Genomic characterization of self-incompatibility ribonucleases (S-RNases) in
loquat (Eriobotrya japonica Lindl.) (Rosaceae, Pyrinae)
Laura Carrera1, Javier Sanzol2, María Herrero1, Jose I. Hormaza3,4
1
Departamento de Pomología, Estación Experimental de Aula Dei - CSIC, Apdo. 202,
50080, Zaragoza, Spain.
2
Unidad de Fruticultura, Centro de investigación y Tecnología Agroalimentaria de
Aragón (CITA). Avda. de Montañana 930, 50059 Zaragoza, Spain.
3
Departamento de Fruticultura Subtropical y Fitopatología, Estación Experimental La
Mayora - CSIC, 29760 Algarrobo-Costa, Málaga, Spain.
4
Corresponding author:
E-mail: ihormaza@eelm.csic.es
Tel.: +34-952548990
Fax: +34-952552677
Keywords: Loquat; Maloideae; Pyrinae; Rosaceae; self-incompatibility; S-allele, S-genotyping; RNase.
Abstract
Loquat (Eriobotrya japonica Lindl) is a fruit tree species of the Pyrinae subtribe of the Rosaceae that
behaves as self incompatible. Since self-incompatibility in the Rosaceae is of the gametophytic type
where a stylar ribonuclease (S-RNase) controls the female function of pollen-pistil recognition, consensus
primers derived from the alignment of S-RNase sequences from other Pyrinae species were used to search
for S-RNases in loquat. As a result, the first four S-RNases were sequenced for this species. The genomic
sequences obtained showed the structural features of Pyrinae S-RNases. Moreover, microscopic
observations of pollen tube growth in the style confirmed the inter- (in)compatibility relationships
predicted from the molecular analyses. Phylogenetic analysis of the deduced amino acid sequences with
other Pyrinae S-RNases confirmed that divergence of S-alleles in loquat and the Pyrinae predated
speciation. This study reports for the first time the genomic characterization of S-RNases in loquat,
providing a sound basis for an appropriate selection of pollinator cultivars and an adequate design of
breeding programs.
1
Introduction
Loquat (Eriobotrya japonica Lindl.) is an evergreen subtropical fruit tree species that belongs to the
former subfamily Maloideae in the Rosaceae. Recent molecular phylogenetic analyses (Potter et al.,
2007) has provided support for the division of Rosaceae into three subfamilies (Dryadoideae, Rosoideae
and Spiraeoideae) further divided into supertribes, tribes, and subtribes. Taxa formerly included within
Maloideae and Prunoideae have been reclassified into the Spiraeoideae where the subfamily Maloideae
now corresponds to the subtribe Pyrinae in the tribe Pyreae and the Prunoideae to the tribe Amygdaleae.
The centre of origin of loquat can be placed in China from where it was introduced in Japan in ancient
times and, more recently, in the Mediterranean basin (in the XVIII century) and America (in the XIX
century) (Lin et al. 1999). While in Western countries loquat is mostly an ornamental plant and still
considered as an incipient crop, in different Asian countries it constitutes a well settled and appreciated
fruit species with clear perspectives of further development (Lin et al. 1999). Loquat total world
production is over 500,000 tons and China, with more than 80% of the world production, is the main
producing country followed by Spain, Turkey, Japan and Pakistan (Lin 2007). An evaluation of pollen
tube arrest following controlled pollinations (Singh and Rajput 1962; Herrero 2002), together with the
gametophytic self-incompatibility (GSI) characteristic of the Rosaceae suggests that a similar selfincompatibility system could operate in loquat.
The Rosaceae family exhibits RNase-based GSI controlled by a multiallelic S-locus (de
Nettancourt 2001). A similar GSI system is also shared by two other distantly related families, the
Solanaceae and the Plantaginaceae (formerly Scrophulariaceae; Albach et al. 2005) (McClure et al. 1989;
Xue et al. 1996). Pollen rejection occurs in the style whenever the S-allele of the haploid pollen matches
one of the S-alleles present in the diploid pistil tissue (for recent reviews see McClure 2006; McClure and
Franklin-Tong 2006). Pollen-pistil recognition involves at least two tightly linked polymorphic genes
located at the S-locus (Kao and Tsukamoto 2004). The gene that controls the female function encodes a
stylar ribonuclease (S-RNase) originally characterized in Nicotiana alata (Bredemeijer and Blaas 1981;
Anderson et al. 1986) and later in other species of the Solanaceae, such as Petunia inflata (Ai et al. 1990),
and Rosaceae, such as Japanese pear (Pyrus pyrifolia L.) (Sassa et al. 1993), apple (Malus x domestica
Borkh.) (Broothaerts et al. 1995), sweet cherry (Prunus avium L.) (Bošković and Tobutt 1996) or almond
(Prunus dulcis Mill.) (Tao et al. 1997). The S-RNase protein is responsible of both pistil recognition
specificity (McClure et al. 1989) and rejection activity (Huang et al. 1994; Lee et al. 1994; Murfett et al.
1994). More recently, pollen specific F-Box genes have been identified as responsible of the pollen
function of GSI in the three families (Rosaceae, Solanaceae and Plantaginaceae). The S haplotype specific
F-box gene (SFB) has been identified as the pollen S in Prunus (Entani et al. 2003; Ushijima et al., 2003;
Sonneveld et al. 2005), whereas the S locus F-box gene (SLF) has been shown to control pollen
specificity in Solanaceae and Plantaginaceae (McClure 2004; Sijacic et al. 2004; Qiao et al. 2004).
Multiple pollen-specific F-box genes (SLF/SFBBs), have been identified as possible candidates of the
pollen S in Malus and Pyrus (Cheng et al. 2006; Sassa et al. 2007) although their role in selfincompatibility is still under investigation (Kakui et al. 2007; Okada et al. 2008).
2
The S-RNase gene is well characterised in several species of the Pyrinae and has been widely
used for assessing allelic diversity of the S-locus both in cultivars of fruit crops such as apple (Broothaerts
et al. 1995), Japanese pear (Ishimizu et al. 1999) and European pear (Pyrus communis L.) (Zuccherelli et
al. 2002), and in wild populations of species such as Sorbus aucuparia and Crataegus monogyna (Raspe
and Kohn 2002). The alignment of the S-RNases of different species of the Pyrinae has identified five
conserved regions (C1, C2, C3, RC4 and C5) and one hypervariable region (HV) responsible of allele
specificity (Sassa et al. 1996; Ishimizu et al. 1998). Furthermore, the characterization of the genomic
region of the gene showed a single intron of variable size located in the HV region (Broothaerts et al.
1995). This last feature has been extensively used in S-allele identification by PCR analysis (Janssens et
al. 1995; Ishimizu et al. 1999; Sanzol and Robbins 2008).
The aim of this work was to identify and characterize the S-RNase gene associated with
gametophytic self-incompatibility in loquat, through genomic PCR and sequencing using primers based
on S-RNase sequences from other species of the Pyrinae. Molecular characterization of the S-locus in
loquat would improve our knowledge on the molecular genetics of self-incompatibility in this species and
in the Pyrinae and will be highly useful to select successful crosses in loquat breeding programmes and to
optimise orchard management.
Materials and methods
Plant material and isolation of genomic DNA
Five loquat cultivars previously evaluated as self-incompatible (Herrero 2002) (‘Algerie’, ‘Amadeo’,
‘Cardona’, ‘Magdal’ and ‘Magdal Rojo’) maintained at the Ruchey Cooperative experimental orchards in
Callosa d´en Sarrià (Alicante, Spain) were used in this study. Two European pear cultivars with known SRNase alleles, ‘Agua de Aranjuez’ (S101S103) and ‘Doyenne du Comice’ (S104S105) (Sanzol et al. 2006;
Goldway et al. in press) were used as control for genomic PCR analyses.
Total genomic DNA for PCR was isolated from young leaves according to Hormaza (2002) with
some modifications: leaf tissue was ground with 400 µl of CTAB extraction buffer, mixed with 400 µl of
chloroform-isoamyl alcohol (24:1) and centrifuged at 10,000 g for 15 min. The upper phase was
recovered and the nucleic acid was precipitated by adding 240 µl of cold isopropanol. The precipitate was
washed in 800 µl of 10 mM ammonium acetate in 76% ethanol for 20 minutes at 4°C and centrifuged at
13,000 g for 5 min; the upper phase was recovered and washed with 70% ethanol and centrifuged again at
13,000 g for 5 min. The pellet was dried and resuspended overnight at 4°C in 100 µl of modified TE
buffer (10 mM of Tris-HCl, 0.1 mM of EDTA, pH 8.0), centrifuged at 14,000 g for 5 min and incubated
with RNase (10 µg/ml) for 2 hours at room temperature. DNA preparations were quantified
spectrophotometrically and diluted to a final concentration of 10 ng/µl in modified TE buffer.
PCR and S-RNase-based genotyping
3
Consensus primers were designed based on the alignment of known S-RNase alleles from other species of
the Pyrinae. Nucleotide sequences were aligned using Clustal X (DNAStar, Madison, USA). The
annealing regions selected for the design of primers were the C1 (MaC1F1) and the C5 (MaC5R1)
conserved regions, and the conserved hexa-peptide ‘IIWPNV’ (MaC2/3R1) located in-between the C2
and the C3 conserved regions (Fig. 1). Primers were used in two different combinations, MaC1F1MaC2/3R1 and MaC1F1-MaC5R1 (Table 1) with expected amplification sizes differing in ~300-bp. Only
full-length S-RNase sequences from apple (Broothaerts 2003) and Japanese pear (Ishimizu et al. 1999)
were used to develop MaC1F1 and MaC5R1, whereas both partial and full-length sequences obtained
from Crataegus monogyna (Raspe and Kohn 2002), Sorbus aucuparia (Raspe and Kohn 2002), apple
(Broothaerts 2003), Japanese pear (Ishimizu et al. 1999) and European pear (Zuccherelli et al. 2002;
Zisovich et al. 2004) were used to develop MaC2/3R1.
A 20 µl of reaction solution containing 80 ng of genomic DNA, 16 mM (NH 4)2SO4, 67 mM TrisHCl pH 8.8, 0.01% Tween20, 1.6 mM MgCl2,, 0.8 mM dNTP, 0.4 µM of each primer, 1.2 units of
BioTaqTM DNA polymerase (Bioline, London, UK) and a drop of mineral oil were used for PCR in a
PTC-200 (MJ Research, Watertown, MA, USA) thermal cycler using the following program: an initial
step of 1 min of denaturation at 94°C, 35 cycles of 30 s at 94°C, 30 s at 55°C and 1 min at 72°C and a
final extension cycle of 5 min at 72°C. Amplified products were resolved in 1x TBE, 1.8% Metaphor
agarose (FMC Bioproducts, Rockland, ME, USA) and stained with ethidium bromide prior to
visualization under UV light (Fig. 2) or by capillary electrophoresis in a CEQTM 8000 capillary DNA
analysis system (Beckman Coulter, Fullerton, CA, USA). For capillary electrophoresis forward primers
were labelled with a WellRED D4 fluorescent dye (Proligo, Paris, France). Samples were denatured at
90ºC during 120 s, injected at 2.0 kV, 30 s and separated at 6.0 kV during 35 min. All the experiments
were repeated at least twice to confirm the reproducibility of the results.
Sequence analysis
After gel electrophoresis, PCR products were excised and purified using the NucleoSpin® Extract II kit
(Macherey-Nagel, Germany). Purified products were cloned into the ‘pGEM-T Easy’ cloning vector
(Promega, Madison, WI, USA), butanol-precipitated, transformed by heat-shock treatment in highefficiency competent cells (supplied with the cloning vector) before sequencing in both forward and
reverse directions using M13 universal primers. Sequences were determined at least from two clones
derived from two independent PCR reactions.
The S1, S2 and S4 alleles were sequenced from PCR amplification products obtained with the
EjC1F-EjC5R primers (see results). The S3 allele could not be sequenced from ‘Amadeo’ with the EjC1FEjC5R primer combination. In a first step, a 530 bp PCR product from this cultivar was sequenced with
the EjC1F-EjC2/3R primer combination. A blast search revealed a high homology between this sequence
and the S42-RNase of Pyrus usuriensis L. (AN: EF643637); thus, a new primer (PyusC5R) was designed
based on the sequence of the S42-RNase of Pyrus usuriensis. Finally, the S3 allele was sequenced from the
PCR amplification product obtained with the EjC1F-PyusC5R primer combination.
4
Pollination and pollen tube growth
To establish the inter-(in)compatibility relationships among the genotypes studied, self and crosspollinations involving the five loquat cultivars were carried out using detached flowers on moist florist
foam in the laboratory. The compatibility of each cross was determined by microscopic observations of
pollen tube growth in the pistil. The pollen used for the pollinations was collected from flowers at the
balloon stage. The anthers were removed and placed on paper at room temperature. After 24 h the pollen
was sieved through a 0.26-mm mesh and used for pollination. A total of 10 flowers of each pollen
recipient genotype were collected at the balloon stage, emasculated, placed on moist florist foam in the
laboratory and pollinated 24 h later. The pollinated pistils were fixed three days after pollination in
formaldehyde:acetic acid:70% ethanol (1:1:18) (Johansen 1940) and stored at 4ºC. Microscopic
observation of pollen tube growth was carried out on squash preparations after washing out the fixative
for 1 h with distilled water 3 times, softening the pistils in 5% sodium sulphite in the autoclave for 10 min
at 1.1 kg cm-2 (Jefferies and Belcher 1974), and staining with 0.1% aniline blue in 0.1 N K 3PO4 (Currier
1957; Linskens and Esser 1957). Preparations were examined under a Leica DM2500 microscope (Leica,
Cambridge, UK) with UV-epiflurescence using a LP 340-380 exciter filter and a LP 425 barrier filter.
The length of the longest pollen tube and the number of pollen tubes at the base of the style were recorded
for each flower. Crosses were considered incompatible when pollen tubes were arrested half their way
through the style (although occasionally some 1-2 pollen tubes per flower could be observed beyond this
point) versus some 10-15 pollen tubes per flower that reached the base of the styles in compatible crosses
(Herrero 2002).
Sequence alignment and comparison to other sequences in the Rosaceae
The amino acid sequences were deduced from the cDNA sequences and they were obtained looking for
the splicing junction points in comparison with closer alleles in the GeneBank database and,
consequently, the putative points of intron insertion could be determined. After removing the introns, the
deduced protein sequences between the C1 and the C5 conserved regions were obtained and aligned using
Clustal X (Thompson et al. 1997) and adjusted manually. The sequences were compared to different
sequences of Pyrinae species (Pyrus x bretschneideri, Malus domestica, Crataegus monogyna and Sorbus
aucuparia) available in the EMBL database (Fig. 5). An unrooted phylogenetic tree with the sequences
studied in this work and 53 additional sequences (50 in the Pyrinae and 3 in the Amygdaleae) was
generated with the program MEGA4 (Tamura et al. 2007) using the Neighbour-Joining (NJ) method with
500 bootstrap replications.
Results
Genomic characterization of S-RNases in loquat
5
A genomic PCR-based approach was used to identify S-RNase related sequences in loquat. Initially, two
pairs of consensus primers derived from the alignment of Pyrinae S-RNases (MaC1F1-MaC2/3R1 and
MaC1F1-MaC5R1, Table 1) were used to amplify genomic DNA from five self-incompatible loquat
cultivars ‘Algerie’, ‘Cardona’, ‘Amadeo’, ‘Magdal’ and ‘Magdal Rojo’ (Fig. 2). Two European pear
cultivars with known S-RNase alleles ‘Agua de Aranjuez’ (S101S103) and ‘Doyenne du Comice’ (S104S105)
were used as control for PCR. PCR products of the expected sizes were obtained with both primer pairs in
the two European pear cultivars.
A polymorphic banding pattern was obtained for the five loquat cultivars analysed, and the size
of the PCR amplifications (360-530 bp) was on the range of known Pyrinae S-RNases: from 321 bp for
PuS30 (EF643641) to 2427 bp for MdS16c (AB126322), although the vast majority (~75%) of known
Pyrinae S-RNases fall into a much narrow range, 321-545 bp (data not shown). The amplification profiles
obtained for loquat cultivars suggested that for each PCR product amplified with the MaC1F1-MaC2/3R1
primer combination, a related product ~300 bp larger in size was obtained with the MaC1F1-MaC5R1
primer combination (Fig. 2). This result was consistent with the location of the primer annealing regions
within the S-RNase gene sequence (Fig. 1).
In order to improve the resolution of the length polymorphisms among PCR amplifications, to
avoid confusion of amplification fragments with similar sizes in ‘Magdal’ and ‘Magdal Rojo’ and to
resolve the case that only a single band could be distinguished after agarose gel electrophoresis in
‘Amadeo’, we used a capillary DNA analysis system for fragment separation. Additionally, to reduce the
background initially obtained using consensus primers we developed three loquat-specific S-RNase
primers based on the sequences obtained with the consensus primers (Table 1): a forward primer localised
in the C1 region (EjC1F) and two reverse primers localised in the C2/3 (Ej2C2/3R) and C5 (EjC5R)
regions (Fig. 1). Overall, four different amplification bands could be discriminated with each primer
combination in the group of cultivars analysed: 355, 385, 520 and 530 bp with the MaC1F1-MaC2/3R1
and 655, 685, 820 and 830 bp with MaC1F1-MaC5R1. The banding pattern obtained after capillary DNA
analysis with the EjC1F-EjC2/3R primer combination resolved two bands for ‘Amadeo’ (523 and 530).
On the other hand, the results confirmed that ‘Magdal’ and ‘Magdal Rojo’ as well as ‘Algerie’ and
‘Cardona’ shared the same bands when evaluated with these primers. Based on these results, the first S
allele composition for loquat cultivars could be established: S1S2 (‘Algerie’ and ‘Cardona’), S1S3
(‘Amadeo’), and S2S4 (‘Magdal’ and ‘Magdal Rojo’) (Table 2).
Microscopic observations of pollen tube growth
To confirm the cross-(in)compatibility relationships predicted from the genomic analysis, pollen tube
growth in the different combinations was analysed at the microscope. The results obtained show that
pollen tubes were arrested in the middle of the style (Fig. 3) in all the self-pollinations and in the crosspollinations of genotypes sharing the same S-alleles whereas cross-pollinations involving genotypes of
different allele composition were compatible (Table 3).
Sequence analysis of the S-RNase gene in loquat and comparison to S-RNase sequences in the Pyrinae
6
The four polymorphic PCR products amplified with the EjC1F-EjC5R primer combination were cloned
and sequenced, showing that they corresponded to the genomic regions of four different S-RNase related
sequences. They were named S1, S2, S3 and S4. Each allele was sequenced from the following cultivars: S1
from ‘Algerie’ and ‘Amadeo’, S2 from ‘Algerie’, S3 from ‘Amadeo’ and S4 from ‘Magdal’ and ‘Magdal
Rojo’ (Table 4). PCR products amplified with the EjC1F-EjC5R primer combination were used for
sequencing S1, S2 and S4 whereas the EjC1F-PyususC5R primer combination was used for sequencing S3.
The intron was found within the hypervariable region in the same position in the four S-RNase
alleles identified (S1, S2, S3 and S4). The length of the intron differed among the four alleles: 326 (S1), 147
(S2), 319 (S3) and 177 (S4) bp (Fig. 1). All the sequences conserve the GT/AG consensus sequence in the
intron splice junctions (Simpson and Filipowicz 1996). Genomic DNA sequences of the four S-RNase
alleles (S1, S2, S3 and S4) have been submitted to NCBI (Accession numbers EU442285, EU442286,
EU442287 and EU442289 for Eriobotrya japonica S1, S2, S3 and S4 RNases respectively).
The deduced amino acid sequences corresponding to the four loquat alleles were aligned with
nine Pyrinae S-RNase sequences downloaded from the EMBL database (Fig. 4). The loquat sequences
had the primary structural features of the Pyrinae S-RNases including the three conserved regions (C2, C3
and RC4) and the HV region at identical position as the other sequences analysed. Moreover, they
exhibited two histidine residues, that have been shown essential for RNase activity in the Solanaceae
(Royo et al. 1994), and eight cysteine residues. Amino acid sequence identity among loquat alleles ranged
from 60% to 71% and confirmed the homology of the four stylar RNase alleles identified in loquat to
Pyrinae S-RNases since identities greater than 95% were obtained (Table 4) with alleles of other Pyrinae
species. The highest homology (98%) was obtained between the loquat S1 allele and both the Sf-RNase
precursor of Malus domestica and the S12-RNase of Pyrus bretschneideri.
Fifty-four Pyrinae S-RNases belonging to six different species, including the four loquat alleles
analysed in this study, were aligned and used to construct a neighbour-joining phylogenetic tree. Three SRNases of the Amygdaleae were also included and used as out-group (Sa of Prunus dulcis, S1 of P. avium
and Sa of P. salicina) (Fig. 5). Loquat alleles unequivocally clustered with Pyrinae S-RNases which
formed a differentiated cluster from the Amygdaleae sequences. Pyrinae S-RNases often displayed closer
relationships to S-RNases of other species than to those of their own species. This trans-specific pattern
was also observed for the newly identified S-RNases of loquat suggesting that S-allele divergence in
loquat predated speciation, in agreement with the general pattern observed for the other Pyrinae species
analysed so far.
Discussion
This study reports for the first time the molecular characterization of S-RNase genomic sequences from
loquat. The use of consensus primers derived from the alignment of other Pyrinae S-RNases allowed the
identification of the S-RNase gene in Eriobotrya japonica and the detection of four different S-RNase
alleles. Microscopic analyses confirmed the molecular results. The deduced amino acid sequences were
7
compared with those of other Pyrinae S-RNases and were used to construct a phylogenetic tree. This
analysis confirmed that loquat alleles exhibit the same trans-specific pattern observed for other Pyrinae SRNases studied so far, where alleles from different species can be more similar than alleles belonging to
the same species (Ioerger et al. 1990).
Self-incompatibility in Eriobotrya japonica
In order to study the incompatibility mechanism operating in E. japonica, consensus primers designed
from the alignment of different Pyrinae S-RNases were used to identify ribonucleases associated to selfincompatibility in loquat. All the S-RNase alleles revealed showed sequences characteristic of S-RNases.
Based on this evidence and on the results obtained after pollen tube growth analyses in the pistil in
controlled crosses, we can conclude that E. japonica shows a gametophytic self-incompatibility (GSI)
system. Consensus primers have also been used successfully to identify S alleles in other species of the
Pyrinae such as apple (Broothaerts 2003), Japanese pear (Ishimizu et al. 1999), European pear
(Zuccherelli et al. 2002) or Sorbus aucuparia and Crataegus monogyna (Raspe and Kohn 2002). The
results obtained in this work support the usefulness of consensus primers to detect S-alleles in related
species.
Structure of the S-RNase gene in E. japonica and comparison with S-RNase alleles in the Pyrinae
The four polymorphic bands obtained in the five cultivars were cloned and sequenced showing that they
correspond to the genomic regions of putative Pyrinae S-RNases. The alignment of the loquat deduced
amino acid sequences of S1, S2, S3 and S4 S-alleles with other sequences of the Pyrinae showed that they
have the primary structural organization of Pyrinae S-RNases (Ishimizu et al. 1998). The C2, C3 and RC4
conserved regions have been identified in loquat and the C2 region appears to be the most conserved
region between loquat cultivars as well as among Pyrinae species (Janssens et al. 1995). The
hypervariable region (RHV) was also highly variable in loquat and, as in the other species of the Pyrinae
studied so far, showed a single intron inserted. Differences in intron size are the main responsible for
allele size polymorphism and this could be used in loquat for the molecular typing of S-alleles as it has
been performed in other species of the Pyrinae such as apple (Broothaerts 2003; Broothaerts et al. 2004;
Matsumoto et al. 2007; Kim et al. 2008; Nybom et al. 2008), Japanese pear (Ishimizu et al. 1999) or
European pear (Sanzol et al. 2006; Takasaki et al. 2006; Mota et al. 2007; Sanzol and Robbins 2008).
The degree of identity of S-RNase alleles among cultivars of the same species is often variable in
the Pyrinae (Ishimizu et al. 1998). In this work, similarity between partial amino acid sequences of the S1,
S2, S3 and S4 alleles of loquat ranged from 60% to 71%, comparable to the range reported in other species
of the Pyrinae such as Japanese pear -59-96% (Ishimizu et al. 1998); 69%-75% (Kim et al. 2007)-, apple 62-74% (Ishimizu et al. 1998), 70-96% (Broothaerts and Van Nerum 2003)- or European pear -55-91%
(Zisovich et al. 2004), 62-90% (Takasaki et al. 2006)-. Often, the identity of Pyrinae S-RNases sequences
can be higher among sequences of different species than among sequences of the same species (Ishimizu
et al. 1998; Takasaki et al. 2004; Kim et al. 2007; Mota et al. 2007; Bokszczanin et al. in press). This is
8
the case in this work where homologies higher than 95% have been found between loquat sequences and
other sequences in the Pyrinae. This is reflected when genetic distances between the nucleotide sequences
of loquat S-RNases and other sequences in the Pyrinae are depicted in a phylogenetic tree where pistil
ribonucleases from loquat are intertwined with those of other species. Three sequences of the
Amygdaleae (from Prunus dulcis, Prunus avium and Prunus salicina) were also included in the analysis
and they formed an outgroup confirming the common phylogenetic origin of Pyrinae S-RNases. These
results support the idea that S-RNase in the Rosaceae form subfamily-specific subgroups instead of
species-specific subgroups (Ishimizu et al. 1998; Ushijima et al. 1998; Yaegaki et al. 2001; Ma and
Oliveira 2002; Zisovich et al. 2004) corroborating the hypothesis that the divergence of S-alleles in the
Pyrinae predates speciation as in most GSI systems in dicots (McClure et al. 1989; Ioerger et al. 1990;
Sassa et al. 1996; Xue et al. 1996; Igic and Kohn 2001; Steinbachs and Holsinger 2002; Sutherland et al.
2008) and, consequently, showing trans-specific S-allele evolution.
In conclusion, the results described in this work provide evidence that the S-RNase alleles
identified are candidates for the pistil S-determinant in loquat. The comparison of the amino acid
sequences of those alleles with the amino acid sequences of alleles in other species of the Pyrinae shows a
similar structure of S-alleles in the subtribe. The identification of S-RNase alleles in loquat will allow the
screening of the S-alleles of different loquat cultivars and the establishment of incompatibility groups in
this species. These results are an important step towards an appropriate selection of the most suitable
pollinator cultivars and the design of crosses in loquat breeding programs and they provide a solid
substrate for further pollen-pistil incompatibility studies with additional loquat cultivars. Finally they
contribute to the understanding of the structure and evolution of S-RNases in the Pyrinae. Further work
will involve the analysis of the expression of the alleles in loquat pistils as well as the study of the pollen
component of the S gene in this species.
Acknowledgements
The authors thank P. Escribano (EELM, Málaga Spain) for help in the molecular analyses and E. Soler
from Coop. Ruchey (Callosa d´Sarrià, Spain) for the plant material. LC was supported by a doctoral
(F.P.I.) fellowship of the Spanish Ministry of Education. Financial support for this work was provided by
the Spanish Ministry of Education (project grants AGL2003-05318-C02-01, AGL2006-13529-C02-02
and AGL2007-60130/AGR), GIC-Aragón 43 and a contract with Coop. Ruchey (Callosa d´Sarrià, Spain).
9
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Table 1. Primers used for S-RNase sequence analysis by PCR in Eriobotrya japonica Lindl.
Primer name Orientation Localisationa 5´-3´ Sequence
Degeneracy (Fold)
MaC1F1b
Forward
C1
att wtc aat tta cgc agc aat aat cag
MaC2/3R1b
Forward
C2/3
gba cgt tyg gcc aaa taa ttn yc
MaC5R1b
Reverse
C5
aaa kas yra yct cra cya att cmg
EjC1F
Forward
C1
att atc aat tta cgc agc aat atc ag
Ej2C2/3R
Reverse
C2/3
gga cgt tcg gcc aaa taa tta cc
EjC5R
Reverse
C5
caa ata cca acc tca acc aat tca g
2
48
256
PyusC5R
caa aga gta acc tca acc aat gca g
Reverse
C5
a
Conserved regions C1, C2/3 and C5
b
Consensus primers were designed based on the alignment of Pyrinae S-RNase alleles. The use of
alternative nucleotides within sequences is indicated by abbreviations: K=G+T, M=A+T, R=A+G,
S=C+G, W=A+T, Y=C+T, B=C+G+T, N=A+C+G+T.
Table 2. S-allele genotype of five loquat cultivars and S-RNase fragment sizes (bp) obtained by PCR
using three different primer pair combinations.
S- Genotype
Loquat cultivars
EjC1F- EjC5R
EjC1F- Ej2C2/3R
EjC1F-PyusC5R
831/652
530/357
n.a./652
831/818
530/523
n.a./818
652/679
357/384
Algerie
Cardona
S1-S3
Amadeo
Magdal
S2-S4
Magdal Rojo
n.a. = no or weak amplification
S1-S2
Table 3. Microscopic observations of self and cross-(in)compatibility relationships between the loquat
cultivars included in this work.
♀
Algerie
Cardona
Amadeo
Magdal
Magdal Rojo
♂
S-Genotype
S1-S2
S1-S2
S1-S3
S2-S4
S2-S4
Algerie
S1-S2
+
+
+
Cardona
S1-S2
+
+
+
Amadeo
S1-S3
+
+
+
+
Magdal
S2-S4
+
+
+
-
Magdal Rojo
S2-S4
+
+
ns
-
(+) = Compatible cross; (-) = Incompatible cross; ns = Cross not scored.
Table 4. Loquat cultivars used to obtain the S-allele sequence and list of the most similar alleles found in
the GenBank database for each of the loquat alleles identified.
S alleles
Cultivars
Primers
Homology Allele
S1
EjC1F-EjC5R
98%
S2
Algerie
Amadeo
Algerie
EjC1F-Ej5R
95%
S3
Amadeo
EjC1F-PyusC5R
97%
S4
Magdal
Magdal Rojo
EjC1F-EjC5R
97%
GenBank accession
number
Sf-RNase precursor Malus x domestica
S12-RNase Pyrus x bretschneideri
S34-RNase Pyrus x bretschneideri
D50837
EU081889
DQ414813
S42-RNase Pyrus ussuriensis
S42-RNase Pyrus x bretschneideri
S17-RNase Crataegus monogyna
EF643637
EF689007
AF504289
15
Figure legends
Fig. 1. Schematic representation of the loquat S-RNase gene with the intron location and size in the four
Eriobotrya japonica S alleles analysed in this work. Position of the primers based on the C1 and C5
conserved regions and the ‘IIWPNV’ consensus protein region of the Pyrinae S-RNase are indicated by
arrows. The signal peptide (SP), the hypervariable region (HV) and the five conserved regions (C1, C2,
C3, RC4 and C5) of the Rosaceae S-RNases are represented by boxes.
Fig. 2. PCR amplification of the 5 S-RNase alleles of 5 loquat cultivars identified in this study, using (a)
MaC1F1-MaC5R1 primer set and (b) MaC1F1-MaC2/3R1 primer set. M = 1 kb DNA ladder (Invitrogen,
Carlsbad, CA, USA), Alg = ‘Algerie’ (S1-S2), Car = ‘Cardona’ (S1-S2), Am = ‘Amadeo’ (S1-S3), Mg =
‘Magdal’ (S2-S4) and MgR = ‘Magdal Rojo’ (S2-S4).
Fig. 3. Incompatible pollen tube of loquat cv. Algerie 3 days after self-pollination arrested 40% down the
way of the style. Bar = 20 μm
Fig. 4. Alignment of the derived amino acid sequences of the four S-RNases from Eriobotrya japonica
analysed in this work compared to nine additional S-RNases of the Pyrinae. Sequences were aligned
manually. Amino acid positions are numbered. The signal peptide, hypervariable region and the five
conserved regions (HV, C1, C2, C3, RC4 and C5) of the Rosaceae S-RNases are underlined. The
conserved amino acid residues are shaded. Gaps are marked by dashes. Conserved cysteines (◊),
histidines (*) in Rosaceae are indicated. The site of the intron is shown by an arrow. Primer sites based on
the C1 and C5 conserved regions and the ‘IIWPNV’ consensus protein region of the Pyrinae S-RNases
are indicated in boxes. Abbreviations are as follows: Ppyr: Pyrus pyrifolia, Pcom: Pyrus communis,
Mdom: Malus domestica. Accession numbers of the nine Pyrinae S-RNases are: AB002139 (PpyrS1),
AB002142 (PpyrS6), AB002143 (PpyrS7), AB236430 (PcomS105), AB236429 (PcomS104), AB236427
(PcomS108), D505837 (MdomS1), U12199 (MdomS2), U12200 (MdomS3).
Fig. 5. Neighbour-joining phylogenetic tree developed in MEGA4 with 500 bootstrap replicates of the
four S-RNase Eriobotrya japonica sequences analysed in this work and 50 additional S-RNase sequences
in the Pyrinae and 3 in the Amygdaleae. The alignment was performed with the derived amino acid
sequences by CLUSTAL_X multiple sequence alignment. Posterior probabilities are indicated above the
branches (values < 75% not shown). Sequence data for the S-RNases included in the alignment are as
follows: 10 sequences from Crataegus monogyna (Cmon) (Raspé and Kohn 2002), 10 from Sorbus
aucuparia (Sauc) (Raspé and Kohn 2002), 15 from Malus domestica (Mdom) using the re-numbering
proposed by Broothaerts (2003), 7 from Pyrus pyrifolia (Ppyr) (Ishimizu et al. 1999), 9 from Pyrus
communis (Pcom) (Takasaki et al. 2006) using the renumbering proposed by Goldway et al. (in press).
Accession numbers of nucleotide sequences of Amygdaleae S-RNases shown are: AB026836 Prunus
dulcis (PdulSa), AB028153 Prunus avium (PaviS1), AB026981 Prunus salicina (PsalSa). S1 to S5
sequences of Eriobotrya japonica (Ejap) are indicated by the  symbol.
16
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