Eleven loci showed polymorphisms (Table 1), and 39 loci were monomorphic (Appendix 2). These 11 polymorphic primers were used in 90 individuals of C. reticulata (four populations) for the population genetic analyses using the same protocol as the initial test. The polymorphic SSR loci were analyzed with POPGENE 32 software (Yeh et al., 2000) and GenAlEx (Peakall and Smouse, 2006) for the number of alleles per locus, observed heterozygosity, and expected heterozygosity (Table 2). Hardy–Weinberg equilibrium by 1000 randomizations and linkage disequilibrium were estimated using POPGENE 32 software (Yeh et al., 2000).

Table 1

Characteristics of 11 polymorphic microsatellite loci developed in Camellia reticulata.a

Locus	Primer sequences (5′–3′)	Repeat motif	Expected allele size (bp)	T a (°C)	Putative function (Organism)	GenBank accession no.
CSSR2	F: GGAATAGGCTCGGATGGT	(TTG)7	186	54	Hypothetical protein TEA_012945 (Camellia sinensis var. sinensis)	FS951581.1
	R: CTCTCTGCTTCTTCACAAATC
CSSR5	F: GCTGTAGGCGAACATGAA	(GGTGCT)6	196	55	Glycine‐rich cell wall structural protein 1.8‐like (Camellia sinensis)	FS952802.1
	R: CACTTCCACTTCCATATCCA
CSSR11	F: GCCCTAACTCTTCACTGTA	(AG)18	123	56	Growth‐regulating factor 4‐like isoform X2 (Camellia sinensis)	FS950234.1
	R: CTATGTCGGCTAGGTTCTT
CSSR17	F: AGAGGAGAGGAGAGGAGAG	(CCTCCA)7	130	54	NONE	GH710908.1
	R: TTTGGAGAGCGACATTGC
CSSR18	F: TCGCTGCTCTCATCTACT	(CTT)9	114	56	Hypothetical protein TEA_017838 (Camellia sinensis var. sinensis)	FS945416.1
	R: TCTACATGGACATGGACTTAG
CSSR19	F: GCTCATGCCATGTCATCC	(CT)12	167	55	sm‐like protein LSM1B (Camellia sinensis)	GH710926.1
	R: TACCCTCATATCAACCTTGTG
CSSR35	F: ATCGCAGACAACAAGAAGA	(TGA)6	105	55	Probable E3 probable E3 ubiquitin‐protein ligase XERICO (Camellia sinensis)	FS947941.1
	R: GGAGGAGATCGTGATGAAG
CSSR36	F: AGGCTTAGGTGTAGATAGGT	(TC)16	117	54	Histone H1‐like protein (Camellia sinensis)	JK711494.1
	R: ACTCCAACTCTTCCACAAC
CSSR38	F: GCTATTGACGCTAATGACC	(TGA)6	117	55	Protein PAT1 2 like (Actinidia chinensis var. chinensis)	FS949009.1
	R: CCAGAAATCATAACGCAACA
CSSR45	F: GTATGACAGATACCATGAACC	(T)21	157	55	Hypothetical protein TEA_005759 (Camellia sinensis var. sinensis)	FF682781.1
	R: TGAAACCAAACCCACACT
CSSR48	F: ATTACCACCACCACTATCAC	(TGG)8	143	55	ABA‐inducible protein PHV A1‐like (Camellia sinensis)	GH738605.1
	R: CCCAAAGAAAGACCAAAGAC

T a = annealing temperature.

All values are based on 90 samples representing populations from southwestern China (N = 18–27 for each); see Appendix 1 for locality and voucher information.

Table 2

Genetic variation in the 11 polymorphic EST‐SSR markers in four Camellia reticulata populations.a

Locus	TC (n = 18)				XD (n = 26)				SM (n = 19)				CX (n = 27)				Total (n = 90)
	A	H o	H e	HWE	A	H o	H e	HWE	A	H o	H e	HWE	A	H o	H e	HWE	A	H o	H e
CSSR2	3	0.222	0.541	0.032	3	0.462	0.503	0.237	2	0.000	0.102	0.000b	2	0.000	0.140	0.000b	3	0.178	0.372
CSSR5	2	0.167	0.322	0.029	3	0.462	0.585	0.000b	3	0.053	0.363	0.000b	3	0.296	0.319	0.008	3	0.267	0.428
CSSR11	4	0.222	0.630	0.000b	4	0.115	0.664	0.000b	5	0.000	0.677	0.000b	4	0.037	0.505	0.000b	5	0.089	0.636
CSSR17	4	0.722	0.589	0.618	5	0.846	0.686	0.000b	3	0.053	0.317	0.000b	4	0.111	0.357	0.000b	7	0.433	0.518
CSSR18	3	0.722	0.643	0.026	5	0.846	0.787	0.000b	4	0.421	0.563	0.000b	3	0.296	0.377	0.144	6	0.567	0.642
CSSR19	2	0.000	0.508	0.000b	2	0.462	0.498	0.705	2	0.000	0.102	0.000b	2	0.148	0.201	0.136	2	0.178	0.422
CSSR35	2	0.056	0.056	1.000	3	0.039	0.112	0.000b	2	0.000	0.102	0.000b	3	0.148	0.322	0.000b	3	0.067	0.166
CSSR36	3	0.111	0.641	0.000b	4	0.039	0.612	0.000b	3	0.000	0.199	0.000b	3	0.037	0.238	0.000b	4	0.044	0.471
CSSR38	3	0.222	0.298	0.000b	4	0.192	0.250	0.000b	2	0.000	0.398	0.000b	5	0.111	0.357	0.000b	5	0.133	0.330
CSSR45	3	0.778	0.560	0.267	4	0.769	0.719	0.012	4	0.158	0.289	0.000b	3	0.444	0.444	0.000b	5	0.544	0.580
CSSR48	3	0.722	0.532	0.220	2	0.000	0.462	0.000b	2	0.000	0.102	0.000b	2	0.074	0.352	0.000b	3	0.167	0.465
Mean	2.909	0.359	0.484		3.546	0.385	0.534		2.909	0.062	0.292		3.091	0.155	0.328		4.182	0.242	0.457

A = number of alleles sampled; H e = expected heterozygosity; H o = observed heterozygosity; HWE = Hardy–Weinberg equilibrium; n = number of individuals sampled.

Locality and voucher information are provided in Appendix 1.

Chi‐square test for Hardy–Weinberg equilibrium. Locus showed significant deviations from Hardy–Weinberg equilibrium (P < 0.001).

Among the 11 polymorphic loci, the number of alleles per locus ranged from two to seven with a mean of 4.182. The levels of observed and expected heterozygosity were 0.044–0.567 and 0.166–0.642, with averages of 0.242 and 0.457, respectively (Table 2). Four SSR markers were able to detect levels of expected heterozygosity above 0.5, indicating a high level of polymorphism in C. reticulata. All 11 polymorphic loci showed deviation from Hardy–Weinberg equilibrium within two or more populations (Table 2) as a result of heterozygosity deficits. This was most likely the result of the reproduction mode, habitat fragmentation, and inbreeding. We found no consistent deviation from linkage disequilibrium for any loci within the populations (P < 0.001). Cross‐species amplification of the 11 polymorphic loci was tested on C. saluenensis, C. pitardii, and C. yunnanensis. All 11 EST‐SSR markers were amplified successfully, using the same PCR protocol for C. reticulata, and were shown to be polymorphic (Table 3).

Table 3

Cross‐amplification and genetic diversity statistics of EST‐SSR markers developed for Camellia reticulata in three related species.a

Locus	Camellia saluenensis				Camellia pitardii				Camellia yunnanensis
	JCPT (n = 13)				JCPL (n = 15)				JCYN (n = 12)
	A	A e	H o	H e	A	A e	H o	H e	A	A e	H o	H e
CSSR2	3	1.476	0.154	0.335	2	1.385	0.067	0.287	2	1.180	0.000	0.159
CSSR5	2	1.257	0.077	0.212	2	1.220	0.067	0.186	1	1.000	0.000	0.000
CSSR11	3	1.660	0.154	0.394	3	1.312	0.067	0.246	3	1.405	0.083	0.301
CSSR17	3	1.733	0.077	0.440	3	1.495	0.133	0.343	2	1.280	0.083	0.228
CSSR18	2	1.451	0.077	0.323	2	1.220	0.067	0.186	2	1.280	0.083	0.228
CSSR19	2	1.451	0.077	0.323	2	1.220	0.067	0.186	1	1.000	0.000	0.000
CSSR35	3	1.751	0.154	0.446	3	1.402	0.133	0.297	3	1.412	0.000	0.304
CSSR36	3	1.808	0.231	0.465	3	1.495	0.067	0.343	3	1.412	0.000	0.304
CSSR38	2	1.257	0.077	0.212	2	1.220	0.067	0.186	2	1.180	0.000	0.159
CSSR45	3	1.660	0.000	0.394	3	1.226	0.067	0.191	2	1.180	0.000	0.159
CSSR48	2	1.257	0.077	0.212	2	1.142	0.000	0.129	1	1.000	0.000	0.000
Mean	2.546	1.514	0.105	0.342	2.455	1.303	0.073	0.235	2.000	1.212	0.023	0.168

A = number of alleles sampled; A e = effective 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.

CONCLUSIONS

The EST‐SSR polymorphic markers developed in this study will add to the existing resources of molecular markers and are expected to be useful for studies on population structure and genetic diversity in C. reticulata. The microsatellite loci described here were successfully cross‐amplified in C. saluenensis, C. pitardii, and C. yunnanensis, suggesting that these markers may also be applicable to the study of genetic diversity in other Camellia species.

Locality and voucher information for Camellia species used in this study.a

Species	Population code	Voucher no.	Location	Geographic coordinates	Elevation (m)	N
C. reticulata Lindl.	TC	CR‐TY‐021	Mazhan, Tengchong, Yunnan, China	25°12′03.65″N, 98°28′34.46″E	1980	18
C. reticulata	XD	CR‐TY‐013	Niujie, Xundian, Yunnan, China	25°52′57.8″N, 102°59′14.4″E	2398	26
C. reticulata	SM	CR‐TY‐004	Baiyi, Songming, Yunnan, China	25°19′21.03″N, 102°53′28.21″E	2121	19
C. reticulata	CX	CR‐TY‐018	Zixi Mountain, Chuxiong, Yunnan, China	24°59′54.33″N, 101°25′10.77″E	2318	27
C. saluenensis Stapf ex Bean	JCPT	CS‐TY‐01	Fuyuan, Songming, Yunnan, China	25°15′53″N, 102°55′18″E	2147	13
C. pitardii Cohen‐Stuart	JCPL	CP‐TY‐01	Junzi Mountain, Shizong, Yunnan, China	24°37′13.58″N, 104°9′28.4″E	2031	15
C. yunnanensis (Pit. ex Diels) Cohen‐Stuart	JCYN	CY‐TY‐01	Machang, Heqing, Yunnan, China	26°28′26.68″N, 100°3′13.53″E	3113	12

N = number of individuals sampled.

All voucher specimens are deposited in the Herbarium of the Kunming Institute of Botany (KUN), Kunming, Yunnan Province, China.

Characteristics of 39 monomorphic microsatellite loci developed in Camellia reticulata.

Locus	Primer sequences (5′–3′)	Repeat motif	Expected allele size (bp)	GenBank accession no.
CSSR1	F: CAAAGCCAAATGGAATTGTC	(A)30	179	FS943489.1
	R: GCCAGTGAATTGTAATACGA
CSSR3	F: TTCCTCCATTTGCGTGAAA	(AG)13	194	FS951626.1
	R: ACCGTCTAGCCTCCAATC
CSSR4	F: TCGTCAATTCCTTCTTGTTG	(CTT)9	128	FS951901.1
	R: TTGGTTACAGATGGAGATGG
CSSR6	F: TGTTCTCAATCCACTCTTCA	(TCA)9	140	FS953739.1
	R: GCGACAATAATAGGCTCTTG
CSSR7	F: AAGATGAAAGTGTGGATTCC	(TG)25	148	GH159087.1
	R: GTAACAACCATCACCAACAT
CSSR8	F: GCAGTAGTTGTTGAAGTTGAG	(A)31	180	GW863559.1
	R: CCAGTGAATTGTAATACGACTC
CSSR9	F: TTGTATGTTCCAAGGCATTG	(A)30	201	GW863563.1
	R: GACTCACTATAGGGCGAATT
CSSR10	F: TGCTGTCAACTACCCTTC	(AG)20	104	GW342632.1
	R: GGTGCTTGAGTCTGTGAT
CSSR12	F: ACCTTGGCTTTGCTCTCT	(AAG)13	135	GO255031.1
	R: TTGACGCCGAAGACTCTC
CSSR13	F: TGCTTGCTATCATACAGTTC	(A)30	192	GW315083.1
	R: GCCAGTGAATTGTAATACGA
CSSR14	F: GGATGTGTGTTTAGGACCAT	(A)30	196	GW863601.1
	R: ACGGCCAGTGAATTGTAAT
CSSR15	F: TCTAATGCCAAGCCTCAAC	(A)30	179	GH734011.1
	R: GACTCACTATAGGGCGAATT
CSSR16	F: TCACTAGACCATGTGCTTA	(A)30	187	GH734178.1
	R: GTGAATTGTAATACGACTCC
CSSR20	F: GCAGCTCTCTACTTGTCAT	(A)31	206	GH709471.1
	R: GCCAGTGAATTGTAATACGA
CSSR21	F: GTTGCTAAATCTGTTGCTAC	(A)30	177	GH709922.1
	R: GCCAGTGAATTGTAAATACGA
CSSR22	F: GAACAATGATGACATCTCCA	(CTCCAG)5	107	GH709760.1
	R: ATAAGGGAGGAGTGATTTGG
CSSR23	F: TTGGACACCTTGAATGACT	(A)28	114	GH612882.1
	R: TAGTGATTAGCGTGGTCG
CSSR24	F: TGTATGATAGCAAGCTGAAG	(A)28	110	GH613058.1
	R: TAGTGATTAGCGTGGTCG
CSSR25	F: GCAGCGAGAACTTTGATG	(A)30	210	GE650217.1
	R: GCCAGTGAATTGTAATACGA
CSSR26	F: TCAGACTGTACTTAGTGGTT	(A)30	126	GH623471.1
	R: TAGTGATTAGCGTGGTCG
CSSR27	F: CAGTGGATGATTGGTAATTTGG	(A)31	176	GW696815.1
	R: AGTGGTATCAGGGCAGAG
CSSR28	F: CACATCTCTCCTGTTGCTA	(A)47	148	FS944961.1
	R: CTTCTTGCTTGTCTTTCTTC
CSSR29	F: GCTGTCTGCTTTGTACGA	(GA)11	163	FE861335.1
	R: TCTCTTCTCTTCCTCCTCTC
CSSR30	F: AGAAAGAAGCTGCAAGGG	(TTCT)5	128	FE861638.1
	R: CGTAGATGAGGCTGGAAG
CSSR31	F: ACGCTGAAGTCCAAATCC	(GCC)6	145	CV699742.1
	R: AGTGGTCTCCTGTGCTAC
CSSR32	F: ACACTCACTCAATCACTGTT	(A)29	207	GH733834.1
	R: TGTAATACGACTCACCATAGG
CSSR33	F: GCAAATGTTGGGCTTGTT	(A)29	172	FS959890.1
	R: GCCAGTGAATTGTAATACGA
CSSR34	F: CATCGCATCGTCGTCATC	(TC)15	128	FS947495.1
	R: GATCCGACACTTGAACTTGA
CSSR37	F: ACCCAAAGCAAAGCCAAT	(AG)13	103	FS948821.1
	R: AACTAGCTGAAGATAGAGGAG
CSSR39	F: TTCATCACAGACACCATCA	(TTTC)8	100	FS949741.1
	R: TCACCAATCACAAATCACAG
CSSR40	F: GGCTATGTACTTGATGTTCTTC	(A)23	197	FS954738.1
	R: CCAGTGAATTGTAATACGACTC
CSSR41	F: CCTCCTCTATCTTCGATCAATA	(GA)10	110	FS949096.1
	R: CGTTAAAGCCATTTCTCTCT
CSSR42	F: GTAACGATTGAATCTGGCAT	(A)20	186	GH623925.1
	R: TCACTATAGGGCGAATTGG
CSSR43	F: CGCTATTTATCCTTGCTGTT	(A)23	133	GH623383.1
	R: GTGGTATCAACGCAGAGTA
CSSR44	F: CCACCATCACATCCTTACA	(CAC)5	157	FS949501.1
	R: GTGGAGGAGGAGATGAGTA
CSSR46	F: GCCGTGAAGATAATGTTGG	(A)64	149	GH623235.1
	R: TAGTGATTAGCGTGGTCG
CSSR47	F: CTAGTGATTAGCGTGGTC	(T)31(T)34	148	GH623319.1
	R: GTTGTGATTACGATCTCTGA
CSSR49	F: TAACCCTATGTAGACCTCAGT	(A)31	215	GW863554.1
	R: CCAGTGAATTGTAATACGACTC
CSSR50	F: TCCATAAAGGAACCTCTAGC	(CT)13	164	JK511141.1
	R: TCCAAACATACTCCCAAACT