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Annals of Botany 92: 749-755, 2003
© 2003 Annals of Botany Company

Association Between Chloroplast DNA and Mitochondrial DNA Haplotypes in Prunus spinosa L. (Rosaceae) Populations across Europe

APARAJITA MOHANTY{dagger},1, JUAN PEDRO MARTÍN*,1, LUIS MIGUEL GONZÁLEZ2 and ITZIAR AGUINAGALDE1

1 Departamento de Biología Vegetal, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Ciudad Universitaria, 28040 Madrid, Spain and 2 Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Centro Nacional de Microbiología, 28220 Majadahonda, Madrid, Spain

* For correspondence. E-mail jpmartin{at}bio.etsia.upm.es
{dagger} Present address: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, 110067 New Delhi, India

Received: 4 April 2003; Returned for revision: 11 July 2003; Accepted: 9 August 2003    Published electronically: 8 October 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) were studied in 24 populations of Prunus spinosa sampled across Europe. The cpDNA and mtDNA fragments were amplified using universal primers and subsequently digested with restriction enzymes to obtain the polymorphisms. Combinations of all the polymorphisms resulted in 33 cpDNA haplotypes and two mtDNA haplotypes. Strict association between the cpDNA haplotypes and the mtDNA haplotypes was detected in most cases, indicating conjoint inheritance of the two genomes. The most frequent and abundant cpDNA haplotype (C20; frequency, 51 %) is always associated with the more frequent and abundant mtDNA haplotype (M1; frequency, 84 %). All but two of the cpDNA haplotypes associated with the less frequent mtDNA haplotype (M2) are private haplotypes. These private haplotypes are phylogenetically related but geographically unrelated. They form a separate cluster on the minimum-length spanning tree.

Key words: cpDNA haplotypes, mtDNA haplotypes, PCR–RFLP, phylogenetic relationship, Prunus spinosa L.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
In most angiosperms, chloroplast and mitochondrial genomes are inherited maternally (Reboud and Zeyl, 1994) and, therefore, are expected to remain completely linked (Schnabel and Asmussen, 1989). There are studies where chloroplast and mitochondrial genomes have been investigated simultaneously (Berthou et al., 1983; Laurent et al., 1993; Shu et al., 1993; Tsunewaki, 1993; Lee et al., 1994; Caha et al., 1998). However, there are few studies describing the association between the two organelle genomes in angiosperms (e.g. Dumolin-Lapègue et al., 1998; Desplanque et al., 2000; Olson and McCauley, 2000; Belahbib et al., 2001).

The chloroplast genome is well characterized and structurally very stable (Clegg et al., 1994). The variations detected in chloroplast DNA (cpDNA) using the PCR–RFLP (polymerase chain reaction–restriction fragment length polymorphism) technique are useful for population genetic studies at both the interspecific and the intraspecific level (McCauley, 1995; Demesure et al., 1996; El Mousadik and Petit, 1996; Dumolin-Lapègue et al., 1997a; King and Ferris, 1998; Newton et al., 1999; Dutech et al., 2000; Fineschi et al., 2000). In some studies, mitochondrial DNA (mtDNA) variations have been very informative. mtDNA polymorphisms are geographically structured at the local scale in Thymus vulgaris (Manicacci et al., 1996), and at the regional scale in Hevea brasiliensis (Luo et al., 1995), Fagus crenata (Tomaru et al., 1998) and Beta vulgaris ssp. maritima (Desplanque et al., 2000). In Theobroma cacao and Glycine soja, mitochondrial haplotypes have widespread geographic distribution, but do not present any geographic structuring (Laurent et al., 1993; Tozuka et al., 1998).

The present investigation was carried out on Prunus spinosa L., a wild shrub of European deciduous forests that is grown as a hedge plant. A preliminary study of seven populations of P. spinosa revealed high cpDNA diversity (Mohanty et al., 2000). In another study, three regions of cpDNA (approx. 8250 bp) were analysed for a population genetic analysis of 25 populations from European deciduous forests. The study revealed low genetic differentiation among populations and an absence of phylogeographic structure (Mohanty et al., 2002). In the present study, 24 populations were analysed with an additional region of cpDNA of approx. 3800 bp and three regions of mtDNA. The main objectives were to study the extent of mtDNA variations and phylogenetic and geographic relationships between cpDNA and mtDNA haplotypes in populations of P. spinosa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material
Twenty-four wild populations, which included 157 individuals of P. spinosa, were sampled from deciduous forests across Europe (Table 1). Only 157 of the 203 samples from a previous study (Mohanty et al., 2002) were analysed by studying an additional region of cpDNA and three regions of mtDNA. All 203 individuals could not be included in the present study as CD fragments could only be amplified in 157 samples.


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  		Table 1. Populations, origin and numbers of Prunus spinosa individuals studied
 
DNA extraction, amplification and digestion
DNA was extracted from frozen leaves following the protocol of Torres et al. (1993), and then standardized (4 ng µl–1). The final results were interpreted by combining data obtained by using four cpDNA primer pairs, HK, K1K2, VL (previous study; Mohanty et al., 2002) and CD (present study), and three mtDNA primer pairs, nad1/B-C, nad4/2–3 and rps14-cob (present study). The four cpDNA primer pairs and three mtDNA primer pairs are described by Demesure et al. (1995) and Dumolin-Lapègue et al. (1997b). The details of amplification with the cpDNA primers are described by Mohanty et al. (2000). The amplifications using mtDNA primers were performed in 30 µl of reaction mixture, consisting of 0·2 µM of each primer, 200 µM of each of the four dNTP, 2 mM MgCl2, 0·5–1·0 U EcoTaq DNA polymerase in the buffer provided by the manufacturers of the enzyme (ECOGEN, S.R.L., Barcelona, Spain), and 12 ng of genomic DNA. The PCR amplifications were carried out in a PTC-100 thermal cycler (MJ Research Inc., Watertown, MA, USA) with a heated lid, using an initial cycle of 4 min at 94 °C, followed by 30 cycles of 45 s at 94 °C, 45 s at 55 °C and 3 min (for nad1/B-C and rps14-cob) or 4 min 30 s (for nad4/2–3) at 72 °C, and finally a 10 min extension at 72 °C.

Amplified products, obtained using cpDNA and mtDNA primers, were digested with the restriction enzymes HinfI and TaqI (Amersham Corporation, Amersham, UK). In addition, AluI was used with the primer pairs VL, nad1/B-C and rps14-cob. The digestion conditions were as detailed by Mohanty et al. (2000). Restriction fragments were separated on 2·6 % agarose gels in Tris-borate-EDTA buffer (x1), run at 3 V cm–1 for 4 h, stained with ethidium bromide and visualized under UV light. The size of the polymorphic bands was analysed using Kodak Digital Science 1D Image Analysis software, and a 50 bp ladder (Pharmacia Biotech, Brussels, Belgium) was used as a molecular size marker.

DNA sequencing
The two detected mtDNA haplotypes were sequenced in order to determine the exact nature of mutation. The amplified fragments obtained using the mtDNA primer pair rps14-cob, for two individuals of each mtDNA haplotype, were cloned into plasmids of pGEM-T (Promega Corporation, Madison, WI, USA). Automated sequencing of the recombinant plasmids was performed using fluorescence-base labelling with the ABI PRISM system (Perkin-Elmer Corporation, Norwalk, CT, USA). The sequencing strategy involved the use of the plasmid-specific SP6 and T7 primers (synthesized by Pharmacia Biotech, Brussels, Belgium), which are located on the vector from both ends of the inserts. The sequencing was performed in both directions until sequences from the two ends overlapped. Analysis of DNA sequences was carried out with the SeqMan and Mapdraw Lasergene programs (DNASTAR Inc., Madison, WI, USA).

Nucleotide sequence data reported in this paper have been submitted to the GenBank, EMBL and DDBJ databases under accession number AF464899 for mtDNA haplotype 1 (M1), and accession number AF464900 for mtDNA haplotype 2 (M2).

Analysis of data
The program HAPLONST (Pons and Petit, 1996) was used to calculate the parameters of cpDNA diversity (HT, total diversity; HS, average intrapopulation diversity; GST, level of population subdivision using unordered alleles; and NST, level of population subdivision using ordered alleles).

The number of mutational differences between haplotypes of wild populations was calculated to produce a minimum-length spanning tree of haplotypes, using the program NTSYS-pc (Rohlf, 1992). The procedure is used to connect points (haplotypes) by direct links having the smallest possible total length (Prim, 1957). Minimum spanning networks are alternatives to Wagner parsimony trees, and convey better the connections between haplotypes (Excoffier and Smouse, 1994).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
cpDNA haplotypes
PCR–RFLP of cpDNA fragments obtained using the primer pair CD, resulted in eight polymorphic fragments (Table 2). The mutations detected using the primer pairs HK, K1K2 and VL (Mohanty et al., 2002) and CD (present study) were combined to define the cpDNA haplotypes C1–C33 (Table 3). Of the 33 cpDNA haplotypes distinguished, nine were shared by two or more populations and the rest (24) were private (as denominated by Slatkin, 1985) or unique haplotypes (Table 3). C20 was the most frequent cpDNA haplotype (frequency, 51 %), being represented in 80 of the 157 individuals studied (Table 3).


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  		Table 2. Major patterns and variants (in bp) in the polymorphic fragments obtained with the primer pair CD and two restriction enzymes (HinfI and TaqI) in Prunus spinosa
 

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  		Table 3. Distribution of cpDNA and associated mtDNA haplotypes in the 24 populations of Prunus spinosa studied (see population code in Table 1)
 
Analysis of diversity using the HAPLONST program revealed high total diversity (HT, 0·76), of which a major portion was located within populations (HS, 0·50). The level of population subdivision using unordered and ordered alleles was GST, 0·34 and NST, 0·46, respectively. The difference between NST and GST was non-significant (U-test, 0·74; P = 0·05; Pons and Petit, 1996).

mtDNA haplotypes
Of the three mtDNA primer pairs used, nad1/B-C and rps14/cob showed good amplification, the sizes of the amplified fragments being approx. 1300 bp and 1280 bp, respectively. There was no amplification with nad4/2–3. The restriction digestions of the amplified fragments with AluI, HinfI and TaqI revealed no polymorphisms in the amplified fragment of nad1/B-C. The combination rps14/cob-TaqI resulted in two mtDNA haplotypes (M1 and M2). Haplotype M1 showed a restricted fragment of approx. 210 bp, and haplotype M2 a 170 bp fragment. Although the mutation appeared to be an indel of approx. 40 bp on the agarose gel, it was actually a restriction site mutation (revealed on sequencing), which appeared as an indel mutation because of the 40 bp fragment migrating out of the gel. Sequencing of mtDNA amplified fragments of the two haplotypes revealed a length of 1286 bp. Sequencing also revealed only a 1 bp substitution at position 72, with a thymine (T) in haplotype M1 and a guanine (G) in haplotype M2, resulting in the gain/loss of a restriction site for the restriction enzyme TaqI. M1 was the more abundant haplotype, being represented in 132 individuals (frequency, 84 %), and M2 the less abundant, being represented in only 25 individuals (frequency, 16 %). Of the 24 populations surveyed, eight (8, 9, 13, 14, 16, 20, 21, 24) were polymorphic, with both mtDNA haplotypes; 14 (1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 15, 17, 18, 22) were monomorphic for haplotype M1, and two (19, 23) were monomorphic for haplotype M2 (Table 3).

Association between cpDNA and mtDNA haplotypes
The cpDNA haplotypes C1–C33 with their corresponding mtDNA haplotypes (M1 and/or M2) are presented in Table 3, and shown in Fig. 1. Twenty cpDNA haplotypes are associated with mtDNA haplotype M1, and 11 cpDNA haplotypes with mtDNA haplotype M2. Two cpDNA haplotypes (C9 and C10) are associated with both mtDNA haplotypes. Of the two individuals representing C9, one has mtDNA haplotype M1 and the other mtDNA haplotype M2 (Table 3). Similarly, of the nine individuals with C10, five have M1 and four have M2 (Table 3). All individuals with haplotypes C9–M2 and C10–M2 are from population 23 (Greece). With the exception of C9 and C10, all other cpDNA haplotypes associated with mtDNA haplotype M2 are private haplotypes (Table 3).



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  		Fig. 1. Minimum-length spanning tree of 33 cpDNA haplotypes of Prunus spinosa with the mtDNA haplotypes plotted on it. The length of segments joining the circles is proportional to the number of mutations between haplotypes. The empty circles, filled circles and half-filled circles represent the cpDNA haplotypes associated with the mtDNA haplotypes M1, M2 and both M1 and M2, respectively.

 
The minimum-length spanning tree showing the phylogenetic relationships of cpDNA haplotypes is presented in Fig. 1. The distribution of mtDNA haplotypes (M1 or/and M2) associated with each cpDNA haplotype are also plotted on the tree. C10 and C20 form two main nodes of the tree. The node represented by C20 harbours a group of cpDNA haplotypes, all of which are associated with mtDNA haplotype M1 (Cluster I). The other node, represented by C10, has two groups of cpDNA haplotypes: Cluster II, associated with mtDNA haplotype M1, and Cluster III, associated with mtDNA haplotype M2, except C9, which is associated with M1 also (Fig. 1). In each cluster, the phylogenetically related cpDNA haplotypes are mostly geographically unrelated.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Prunus spinosa is a wild allotetraploid shrub (Reynders-Aloisi and Grellet, 1994) representing one of the possible ancestors of P. domestica (Watkins, 1976, 1981). The shrub has a wide range of environmental adaptability, including resistance to calcareous soils and drought. Its fruits are used in the preparation of an alcoholic drink (Pacharan) in Spain. All these attributes of P. spinosa are important for the improvement of rootstocks and/or varieties through interspecific hybridization. For such improvement programmes, assessment of genetic variability in populations is useful. A population genetic analysis of this shrub using cpDNA markers has already provided some information about genetically diverse populations (Mohanty et al., 2002). In the present investigation, cpDNA and mtDNA diversity and phylogenetic and geographic relationships between them, which can be useful for identifying populations for conservation and to formulate their management strategies, were studied.

In the previous investigation (Mohanty et al., 2002), three regions of cpDNA (approx. 8250 bp in total) were analysed for a population genetic study. In the present study, another region of the chloroplast genome (approx. 3800 bp) was analysed and found to be very polymorphic, with eight polymorphic fragments when restricted with two restriction enzymes. The combination of all the mutations detected in the regions HK, K1K2, VL (previous study), and CD (present study) resulted in 33 haplotypes in 157 individuals from 24 populations, implying a mean of 1·38 haplotypes per population. Of the 33 cpDNA haplotypes, only nine are shared between two or more populations and 24 are private. Only one haplotype (C20; frequency, 51 %) is abundant and geographically widespread. This cpDNA haplotype may correspond to the ancestral type. Analysis of diversity showed that differentiation among the populations was low (GST, 0·34), which is very close to that obtained when only three regions of cpDNA were analysed (GST, 0·33; Mohanty et al., 2002). The NST value was not significantly higher than the GST value, indicating an absence of phylogeographic structure. Thus, the additional analysis of another fragment CD of cpDNA showed several more polymorphisms but did not change the overall result (i.e. low genetic differentiation among populations and absence of phylogeographic structure) from that obtained in the previous study (Mohanty et al., 2002). Absence of phylogeographic structure could be due to efficient seed dispersal by mammals and birds which ingest the fruits of P. spinosa. Intensive seed movements can decrease genetic heterogeneity among populations and erase phylogeographic structure (Mohanty et al., 2002).

In contrast to high cpDNA diversity (33 haplotypes), the mtDNA showed lower levels of variation (only two haplotypes). The low variation of mtDNA may be explained by the fact that the rate of nucleotide substitution is at least three times slower in mtDNA than in cpDNA (Wolfe et al., 1987; Palmer, 1992). However, there are studies where lower cpDNA variations and higher mtDNA variations have been observed (Laurent et al., 1993; Caha et al., 1998).

Of the 33 cpDNA haplotypes, 15 are shared between two or more individuals and 18 are represented in one individual each. Of the 15 shared cpDNA haplotypes, 13 show strict association with their mtDNA haplotype. Most prominent is C20 (represented in more than 50 % of the individuals studied, and also geographically the most widespread), which is always coupled with mtDNA haplotype M1. The strong association between the two genomes suggests the same inheritance pattern for both organelles, which is assumed to be maternal. In P. spinosa there is no previous study demonstrating maternal inheritance of the two cytoplasmic genomes, but maternal inheritance of cpDNA has been demonstrated in P. cerasus (Brettin et al., 2000). So, if C20 represents an ancient haplotype, then M1 (which is strictly associated with C20) may also be considered to be of older origin than M2.

There are only two cases of uncoupling of cpDNA with its corresponding mtDNA. The dissociations are in the cpDNA haplotypes C9 and C10. C10 is associated with M1 in five individuals and with M2 in four individuals. C9 is associated with M1 in one individual of population 3 and with M2 in one individual of population 23 (Greece). There are three possible explanations. The first is homoplasy in mtDNA, although this seems unlikely because of the very low polymorphism observed in mtDNA (implying a low mutation rate). The second is a mtDNA mutation (a substitution) which must have occurred only once, thereafter C9 was derived twice independently from C10. This hypothesis is more likely because only two indel mutations distinguish C9 from C10. It appears that a recurrent mutation event has occurred in cpDNA (since the two mtDNA haplotypes are both found in two cpDNA types). These cpDNA haplotypes are probably similar by state but not by descent. The third is paternal leakage of either cpDNA or mtDNA may have occurred causing dissociation of cpDNA and mtDNA.

The minimum-length spanning tree of cpDNA haplotypes, with the mtDNA haplotypes plotted on it shows three clusters: cpDNA haplotypes coupled to mtDNA haplotype M1 are distributed in Cluster I and Cluster II, whereas Cluster III consists of all cpDNA haplotypes coupled to mtDNA haplotype M2, except C9 which is associated with both M1 and M2. C20 represents a node in Cluster I, while Clusters II and III arise from the node represented by C10. With the exceptions of C9 and C10, all other cpDNA haplotypes associated with M2 are private haplotypes. Although these private cpDNA haplotypes are unrelated geographically (phylogeographic structure is absent), they are related phylogenetically and have the same mtDNA haplotype, M2. Cluster II does not separate from Cluster III in the absence of a mtDNA marker (a non homoplasious marker in the present study). All three clusters have phylogenetically related, but mostly geographically unrelated, cpDNA haplotypes.


   ACKNOWLEDGEMENTS
 
We thank Dr Remy J. Petit for providing significant support as a coordinator during this project, and for helpful suggestions and valuable comments on the manuscript. The research was supported by the European Community research programme FAIR5-CT97-3795.


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 MATERIALS AND METHODS
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 DISCUSSION
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