Heredity (2006) 97, 283–290 & 2006 Nature Publishing Group All rights reserved 0018-067X/06 $30.00 www.nature.com/hdy Evolution of the self-incompatibility system in the Brassicaceae: identification of S-locus receptor kinase (SRK ) in self-incompatible Capsella grandiflora M Paetsch, S Mayland-Quellhorst and B Neuffer Department of Systematic Botany, University of Osnabrueck, Barbarastrae 11, Osnabrueck 49076, Germany Self-incompatibility (SI) has been well studied in the genera sequence and structure. Our phylogenetic analysis supports Brassica and Arabidopsis, which have become models for the scenario of differing SI evolution along the two lineages investigation into the SI system. To understand the evolution (The Brassica lineage and Arabidopsis/Capsella lineage). of the SI system in the Brassicaceae, comparative analyses Our results also argue that the ancestral S-locus lacked the of the S-locus in genera other than Brassica and Arabidopsis SLG gene (S-locus glycoprotein) and that the diversification are necessary. We report the identification of six putative of S-alleles predates the separation of Arabidopsis and S-locus receptor kinase genes (SRK) in natural populations Capsella. of Capsella grandiflora, an SI species from a genus which is Heredity (2006) 97, 283–290. doi:10.1038/sj.hdy.6800854; closely related to Arabidopsis. These S-alleles display published online 14 June 2006 striking similarities to the Arabidopsis lyrata SRK alleles in Keywords: self-incompatibility; Capsella; S-locus receptor kinase; S-alleles; Brassicaceae; natural populations Introduction S-locus is a complex locus spanning about 20 kb up to 200 kb and containing several physically linked tran- In more than 50% of angiosperms species, outbreeding scriptional units that cosegregate with SI phenotype. A is promoted by the action of a self-incompatibility (SI) subset of genes within the S-locus-complex is highly system (Brewbaker, 1959), which allows the stigma to polymorphic as would be expected for genes involved in reject self-pollen. The Brassicaceae are characterised by a recognition. To reflect this complexity, the ‘S-alleles’ of sporophytic SI system; the SI phenotype is determined classical SI genetics are sometimes referred to as ‘S by the diploid genotype of the parent plant. The genetics haplotypes’ (eg Nasrallah and Nasrallah, 1993). Com- of SI in the Brassicaceae were deciphered in the early parative mapping of different S haplotypes has demon- 1950s by Bateman (1955) who described control by a strated that they vary not only in the sequence of their SI single Mendelian locus, the S-locus, which exists as genes but also in their order, relative orientation, and multiple alleles, each of which encodes a distinct mating spacing between the genes (Boyes and Nasrallah, 1993; specificity. Starting in the 1980s, molecular methods were Cui et al, 1999; Suzuki et al, 1999). Three highly applied to the study of SI, focussing on the genera polymorphic genes are involved in the SI response: Brassica (reviewed by Kemp and Doughty, 2003) and SLG (S-locus glycoprotein), SCR/SP11 (S-locus cystein Arabidopsis (Kusaba et al, 2001; Schierup et al, 2001; rich), and SRK (S-locus receptor kinase). The genes are Charlesworth et al, 2003a, b; Mable et al, 2003) as model tightly linked and behave as a single Mendelian locus plants for sporophytic SI. Recent molecular knowledge (S-locus). SRK is a plasmamembrane-spanning protein strongly argues for an ancient and monophyletic origin localised in stigmas and has been found to be the female of SI in the Brassicaceae (reviewed by Fobis-Loisy et al, determinant (reviewed by Kemp and Doughty, 2003). Its 2004). Thus it appears, that SI is the ancestral system in extracellular domain (Exon 1: ‘S-domain’) is highly the Brassicaceae, that self-fertility is a derived condition, polymorphic between S-alleles. SCR/SP11 has been and that self-compatible species (SC) have arisen via identified as the male determinant of the SI response mutation of the genes important for SI. (Schopfer et al, 1999; Suzuki et al, 1999). It encodes a small cystein-rich protein which is secreted to the surface of the pollen grain and shows an even higher degree of The S-locus in Brassica and Arabidopsis polymorphism than SRK.InBrassica, the majority of Molecular analyses of the S-locus region in the Brassica- polymorphisms in the S-domain are concentrated in ceae, namely in the genus Brassica, have shown that the three hypervariable (HV-) regions which have been suggested to play a role in determining allele-specificity Correspondence: M Paetsch, Department of Systematic Botany, University and thus might be an important factor in the generation of Osnabrueck, Barbarastrae 11, Osnabrueck 49076, Germany. E-mail: [email protected] of new S-haplotypes (Miege et al, 2001). HV-regions in Received 12 July 2005; accepted 3 May 2006; published online 14 Arabidopsis are not so clearly distinctive; polymorphic June 2006 sites are distributed more evenly over the complete Identification of S-alleles in Capsella M Paetsch et al 284 sequence (Schierup et al, 2001). SLG is similar to the Materials and methods extracellular domain of SRK and probably originated by a duplication of the SRK S-domain (eg Nasrallah, 1994). Plant material and DNA preparation It is found in Brassica and Raphanus (Sakamoto et al, C. grandiflora seed material was individually collected on 1998), but not in Arabidopsis lyrata (Kusaba et al, 2001; the mainland of Greece and the Greek island of Corfu, C. Schierup et al, 2001). It appears not to be essential for the rubella from different Mediterranean countries (Table 1). SI response but may have an accessory function in Progeny of 5–10 individuals of each population were stabilising the active S-receptor complex (reviewed in grown in the greenhouse. Voucher specimens are Kemp and Doughty, 2003). The current model of SI deposited in the Herbarium OSBU. DNA was extracted response is that SCR/SP11 interacts with the extracellular from young leaves (0.5–1.0 g) using the following CTAB domain of the SRK protein, and the binding of the two method (Doyle and Doyle, 1987; modified). Leaves were proteins induces phosphorylation of SRK which initiates homogenised in liquid nitrogen, mixed with 2 Â CTAB a signalling cascade leading to inhibition of self-pollen. and b-mercaptoethanol and incubated for 1 h at 601C. DNA was extracted with chloroform in two steps, and precipitated through incubation with ice-cold The genus Capsella isopropanol for 30 min. After centrifugation, pellets were The genus Capsella comprises three species, two diploids washed twice with 70% ethanol, dried, resuspended in and one tetraploid. Distribution of Capsella grandiflora 100 ml water (autoclaved HPLC-water), and stored (FAUCHEZ &CHAUB)BOISS.(2Â ) is restricted to Western at 41C. Greece, Albania and northern Italy. C. rubella REUTER. (2 Â ) is distributed in the Mediterranean, the Middle East and followed European settlers occasionally into the New World and Australia (Figure 1) (Hurka and Neuffer, Table 1 Origin of Capsella seed material 1997; H Hurka, personal communication). C. grandiflora Pop. no. Provenance Elevation represents the ancestral obligate outbreeding species (SI) a.s.l. from which two self-compatible species, C. rubella and Capsella bursa-pastoris (L.) MED.(4Â ), originated (Hurka C. grandiflora and Neuffer, 1997). The breakdown of the SI system in 900 Greece: Corfu: Acharavi 3 m 391470N, 191480E Capsella SC-species coincides with reduction in flower 923 Greece: W-Peloponnes: Vrosina 90 m size (Figure 1) (Hurka and Neuffer, 1997; Neuffer and 391390N, 201310E Hurka, 1999). Capsella appears to be closely related to 924 Greece: W-Peloponnes: Botzaras 250 m Arabidopsis (Koch et al, 2000, 2001) on the basis of 391400N, 201350E sequence data from various nuclear genes and cpDNA. 926 Greece: W-Peloponnes: Votonosi 750 m 0 0 Comparative genome analysis has revealed extensive 39146 N, 21107 E 927 Greece: W-Peloponnes: Metsovo 1150 m conservation of genome organisation among Arabidopsis 391460N, 211100E thaliana and C. rubella (Acarkan et al, 2000; Koch and 934 Greece: W-Peloponnes: Metsovo 1350 m Kiefer, 2005). Owing to this close relationship it should 391460N, 211100E be possible to make use of the present knowledge of Arabidopsis SI in research on Capsella. It is therefore a C. rubella promising new object for further investigations on the 774 Italy: Mt. Gargano; 800 m 411500N, 161000E Brassicaceae S-locus in natural populations. 1241 Spain: Sierra de Guadalupe: 511 m The overall objective of our research is to better monastery; understand the evolution of the SI system within the 391270N, 51190W Brassicaceae. There is urgent need to analyse more wild 1249 Portugal: Sintra near Lisbon; 143 m species for comparative analyses. The aim of the present 381240N, 71230W 1504 Spain: Canary Islands: La Palma; 750 m study was to identify S-locus genes in the genus Capsella, 1 0 1 0 beginning with the self-incompatible C. grandiflora, and 28 40 N, 17 52 W to relate the SI systems of Capsella to Arabidopsis and Pop. no. ¼ Population numbers in collection of Capsella Seed Bank Brassica. (University of Osnabrueck). Capsella grandiflora 2x; self-incompatible 0.5 cm Capsella rubella 0.5 cm 2x; self-compatible Figure 1 Distribution and flower morphology of diploid Capsella species. The outcrossing C. grandiflora presents fragrant and attractive flowers due to dependence on pollinators and is restricted to a small area (black asterisk). In the selfing C. rubella breakdown of SI system coincides with reduction of flower size, loss of fragrance and colonising ability (hatched dots). Heredity Identification of S-alleles in Capsella M Paetsch et al 285 Amplification, cloning and sequencing Capsella sequences were aligned with the most similar For PCR amplification primers SLGf(50-AGAACCTATG sequences indicated by BLAST (for accession numbers CATGGGTTGC-30) and SLGr(50-ATCTGACATAAA see Table 2).