SRAP markers for genetic diversity assessment of lavender (Lavandula angustifolia mill.) varieties and breeding lines

Abstract

Lavender (Lavandula angustifolia Mill.) is an essential oil-bearing and medicinal plant of great economic and social importance to Bulgaria, which is the world largest producer of lavender oil nowadays. Currently, the industrial cultivation of lavender in Bulgaria employs up to seven varieties established during the last century. Despite the growing industrial lavender cultivation, few molecular markers have been applied for characterization of lavender genetic resources and breeding. The present study employed sequence-related amplified polymorphism (SRAP) markers to characterize the genetic resources and genetic diversity within and between two groups of ten Bulgarian and five foreign lavender varieties and breeding lines. The data generated following analysis with 51 SRAP primer pairs showed amplification of a high number (4697) and ratio (77.2%) of polymorphic SRAP fragments resulting in relatively high value of the polymorphism information content, with an average of 0.27 ± 0.03 for the used primer pairs. Analysis of molecular variance of the SRAP data further revealed high level of genetic diversity (96.6% of the total variations) within the analyzed groups of Bulgarian and foreign varieties and rather low diversity (3.4% of total variations) between the two groups. The possibilities for further applications of SRAP markers in lavender breeding and cultivation are discussed.

Keywords:

• SRAP markers
• lavender
• Lavandula angustifolia
• varieties
• genetic diversity
• genetic resources

Introduction

Lavender (Lavandula angustifolia Mill.) is an important oil-bearing plant belonging to the mint family (Lamiaceae), which is cultivated as medicinal and garden plant, and for essential oil production worldwide [1]. Large and increasing volumes of lavender essential oil are applied in a wide range of home and personal care products, perfumery, aroma-therapy and alternative medicine [2]. During the last decade Bulgaria has emerged as the world’s largest producer and exporter of lavender essential oil [2, 3]. This was related to expansion of the lavender growing areas, including establishment of new lavender fields in the country regions with diverse soil and climatic conditions, which have been traditionally used for growing other crops. All these increased the demand for better characterization and utilization of the existing lavender genetic resources and initiation of a lavender breeding programme supported by marker-assisted selection for development of new varieties [3].

So far, molecular genetic research related to Lavandula species has been focused mainly on cloning and characterization of terpene synthase genes involved in biosynthesis of the essential oil compounds [4–9] and draft genome sequence [10]. However, molecular marker development and application for characterization of the genetic resources and biodiversity of lavender and other Lavandula species has been restricted to a few reported studies. Curto et al. developed molecular markers from single-copy nuclear genes for phylogenetic analyses at the species level in the Lamiaceae family [11] and Hind et al. were able to discriminate the cultivated L. latifolia from that of L. angustifolia and L. x intermedia using DNA barcoding [12]. Hnia et al. reported characterization of the genetic diversity of Tunisian L. multifida populations using RAPD markers [13]. Adal et al. developed the first set of SSR markers for Lavandula sp. based on sequences from the L. angustifolia and L. x intermedia EST databases [14]. However, the efficiency of their application in the characterization of closely related sets of lavender genetic resources remains to be evaluated. Thus, in spite of the increased interest on lavender cultivation and essential oil production, the genetic diversity of the employed lavender genetic resources remains poorly studied and no reliable molecular marker methods are currently applied for testing the genetic authenticity and homogeneity of the industrially used lavender planting material.

Sequence-related amplified polymorphism (SRAP) markers developed by Li and Quiros [15] have been widely applied in a number of genetic studies due to their simplicity, reliability, effectiveness and genome-wide coverage [16, 17]. Although the results from applications of SRAP markers in taxonomic studies could be influenced by anthropogenic or other types of selective pressure [17], during the last decade SRAP markers have been successfully applied in a number of studies on characterization of plant genetic resources and natural populations, including species from the Lamiaceae family outside the genus Lavandula [18–23].

In this study, we report SRAP markers for characterization of the genetic diversity in a set of lavender genetic resources including seven Bulgarian industrially cultivated lavender essential oil varieties, three breeding essential oil lines and five lavender cultivars grown in other countries. The potential of SRAP markers in lavender breeding and cultivation are discussed.

Materials and methods

Plant material

The lavender genotypes used in the present study included (a) the seven Bulgarian essential oil varieties cultivated currently: ‘Yubileina’, ‘Hemus’, ‘Hebar’, ‘Raya’, ‘Sevtopolis’, ‘Druzhba’ and ‘Karlovo’; (b) three Bulgarian superior essential oil breeding lines: ‘D1’, ‘S1’, ‘Y1’ and (c) five lavender varieties grown in other countries: ‘Blue ice’, ‘Hidcote’, ‘Mellisa Lilac’, ‘Munstead’ and ‘Rosea’. The Bulgarian varieties and breeding lines were maintained in the genetic resources collections of the Institute of Rose and Aromatic Plants, Agricultural Academy, Kazanlak, Bulgaria, and ZP Janeta Staneva Nursery, Kazanlak, Bulgaria. The rest of the lavender varieties were purchased from Peter Beales Roses Ltd., Norwich, UK. Leaves from single lavender bushes were collected, immediately frozen in liquid nitrogen and stored at –80 °C until DNA extraction.

DNA extraction

Lavender leaves were ground into fine powder using liquid nitrogen frozen stainless steel jars and TissueLyser II (QIAGEN GmbH), at 30 Hz for 2 min. Genomic DNA was extracted using a CTAB protocol [24]. The purified total DNA was quantified spectrophotometrically using Nanodrop 2000 (Thermo Scientific), diluted to 25 ng/μL and stored at –20 °C until use.

PCR amplification and fragment analysis

The SRAP primers used in the study were designed according to Li and Quiros [15] and are shown in Table 1. The forward ‘ME’ primers were 5′ end labeled with FAM dye. PCR reactions were carried out in a volume of 25 μL containing: 2 μL of DNA template (25 ng/μL), 0.1 μmol/L of each forward and reverse primer, 12.5 μL of MyTaq HS DNA polymerase (Bioline Ltd., UK). Samples were PCR amplified using the following thermal profile: 5 min at 94 °C; 3× cycles of 1 min at 94 °C, 1 min at 35 °C and 1 min at 72 °C; 35× cycles of 1 min at 94 °C, 1 min at 50 °C and 1 min at 72 °C and a final elongation step of 3 min at 72 °C. PCR products were diluted tenfold. For electrophoresis, 1 μL of the diluted PCR amplified product was mixed with 0.5 μL of the GeneScan 600 LIZ Size Standard and 9 μL of Hi-Di Formamide (Thermo Fisher Scientific Inc.). The mixture was denatured and analyzed on an Applied Biosystems 3130 Genetic Analyzer using 3130 POP-7 Polymer and 36 cm capillary array (Thermo Fisher Scientific Inc.).

Table 1. Sequences of SRAP primers used in the analysis.

Forward primer (5′–3′)		Reverse primer (5′–3′)
ME1	tgagtccaaaccggaaa	EM1	gactgcgtacgaattaaa
ME2	tgagtccaaaccggagc	EM2	gactgcgtacgaatttgc
ME3	tgagtccaaaccggtaa	EM3	gactgcgtacgaattatt
ME4	tgagtccaaaccggacc	EM4	gactgcgtacgaattata
ME5	tgagtccaaaccggaag	EM5	gactgcgtacgaattcaa
ME6	tgagtccaaaccggaga	EM6	gactgcgtacgaattttt
ME7	tgagtccaaaccggtcc	EM7	gactgcgtacgaattagc
ME8	tgagtccaaaccggctg	EM8	gactgcgtacgaattcac
ME9	tgagtccaaaccggttc	EM9	gactgcgtacgaattaat
ME10	tgagtccaaaccggtga	EM10	gactgcgtacgaatt tca

Data analysis

SRAP fragment analysis was performed with the GeneMapper Analysis Software v4.0 (Thermo Fisher Scientific Inc.). The threshold for peaks assignation was set to 350 relative fluorescence units. Fragment analysis was carried out for fragment sizes in the range of 50–950 bp. All clearly distinguished SRAP fragments were scored as (1) present or (0) absent, and the obtained data were used for construction of a SRAP data matrix. The polymorphism information content (PIC) values were calculated using the ‘GDdom’ online tool [25]. Phylogenetic analysis and analysis of molecular variance (AMOVA) analysis were performed with the FAMD software [26].

Results and discussion

A total of 51 SRAP primer pairs were selected after preliminary testing of all possible 100 EM1/EM10 and ME1/ME10 primer combinations (Table 1). The primer pairs were selected for their ability to prime PCR amplification from DNA of lavender var. Druzhba and after assessment of the SRAP peaks quality in the obtained electropherograms. The selected primer pairs were further used for PCR amplifications of DNA isolated from seven Bulgarian essential oil lavender varieties, three breeding lines and five foreign lavender varieties. The results from the comparative analysis of the amplified SRAP fragments across all studies genotypes are summarized in Table 2. The tested 51 primer pairs amplified a total of 6088 different SRAP fragments with sizes between 50 and 950 bp, represented by well-resolved peaks on the electropherograms. Of them, 4697 fragments showed polymorphisms after SRAP analysis of the studied genotypes. The data analysis further showed that the number of the amplified SRAP fragments varied significantly among the tested primer pairs and studied genotypes, ranging between 38 and 220 fragments per primer pair (Table 2). Accordingly, 29%–97% of the amplified fragments appeared to be polymorphic in the studied genotypes pool, as the average percentage of polymorphic fragments was 74.3 ± 13.3. The average number of the polymorphic SRAP fragments per one genotype also varied significantly depending on the primer pair, ranging from 6.3 ± 1.6 polymorphic fragments per genotype for primer pair ЕМ7/МЕ9 to 52.6 ± 13.3 polymorphic fragments for primer pair ЕМ2/МЕ10. The average number of polymorphic SRAP fragments amplified by one primer pair per one genotype for the entire study was 25.5 ± 12.8. The calculated PIC values were relatively high for all primer pairs, varying from 0.22 to 0.39, with an average of 0.27 ± 0.03 for the study. These data and the calculated PIC values demonstrated a relatively high level of information of the SRAP markers, with a large number of loci producing unique fingerprints.

Table 2. Summary of SRAP fragments data following SRAP analysis of all studied genotypes.

Primer pair	SRAP fragments					Primer pair	SRAP fragments
	T	TP	PPF (%)	APFG	PIC		T	TP	PPF (%)	APFG	PIC
ЕМ1_МЕ1	93	78	84%	15.6 ± 6.3	0.26	ЕМ6_МЕ1	48	34	71%	10.3 ± 3.2	0.30
ЕМ1_МЕ2	70	42	60%	14.2 ± 3.7	0.29	ЕМ6_МЕ4	173	151	87%	26.4 ± 4.3	0.25
ЕМ1_МЕ3	135	115	85%	26.9 ± 6.2	0.25	ЕМ6_МЕ5	78	59	76%	46.6 ± 7.5	0.33
ЕМ1_МЕ6	115	91	79%	27.7 ± 6.5	0.30	ЕМ6_МЕ6	134	109	81%	23.8 ± 4.6	0.26
ЕМ1_МЕ10	98	71	72%	15.9 ± 9.9	0.25	ЕМ6_МЕ10	201	180	90%	44.0 ± 10.8	0.27
ЕМ2_МЕ3	220	156	71%	50.2 ± 11.3	0.28	ЕМ7_МЕ5	101	74	73%	21.5 ± 6.0	0.25
ЕМ2_МЕ7	164	108	66%	20.6 ± 8.8	0.22	ЕМ7_МЕ9	49	14	29%	6.3 ± 1.6	0.39
ЕМ2_МЕ9	136	104	76%	44.4 ± 5.8	0.26	ЕМ7_МЕ10	205	182	89%	38.1 ± 9.1	0.23
ЕМ2_МЕ10	209	185	89%	52.6 ± 13.3	0.26	ЕМ8_МЕ1	156	127	81%	39.5 ± 7.3	0.24
ЕМ3_МЕ1	164	138	84%	34.1 ± 7.9	0.27	ЕМ8_МЕ2	104	87	84%	31.8 ± 4.2	0.22
ЕМ3_МЕ3	112	90	80%	18.9 ± 4.7	0.25	ЕМ8_МЕ6	38	29	76%	13.5 ± 3.1	0.31
ЕМ3_МЕ6	62	36	58%	8.5 ± 4.8	0.26	EM8_ME7	99	82	83%	26.5 ± 6.0	0.27
ЕМ3_МЕ10	109	93	85%	18.6 ± 4.5	0.24	ЕМ8_МЕ8	94	63	67%	20.4 ± 4.6	0.31
ЕМ4_МЕ1	213	207	97%	47.6 ± 15.5	0.26	ЕМ8_МЕ10	93	77	83%	20.9 ± 5.6	0.25
ЕМ4_МЕ3	154	119	77%	26.1 ± 9.4	0.27	ЕМ9_МЕ2	107	92	86%	23.6 ± 6.8	0.29
ЕМ4_МЕ5	76	47	62%	13.1 ± 2.4	0.25	ЕМ9_МЕ4	86	57	66%	14.6 ± 3.4	0.26
ЕМ4_МЕ6	99	78	79%	21.8 ± 5.1	0.25	ЕМ9_МЕ5	151	117	77%	29.6 ± 7.6	0.26
ЕМ4_МЕ10	82	41	50%	11.4 ± 2.1	0.26	ЕМ9_МЕ9	110	84	76%	21.3 ± 5.6	0.25
EM5_ME1	95	64	67%	26.4 ± 2.5	0.30	ЕМ9_МЕ10	165	126	76%	28.3 ± 7.2	0.24
ЕМ5_МЕ2	88	40	45%	18.0 ± 2.7	0.28	ЕМ10_МЕ1	87	66	76%	23.6 ± 11.1	0.32
ЕМ5_МЕ3	130	114	88%	25.4 ± 6.0	0.25	ЕМ10_МЕ5	107	82	77%	25.3 ± 5.6	0.26
ЕМ5_МЕ4	184	144	78%	36.6 ± 7.2	0.26	ЕМ10_МЕ8	151	134	89%	29.6 ± 5.3	0.24
ЕМ5_МЕ5	87	40	46%	16.6 ± 2.8	0.29	ЕМ10_МЕ9	149	126	85%	15.4 ± 4.2	0.24
ЕМ5_МЕ6	76	47	62%	15.9 ± 4.6	0.30	ЕМ10_МЕ10	117	92	79%	27.4 ± 5.7	0.27
ЕМ5_МЕ7	82	46	56%	20.5 ± 9.8	0.33	Total	6088	4697	77.2%
ЕМ5_МЕ8	100	58	58%	19.9 ± 4.0	0.29	Average	119.4 ± 44.9	92.1 ± 44.5	74.3% ± 13.3%	25.5 ± 12.8	0.27 ± 0.03
ЕМ5_МЕ10	132	101	77%	27.4 ± 7.4	0.27

T, total number of different SRAP fragments observed for the tested primer pair in all analyzed genotypes; TP, total number of different polymorphic SRAP fragments observed for the tested primer pair in all analyzed genotypes; PPF, percent of the polymorphic fragments for the tested primer pair; APFG, average number of polymorphic fragments for the tested primer pair observed for one genotype; PIC, polymorphism information content.

The obtained SRAP data were further used to determine the genetic relatedness among the studied lavender genotypes. The genetic similarity estimates based on the Jaccard similarity coefficients ranged from 0.22 to 0.395 for the tested genotypes and the overall similarity for the tested genotypes was 0.30 ± 0.04. The constructed phylogenic tree shows that all foreign varieties form a distinct cluster and were embedded among the clusters formed by the Bulgarian essential oil varieties and breeding lines suggesting relatively higher level of genetic diversity within the Bulgarian varieties (Figure 1). The AМOVA results of the SRAP data for the two groups of Bulgarian essential oil and foreign varieties provided further support for this. The AМOVA revealed that only 3.4% of the overall SRAP variations in the study are related to differences between the two groups of Bulgarian essential oil and foreign lavender varieties, as the remaining 96.6% of the variations are due to the genetic diversity within the studied groups. This supports our earlier results from the analysis of flower volatiles composition, showing high level of variation in volatiles related to the essential oil of Bulgarian lavender varieties [27]. Although the present study involves a restricted number of lavender varieties and breeding lines, the obtained results suggest that the level of genetic diversity of Bulgarian essential oil varieties and breeding lines is relatively high and the genetic resources available in the country (analyzed here and an additional set of breeding lines) could be used as a basis for further lavender breeding without an immediate need for expansion of the genetic resources pool.

Figure 1. Neighbour-joining phylogenetic tree constructed from SRAP data following analysis of Bulgarian industrially cultivated lavender essential oil varieties and breeding lines, and foreign lavender cultivars. The names of the analyzed varieties and breeding lines are shown on the right. The cluster formed by foreign varieties is designated with the abbreviation ‘FV’.

The results of the present study clearly demonstrate that the SRAP markers are a powerful tool for studying genetic diversity in lavender. SRAP markers offer a non-specific genome wide coverage which could well complement the use of other markers related to single or small number of loci, e.g. SSR and SNP markers. The reported SRAP markers could be successfully applied in different areas of lavender research and cultivation. The SRAP analysis of genetic diversity could be readily applied for (re-)evaluation of the full set of lavender genetic resources including a number of breeding lines developed during the last five decades. The large number of polymorphic SRAP fragments/markers described in the study suggests they could be used as affordable and efficient tool for development of the first lavender genetic map and initiation of breeding programmes based on marker-assisted selection. One of the current problems related to the market of lavender planting material for industrial cultivation is the lack of efficient routine methods for quality control. The planting of homogenous vegetatively propagated lavender planting material is essential for the high yield and quality of the produced essential oil [3]. The high number of polymorphic SRAP fragments generated by a single primer pair, accounting to an average of 25.5 ± 12.8 (Table 2), suggests that SRAP markers could offer an affordable method for routine quality testing of the origin and homogeneity of the vegetatively propagated planting material used for establishment of new plantations. Indeed, the analysis of vegetatively propagated lavender plantlets using one or few SRAP primers pairs could be sufficient to confirm or reject their declared origin via comparison of their SRAP pattern with those of a reference sample from the corresponding lavender variety. Our preliminary study involving SRAP analysis with three primer pairs of 24 vegetatively propagated plants from var. Hemus shows that all of them display one and the same pattern of SRAP fragments confirming the reproducibility of the results from SRAP marker application, which is mandatory for development of a routine testing procedure.

Conclusions

The overall results from the present study demonstrate that SRAP markers are a powerful and efficient tool to study the lavender genetic diversity and exhibit high potential for a wide range for further applications in lavender breeding and cultivation including: characterization of lavender genetic resources pool, characterization of lavender segregating populations and genetic mapping studies and marker-assisted selection. Additionally, the tested SRAP markers could be used as a basis for development of an affordable and routine procedure for quality testing of vegetatively propagated lavender planting material. The SRAP data analysis demonstrates a relatively high level of genetic diversity within the tested group of Bulgarian industrially cultivated lavender essential oil varieties and breeding lines, suggesting the currently available genetic resources pool is a sufficient basis for initiation of a long-term lavender breeding programme.

Disclosure statement

The authors report no conflict of interest

Additional information

Funding

This work was supported by the National Science Fund of Bulgaria under Grant number KP-06-M26/1 from 30.11.2018.