Development and characterization of microsatellite markers for the French endemic Angelica heterocarpa (Apiaceae) and congeneric sympatric species.

OBJECTIVE: Angelica heterocarpa (Apiaceae) is a wild endemic French species with special conservation interest in the European Union. It belongs to Angelica complex genus which is widespread throughout the north temperate zone, and is sympatric with other congeneric species. The objective of this work is to develop and characterize microsatellite markers as a new tool for understanding the ecology and evolution of Angelica species complex.
RESULTS: We identified simple sequence repeat (SSR) regions in a microsatellite-enriched library from A. heterocarpa and A. sylvestris. All 16 selected SSR regions were found to amplify in these species and were highly polymorphic. Marker transferability was validated in A. razulii and A. archangelica. These markers will help us to better understand the evolutionary dynamic between rare endemics and widespread sister species, and be useful for conservation of the endemic species. Moreover, they can provide new tools for studying the numerous traditional medicinal herbs of the Angelica genus.

Introduction

Angelica L. is a large complex genus comprising approximately 110 species confined in the northern hemisphere, with the majority in Eurasia [1]. In this paper, four French native congeneric Angelica species are considered. Angelica heterocarpa Lloyd is endemic to southwestern French estuary banks. It is protected at national level in France and is listed as priority species in the Habitats Directive of European Union [2]. Angelica sylvestris L. is common in open and forest habitats and is widely distributed among Europe and Asia. Despite different ecological niches, A. heterocarpa and A. sylvestris can live in neighboring riverbank locations. The observation of morphological intermediates questions the taxonomic relationship and potential for hybridization between these two species. Two other species are present in southwestern France: Angelica razulii Gouan, a Pyrenean endemic that shares hydrographic zones with A. heterocarpa and A. sylvestris, and Angelica archangelica L. that occurs naturally in estuaries from northern France and Europe and is cultivated in southwestern France for aromatic and pharmacological interests.

For the effective conservation of A. heterocarpa, genetic markers providing resolution at the population level are essential although, until now, not available. Here, we report the characterization of 16 new polymorphic microsatellite markers for A. heterocarpa and A. sylvestris and test their cross‐species transferability in A. razulii and A. archangelica.

Main text

Methods

Plant material was collected across natural populations in France or Germany: two for A. heterocarpa (N = 78), three for A. sylvestris (N = 98) and one for A. razulii (N = 50) and A. archangelica (N = 3) (Table 4 in Appendix 1). One or two leaflets were collected from each plant and preserved dried in silica gel. DNA was extracted with the Invitek extraction kit (Invitek, Berlin, Germany). Microsatellites markers were developed from sequences obtained from A. heterocarpa and A. sylvestris after enrichment by both traditional cloning and high throughput sequencing (GenoScreen, Lille, France) of microsatellite-enriched library [3]. Sequences containing microsatellites were identified using the QDD software [4] and primers were designed using the Primer 3 software [5] using default parameters with 56 °C as annealing temperature. A total of 119 primers pairs for these SSR loci were tested for amplification and genotyping of 4 of each A. heterocarpa and A. sylvestris individuals using primer extended with M13 sequence for fluorescent labeling [6]. All of them were found to amplify in the both species and among them, 16 showing polymorphisms and consistent peak profile were selected in the final genotyping protocol (Table 1).

Table 4

Taxonomic, geographic information (municipality, department number, country) and GPS central coordinates of Angelica sp. populations sampling (n = number of individuals) represented in this study

Species	Location, country	n	Geographic coordinates	Population name
A. heterocarpa	Isle-Saint-Georges (33). FR	38	N44°43′30″-W0°27′28″	Isle St George
A. heterocarpa	Rézé (44). FR	40	N47°11′28″-W1°36′6″	Rézé
A. sylvestris	La Brède (33). FR	30	N44°40′21″-W0°33′17″	La Brède
A. sylvestris	Aurice (40). FR	38	N43°48′38″-W0°35′36″	Aurice
A. sylvestris	Le Bonhomme (68). FR	30	N48°8′10″-E7°5′19″	Le Bonhomme
A. razulii	Bagnères de Bigorre (64). FR	50	N42°59′48″-E0°7′29″	Bagnères
A. archangelica	Hambourg. DE	3	N53°34′18″-E10°00′09″	Hambourg

FR, France; DE, Germany

Table 1

Characteristics of 16 primer pairs for microsatellites loci developed in A. heterocarpa and A. sylvestris

Locus	Primer sequences (5′ → 3′)		Motif	Range (bp)	Label	Multiplex	GenBank
An23	F:	GAAACAAAATCAAATAGTAATCGCA	AC	75–95	PET	1	MZ065562
	R:	AAATGTAATCTGCACGCGGT
An35	F:	GGTTGCAACTCAGATGCTGG	GT	175–189	FAM	1	MZ065564
	R:	ACCCTGTGCGTATTGCCTAC
An69	F:	AGCAAGTGAGGCAAGACCAT	AG	144–176	FAM	2	MZ065568
	R:	CAAACTCTCCTTCACCCCAA
An71	F:	GAGCATCCTCGAAGAGATCAA	AG	216–262	FAM	1,2	MZ065569
	R:	GGGACCACTCAGAATTGGAA
An72	F:	TTGGATTCAGGAGAGGACCA	GAT	66–114	FAM	2	MZ065570
	R:	CTTCTCCCAACCAGGATCAG
An74	F:	GCATTGTAGCCTACTGGAGGG	GT	80–134	VIC	2	MZ065571
	R:	GGCAACAGAGTAGACCTTAACCTG
An89	F:	GCAACCGCAGCTCATTTTAT	GT	88–106	PET	2	MZ065572
	R:	AAACAAAGGACCAGCCGAC
An91	F:	CTCGCGTTGACAGGGTTTA	GT	129–143	PET	2	MZ065573
	R:	TCGAGTTCTAGTTGGACAGGG
An93	F:	GGCAGCTAAGTGAAGCAAAA	AC	170–190	VIC	2	MZ065574
	R:	TGCGCATGTTACTAAGGCTG
S216	F:	TCTCTGAGTATATATTTTTGGGTGTG	CT	167–179	PET	1	MZ065559
	R:	TCGCAAAATACCCTCATCTC
An11	F:	GGGACATTCAACACAACATCA	AG	96–128	VIC	1	MZ065561
	R:	CTCTTTCTTGCACCCTTCCA
An39	F:	TTGGCTGCACTTACATTTGC	CT	212–284	VIC	1	MZ065565
	R:	ATGATAAACCCGGTTGCTTG
An68	F:	TCCAAAATGCACAGATCCAG	AG	129–171	NED	2	MZ065567
	R:	CTCGTCGAGTTCTACGCTCC
An32	F:	TGGGTTCATCAAGATTCAAGG	AG	118–174	NED	1	MZ065563
	R:	GTGTGGTCACTGCAAGCATC
An41	F:	GGGAAACTGAATTAACCGAGC	AG	263–324	PET	1	MZ065566
	R:	CCACTTGTGGTCTCTAACATGG
S37	F:	CAAAAGTGGATACTAGTTGTGTG	CT	192–224	NED	1	MZ065560
	R:	GCTCTACCATTAGCAAAACC

Label: fluorophore dye labeling at 5′ end of forward primers

F, forward primer sequence; R, reverse primer sequences; bp, base pair

Multiplexed PCR were achieved using Qiagen Microsatellite Type-It master mix (Qiagen, Hilden, Germany) and cycling conditions were: 15 min at 95 °C; 30 cycles of 30 s at 94 °C, 60 s at 56 °C, 45 s at 72 °C; and 60 °C for 30 min. The fluorescent‐labeled PCR products were run on an ABI 3730 DNA Analyzer (Applied Biosystems, Waltham, MA, USA) at the Genome Transcriptome Facility of Bordeaux. Genotype calling was carried out manually using GeneMapper Software (Applied Biosystems, Waltham, MA, USA).

Results and discussion

Overall, the developed microsatellite loci were highly polymorphic with a number of alleles per locus ranged from 2 to 21 with a mean of 8 alleles per locus in the five studied A. heterocarpa and A. sylvestris populations (Table 2). At the population level, the observed heterozygosity ranged from 0.54 to 0.60, and the expected heterozygosity ranged from 0.66 to 0.74. Four out of the 16 loci showed significant deviations from Hardy–Weinberg equilibrium (HWE) in more than one population after correcting for multiple testing (P < 0.001; Table 2).

Table 2

Genetic diversity characteristics of 16 microsatellites loci for A. heterocarpa and A. sylvestris populations

	A. heterocarpa—Rézé (N = 40)				A. heterocarpa—Ile-St-Georges (N = 38)				A. sylvestris—Aurice (N = 38)				A. sylvestris—La Brède (N = 30)				A. sylvestris—Le Bonhomme (N = 30)
Loci	NA	HE	HO	FIS	NA	HE	HO	FIS	NA	HE	HO	FIS	NA	HE	HO	FIS	NA	HE	HO	FIS
An23	6	0.75	0.56	0.25	10	0.82	0.68	0.17	5	0.75	0.76	− 0.02	7	0.70	0.50	0.29	4	0.58	0.13	0.79a
An35	5	0.67	0.58	0.14	4	0.53	0.55	− 0.05	5	0.54	0.32	0.42	6	0.65	0.70	− 0.08	4	0.56	0.57	− 0.01
An69	3	0.33	0.30	0.09	9	0.83	0.79	0.05	15	0.88	0.76	0.13	5	0.46	0.33	0.29	6	0.47	0.40	0.15
An71	9	0.76	0.32	0.58a	18	0.91	0.58	0.36a	14	0.88	0.31	0.65a	13	0.89	0.72	0.19	15	0.88	0.59	0.34a
An72	8	0.80	0.69	0.13	8	0.77	0.76	0.01	11	0.79	0.85	− 0.07	8	0.85	0.83	0.02	13	0.86	0.87	− 0.01
An74	2	0.43	0.10	0.77	4	0.45	0.23	0.49a	4	0.55	0.18	0.67a	4	0.23	0.18	0.24	2	0.04	0.04	0.00
An89	6	0.74	0.71	0.05	5	0.53	0.27	0.49a	8	0.82	0.33	0.60a	6	0.71	0.44	0.39	6	0.65	0.53	0.18
An91	6	0.59	0.53	0.12	6	0.51	0.45	0.12	6	0.61	0.58	0.05	6	0.62	0.73	− 0.18	6	0.74	0.69	0.07
An93	5	0.54	0.29	0.47	7	0.69	0.32	0.54a	7	0.77	0.21	0.74a	7	0.79	0.48	0.40a	10	0.89	0.52	0.42a
S216	4	0.58	0.58	0.01	4	0.55	0.50	0.10	4	0.64	0.61	0.05	5	0.70	0.43	0.39	3	0.50	0.53	− 0.07
An11	8	0.77	0.75	0.03	9	0.87	0.81	0.07	12	0.84	0.75	0.11	11	0.81	0.67	0.18	8	0.79	0.72	0.09
An39	12	0.82	0.88	− 0.07	21	0.92	0.92	0.00	11	0.76	0.69	0.08	12	0.90	0.76	0.16	7	0.78	0.71	0.09
An68	8	0.77	0.72	0.07	11	0.83	0.79	0.05	15	0.83	0.86	− 0.03	8	0.77	0.83	− 0.09	8	0.74	0.66	0.11
An32	9	0.68	0.74	− 0.09	4	0.29	0.19	0.34	8	0.76	0.50	0.34a	6	0.65	0.69	− 0.06	8	0.82	0.60	0.27
An41	12	0.89	0.86	0.04	19	0.91	0.66	0.28a	–	–	–	–	10	0.86	0.88	− 0.03	–	–	–	–
S37	8	0.84	0.57	0.33	8	0.77	0.24	0.69a	–	–	–	–	6	0.74	0.48	0.36	–	–	–	–
Mean	6.9	0.69	0.57	0.18	9.2	0.70	0.55	0.23	8.9	0.74	0.55	0.27	7.5	0.71	0.60	0.15	7.1	0.66	0.54	0.17

N, number of samples; NA, number of alleles (NA); HO, observed heterozygosity; HE, expected heterozygosity; FIS, inbreeding coefficient; –, unavailable data

aSignificant deficit in heterozygotes (P < 0.01)

The cross‐species transferability and polymorphism of the developed SSR markers were further tested in the two other congeneric species (Table 3). In A. razulii, only 7 out of the 16 loci amplified consistently and were polymorphic. On the 12 markers tested in A. archangelica, 7 amplified successfully and were polymorphic. Note that additional markers amplified inconsistently, suggesting presences of null alleles or limited transferability (3 markers for A. razulii and 4 for A. archangelica, Table 3).

Table 3

Number of alleles and allele range of microsatellite loci in A. razulii and A. archangelica

Loci	A. razulii (N = 50)		A. archangelica (N = 3)
	NA	Range (bp)	NA	Range (bp)
An23	0	0	1	79
An35	3	177–185	3	169–179
An69	7	140–152	2	142–150
An71	5	212–220	2	216–230 (2)
An72	8	63–96	2	69–72
An74	0	0	1	82 (1)
An89	5	92–102 (20)	1	92 (1)
An91	9	125–143	2	133–135 (1)
An93	0	0	2	170–178
S216	0	0	2	169–179
An11	0	0	2	132–138
An39	2	220–224	3	188–232
An68	0	0	–	–
An32	12	124–156	–	–
An41	2	267–271 (12)	–	–
S37	3	151–171 (3)	–	–

Values in () indicates the number of individuals with at least one PCR amplified signal

N, number of samples; NA, number of alleles; bp, base pairs; –, unavailable data

These new polymorphic markers will allow investigating population genetic structure, reproductive system, and potential hybridization within the Angelica species complex. Moreover, as numerous Angelica species are traditional medicine herbs all over the Eurasia continent, including A. sylvestris and A. archangelica [7], these microsatellite markers complement the molecular markers previously developed for other congeneric species of particular pharmacological interest [8-10].

Limitations

Deviation from HWE: Loci showing marked deficits in heterozygotes in most populations are likely affected by null alleles. However, this should be further investigate with larger sample size because the restricted sampled size of the present study hampered additional statistical test. In addition, more biological insight to support the fact that the species is expect to follow HWE is needed because population substructure or hybridization are likely affecting Angelica population and would resulted in departure from HWE.

Sample size limitation for A. archangelica: only 3 individuals have been included to test for transferability and polymorphism for this species. Amplification success and polymorphism should be assessed in a larger sample size to confirm monomorphic loci.