Experimental Farm, Graduate School of Agriculture, Kyoto University, Takatsuki , Japan 3

Full text

(1)

40 Available online at www.jstage.jst.go.jp/browse/jjshs1

JSHS © 2009

Characterization of a Novel Self-compatible S3' Haplotype Leads to the Development of a Universal PCR Marker for Two Distinctly Originated

Self-compatible S haplotypes in Japanese Apricot (Prunus mume Sieb. et Zucc.)

Hisayo Yamane1, Kyoko Fukuta1, Daiki Matsumoto1, Toshio Hanada1, Gao Mei1, Tomoya Esumi1, Tsuyoshi Habu2, Yoshiro Fuyuhiro3, Shinichiro Ogawa3,

Hideaki Yaegaki4, Masami Yamaguchi4 and Ryutaro Tao1*

1Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

2Experimental Farm, Graduate School of Agriculture, Kyoto University, Takatsuki 569-0096, Japan

3Fukui Prefectural Horticultural Experiment Station, Fukui 919-1123, Japan

4National Institute of Fruit Tree Science, Tsukuba 305-8605, Japan

Japanese apricot (Prunus mume Sieb. et Zucc.) exhibits the S-RNase-based gametophytic self-incompatibility (SI) system, which is controlled by a single polymorphic locus called the S locus containing the pistil S (S-RNase) and pollen S genes (SFB/SLF for S haplotype-specific F-box gene/S locus F-box gene). This study was conducted to elucidate the unknown molecular basis of self-compatibility (SC) in a selection line 1K0-26, an offspring of

‘Benisashi’ × ‘Koshinoume’, to explore the possible use of this line for SC breeding in Japanese apricot. Controlled pollination and segregation analyses demonstrated that 1K0-26 has an intact SI S7 haplotype and a pollen-part mutant SC S3' haplotype. Cloning and DNA sequence analysis of the S3' locus revealed a 7.1 kb insertion in the pollen determinant SFB3', which makes a premature stop codon to produce transcripts for truncated dysfunctional SFB, just like the mode of mutation in SFBf, another SC S haplotype in Japanese apricot. The inserted sequence appeared to be a non-autonomous retroposon with long terminal repeats, a part of which is identical to part of the inserted sequence to SFBf. Taking advantage of the similarity of the inserted sequences to the two SC S haplotypes of distinct origin, S3' and Sf, we have developed a universal PCR marker for the marker-assisted selection for SC in Japanese apricot. We surveyed 86 Japanese apricot cultivars and lines using the PCR marker, and it appeared that 38, 1, and 3 appeared to have only Sf, only S3', and both Sf and S3', respectively. Marker- assisted selection for SC in Japanese apricot and the possible use of these cultivars and lines for future SC Japanese apricot breeding programs are discussed.

Key Words: marker-assisted selection, self-incompatibility, SFB, S-RNase, transposable element.

Introduction

Gametophytic self-incompatibility (GSI) is a wide- spread mechanism in flowering plants which prevents self-fertilization and promotes out-crossing. Japanese apricot (Prunus mume Sieb. et Zucc.), which belongs to the Rosaceae, exhibits the S-RNase-based GSI (Sugiura

and Tao, 2002; Tao et al., 2000, 2002a, 2002b; Yaegaki et al., 2001), as do most other Prunus fruit tree species (Burgos et al., 1998; Tao et al., 1997, 1999; Ushijima et al., 1998; Yamane et al., 1999, 2001). GSI in Rosaceae is controlled by a single polymorphic locus called the S locus, which contains the pistil S and pollen S genes. In Prunus, the former is S-RNase for the S-ribonuclease gene (Tao et al., 1997, 1999; Ushijima et al., 1998) and the latter is SFB/SLF for the S haplotype-specific F-box gene/S locus F-box gene (Entani et al., 2003; Romero et al., 2004; Ushijima et al., 2003; Yamane et al., 2003a).

Although both self-compatible (SC) and self- incompatible (SI) cultivars of Japanese apricot are commercially grown for fruit production in Japan, SC

Received; January 25, 2008. Accepted; May 12, 2008.

This work is supported in part by a Grant-in-Aid (no. 17380021) for Scientific Research (B) and a Grant-in-Aid (no. 20248004) for Scien- tific Research (A) to Ryutaro Tao from the Japan Society for the Pro- motion of Science (JSPS).

* Corresponding author (E-mail: rtao@kais.kyoto-u.ac.jp).

(2)

cultivars have a horticultural advantage over SI cultivars because no cross-pollinizer is required. Consequently, one of the major breeding goals for Japanese apricot is to produce SC cultivars of good horticultural quality.

We have demonstrated that SC cultivars of Japanese apricot have a common S-RNase gene, designated as Sf- RNase (Tao et al., 2000). Cosegregation of Sf-RNase and SC has been confirmed by segregation analyses using various combinations of crosses (Tao et al., 2002a).

Furthermore, recent molecular characterization of the Sf haplotype revealed that SC conferred by Sf haplotype was caused by insertion of a non-atutonomous retroposon to the SFBf coding sequence (Ushijima et al., 2004).

Since SC in Japanese apricot cultivars that have been analyzed so far, except for a selection line 1K0-26, is conferred by the Sf haplotype (Tao et al., 2002a), several molecular markers for Sf haplotype and thus for SC have been developed and used for SC breeding programs of Japanese apricot (Habu et al., 2006; Tao et al., 2000, 2003; Yamane et al., 2003b). Since the marker-assisted selection of SC progenies soon after germination is possible, the time required for breeding of SC Japanese apricot could be considerably shortened.

Our previous study indicated that there is at least an additional different SC mechanism than that found in Sf haplotype, which confers SC in Japanese apricot. During our study to confirm cosegregation of Sf haplotype and SC in Japanese apricot, we found that a selection line 1K0-26, an offspring of ‘Benisashi’ × ‘Koshinoume’, showed SC even without Sf haplotype (Tao et al., 2002a).

As 1K0-26 does not have the Sf haplotype, SC in this line is supposed to be conferred by dysfunction of the S locus genes or a general factor, a so-called “modifier gene”.

In this study, we investigated the pollen and pistil functions for the SI reaction in 1K0-26 by controlled pollination study and showed that 1K0-26 has a pollen mutant SC S3' haplotype. Cloning and characterization of the S locus of S3' haplotype revealed a transposable element insertion to SFB3. As it appeared that part of the inserted sequence to SFB3' is identical to part of the inserted sequence to SFBf, we could develop a universal PCR marker for the two SC S haplotypes of distinct origin, S3' and Sf, for marker-assisted selection for SC in Japanese apricot. Marker-assisted selection for SC in Japanese apricot is discussed.

Materials and Methods Plant materials and DNA extraction

A total of 86 Japanese apricot (Prunus mume Sieb. et Zucc.) cultivars and lines were used in this study (Table 1), including both SI and SC cultivars and lines for fruit production and ornamental usage. We also used 27 and 31 seedlings obtained from selfed progenies of 1K0-26 and ‘Koshinoume’, respectively. Total DNA was isolated from young leaves, which were ground to a powder using a mortar and pestle in liquid nitrogen, and

homogenized in homogenization buffer (Zhang et al., 1995). The homogenate was centrifuged (1800 × g, at 4°C, for 15 min) to collect the pellet. The pellet was resuspended in homogenization buffer and centrifuged again. After repeated resuspension and centrifugation, genomic DNA was isolated from the pellet using Nucleon Phytopure for a Plant DNA extraction kit (GE Healthcare UK Ltd, Buckinghamshire, England), and further purified by phenol/chloroform extraction.

Pollination and pollen-tube growth test

Pollen tube growth tests were performed for self and reciprocal crosses of two selection lines, 1K0-26 (we used S3'S7 in this study rather than the previously scored S3S7; see below) and 1C1-10 (S3S7), progenies of

‘Benisashi’ (S7Sf) × ‘Koshinoume’ (S3'Sf) and ‘Benisashi’

(S7Sf) × ‘Kairyou Uchidaume’ (S3S4), respectively. Pollen viability of the two selection lines was confirmed by pollen tube growth in the pistil of ‘Kotsubunanko’

(S7S10). The pollen tube growth test was performed as described previously (Tao et al., 2002a). Briefly, emasculated flowers on the branches of the two selections were hand-pollinated with pollen grains in the laboratory at 20°C, when the pistil of the flowers became receptive (24 h after emasculation). The pollinated pistils were collected 72 h after pollination, placed in a fixing solution (chloroform : ethanol : glacial acetic acid, 1 : 3 : 1 (v/v)) for 24 h, transferred into 100% ethanol, and stored at 4°C until stained. For fluorescent staining of pollen tubes, the pistils were washed thoroughly under running tap water, incubated in 10 N NaOH for 6 h to soften the tissues, and soaked in 0.1% aniline blue solution with 33 mM K3PO4. Pollen tubes were observed by ultraviolet fluorescence microscopy (Bx60, Olympus, Tokyo, Japan) equipped with a digital camera (DP50, Olympus).

Segregation analysis

Selfed progenies of 1K0-26 (S3'S7) and ‘Koshinoume’

(S3'Sf) were obtained with controlled pollination at the Kyoto Farmsted of the Experimental Farm of Kyoto University. Flowers that were emasculated one to two days before anthesis were hand-pollinated with pollen grains collected in the previous year that had been stored with desiccant at 4°C. The resultant fruits were harvested at maturity and the seeds were collected and stored at 4°C for four months. The seeds were then sowed in soil in a greenhouse and leaves were collected from the seedlings. S-RNase genotype of the seedlings was determined by S-RNase gene-specific PCR with a universal Prunus S-RNase primer set, Pru-C2 and Pru- C4R (Tao et al., 1999). PCR condition was identical to that described previously (Tao et al., 1999).

Construction and screening of the genomic libraries Fosmid libraries were constructed from the genomic DNA of the two Japanese apricot selection lines, 1K0-

(3)

Table 1. Characteristics of Japanese apricot cultivars used in this study.

Cultivars and lines Presence of S3'/Sf SU/SI/SC/SFz Male sterility Types Reference

1C1-10 no SI no fruit production Tao et al. (2002a)

1K0-26 S3' SF/SC no fruit production Tao et al. (2002a)

Akananiwa no SU no ornamental Yaegaki et al. (2002b)

Akebono no SU MS fruit production Yaegaki et al. (2002a, 2002b)

Baigou no SU no fruit production Yaegaki et al. (2002a, 2002b, 2003)

Benisashi Sf SF no fruit production Yaegaki et al. (2002b)

Bungo (Hiratsuka) no SU MS fruit production Yaegaki et al. (2002a, 2002b)

Bungo (Kurume) Sf SF no fruit production Yaegaki et al. (2002b)

Bungo × Kousyuu Oujyuku no SU MS fruit production Yaegaki et al. (2002a, 2002b)

Chouhanagata no SU MS ornamental Yaegaki et al. (2002a, 2002b)

Fujibotan Sf SU no ornamental Yaegaki et al. (2002b)

Fujiedatankoubai Sf SU MS fruit production Yaegaki et al. (2002a, 2002b)

Fujinoume Sf SF no fruit production Yaegaki et al. (2002b)

Futono no SU no fruit production Yaegaki et al. (2002b)

Gecchibai Sf SU no fruit production Yaegaki et al. (2002b, 2003)

Gessekai no SU no fruit production Yaegaki et al. (2002b, 2003)

Gojirou no SU MS fruit production Yaegaki et al. (2002a, 2002b)

Gyokuei no SU MS fruit production Yaegaki et al. (2002a, 2002b, 2003)

Hachirou Sf SF no fruit production Yaegaki et al. (2002b, 2003)

Hanakami Sf SU no fruit production Yaegaki et al. (2002b)

Ihara no SU no fruit production Yaegaki et al. (2002b)

Inazumi Sf SF no fruit production Yaegaki et al. (2002b, 2003)

Inkyo no SU no fruit production Yaegaki et al. (2002b)

Ishikawa Oomiume Sf SU no fruit production Yaegaki et al. (2002b)

Issunnbai Sf SU no fruit production Yaegaki et al. (2002b)

Jizouume Sf SF no fruit production Yaegaki et al. (2002b, 2003)

Jyousyuushiro no SU no fruit production Yaegaki et al. (2002b)

Jyuurou no SU no fruit production Yaegaki et al. (2002b, 2003)

Kagajizou no SU MS fruit production Yaegaki et al. (2002a, 2002b, 2003)

Kairyou Uchidaume no SU no fruit production Yaegaki et al. (2002b, 2003)

Kankoubai Sf SU no ornamental Yaegaki et al. (2002b)

Kasugano no SU MS ornamental Yaegaki et al. (2002a, 2002b)

Kenkyou no SU no ornamental Yaegaki et al. (2002b)

Kensaki Sf SF no fruit production Yaegaki et al. (2002b)

Kichirobei no SU no fruit production Yaegaki et al. (2002b)

Kinsujiume no SU MS ornamental Yaegaki et al. (2002a, 2002b)

Komukai no SU MS fruit production Yaegaki et al. (2002a, 2002b)

Koshinoume S3', Sf SF/SC no fruit production Tao et al. (2002a), this study

Koubai Sf SU no ornamental Yaegaki et al. (2002b)

Koume Sf SU no fruit production Yaegaki et al. (2002b)

Koushuu Oujyuku Sf SU no fruit production Yaegaki et al. (2002b)

Koushuu Saishou Sf SF no fruit production Yaegaki et al. (2002b)

Koushuu Shinnko Sf SF no fruit production Yaegaki et al. (2002b)

Makitateyama Sf SU MS ornamental Yaegaki et al. (2002a, 2002b)

Mangetushidare no SU no ornamental Yaegaki et al. (2002b)

Michishirube Sf SU MS ornamental Yaegaki et al. (2002a, 2002b)

Muroya Sf SF no fruit production Yaegaki et al. (2002b)

Naniwa no SU no fruit production Yaegaki et al. (2002b)

Nankou no SU no fruit production Yaegaki et al. (2002b, 2003)

Natsuka Sf SF no fruit production Yaegaki et al. (2002b)

Okituakabana no SU MS ornamental Yaegaki et al. (2002a, 2002b)

Orihime Sf SF no fruit production Yaegaki et al. (2002b, 2003)

Oushuku no SU no fruit production Yaegaki et al. (2002b)

Oushukubai no SU no ornamental Yaegaki et al. (2002b)

(4)

26 (S3'S7) and 1C1-10 (S3S7), using the CopyControl Fosmid Library Production Kit (Epicentre, Madison, WI, USA). The libraries were screened with a DIG-dUTP- labeled (Roche, Basel, Switzerland) probe synthesized from S3-RNase (Acc. No. AB047102) (Yaegaki et al., 2001) of ‘Koshinoume’ (S3'Sf) using Pru-C2 and Pru- C4R primers (Tao et al., 1999). Positive clones were subsequently used as templates for PCR with the S- RNase gene-specific primer set (Pru-C2 and Pru-C4R) and the SFB gene-specific primer set (SFB-C2F and SFB-C5R) (Ikeda et al., 2004; Yamane et al., 2003a) to confirm the presence of S3-RNase and its linked SFB allele, respectively. Nucleotide sequences of S-RNase and SFB in the clones were determined by primer walking using the CEQTM8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA).

Development of a universal PCR marker for SC A reverse primer, S3' insR1 (5'-GTT CCC AAC CCA

GAA GTT AC-3'), was designed from the identical sequence present in the long terminal repeat (LTR) regions of the inserted sequences to SFB3' (+1364 to +1383) and SFBf (+957 to +976) (Fig. 2A). Pm-SFB- C2F (5'-CCT ATA CAC ATA TGG AAC CCA-3'), one of the four components of the mixed primer SFB-C2F primer (5'-CCW ATA CAM ATA TGG AAC CC-3') previously designed from the conserved region of PmSFBs (Ikeda et al., 2004), was used as a forward primer. PCR with this primer set is supposed to amplify DNA fragments of 995 bp and 587 bp from SFB3' and SFBf, respectively. PCR was performed using a program of 28 cycles at 94°C for 20 sec, 58°C for 20 sec, and 72°C for 30 sec with initial denaturing at 94°C for 3 min and final extension at 72°C for 5 min. The PCR reaction mixture contained 1 × ExTaq buffer (TaKaRa BIO Inc., Ohtsu, Japan), 200μM each of dNTPs, 400 nM each of primers, 50 ng of template DNA, and 1 U of TaKaRa Ex Taq polymerase (TaKaRa BIO Inc.) in a 50-μL

Table 1. (Continued).

z SU = self-unfruitful (less than 10% of fruit set after self-pollination), SI = self-incompatible, SC = self-compatible, and SF = self-fruitful (10% or more than 10% of fruit set after self-pollination).

Cultivars and lines Presence of S3'/Sf SU/SI/SC/SFz Male sterility Types Reference

Rinshuu Sf SF no fruit production Yaegaki et al. (2002b)

Ryuukyou Koume Sf SF no fruit production Yaegaki et al. (2002b, 2003)

Saju no SU MS ornamental Yaegaki et al. (2002a, 2002b)

Sarasa no SU no ornamental Yaegaki et al. (2002b)

Seiyoubai no SU MS fruit production Yaegaki et al. (2002a, 2002b)

Shimosukeume Sf SF no fruit production Yaegaki et al. (2002b)

Shiratamaume Sf SF no fruit production Yaegaki et al. (2002b)

Shirobotan no SU MS ornamental Yaegaki et al. (2002a, 2002b)

Shirokaga no SU MS fruit production Yaegaki et al. (2002a, 2002b, 2003)

Shukou Sf SF no fruit production Yaegaki et al. (2002b)

Sugita no SU no fruit production Yaegaki et al. (2002b)

Sumomoume no SU MS fruit production Yaegaki et al. (2002a, 2002b)

Suzukishiro Sf SU MS fruit production Yaegaki et al. (2002a, 2002b)

Taihei Sf SU MS fruit production Yaegaki et al. (2002a, 2002b)

Tairinryokugaku no SU no ornamental Yaegaki et al. (2002b)

Taisyouume Sf SF no fruit production Yaegaki et al. (2002b)

Takadaume Sf SU no fruit production Yaegaki et al. (2002b)

Tamabotan Sf SU no ornamental Yaegaki et al. (2002b)

Tamagakishidare no SU MS ornamental Yaegaki et al. (2002a, 2002b)

Tamaume no SU no fruit production Yaegaki et al. (2002b)

Tamaume × Koushuu Saisyou no SU no fruit production Yaegaki et al. (2002b)

Tobiume no SU no ornamental Yaegaki et al. (2002b)

Tougoro S3', Sf SF no fruit production Yaegaki et al. (2002b)

Touji no SU no ornamental Yaegaki et al. (2002b)

Tsukasashibori no SU no ornamental Yaegaki et al. (2002b)

Umetukuba No. 4 Sf SF no fruit production Yaegaki et al. (2003)

Yaezakikankou Sf SU MS ornamental Yaegaki et al. (2002a, 2002b)

Yakushiume no SU no fruit production Yaegaki et al. (2002b)

Yatsubusa (Akita strain) Sf SU no fruit production Yaegaki et al. (2002b)

Yatsubusa (Shimada strain) S3', Sf SF no fruit production Yaegaki et al. (2002b)

Yourou no SU no fruit production Yaegaki et al. (2002b)

Youseiume Sf SF no fruit production Yaegaki et al. (2002b)

(5)

reaction volume. After PCR, the PCR mixture was run on 1% agarose gel and DNA bands were visualized by ethidium bromide staining.

Results

Pollen tube growth test and segregation analysis Pollen tubes from 1K0-26 (S3'S7) and 1C1-10 (S3S7) grew down the full-length of the ‘Kotsubunanko’ (S7S10) pistil (Figs. 1A, 1B). 1K0-26 pollen tubes grew down the full-length of the 1K0-26 pistil (Fig. 1C), while 1C1- 10 pollen tube growth was arrested halfway down the 1C1-10 pistil (Fig. 1D). When 1K0-26 and 1C1-10 were reciprocally crossed, 1K0-26 pollen tubes grew down the full length of the 1C1-10 style (Fig. 1E), while 1C1- 10 pollen tube growth was arrested halfway down the 1K0-26 style (Fig. 1F).

Segregation analysis

Selfed progeny of 1K0-26 (S3'S7) segregated into two S-RNase genotypes, S3'S3' and S3'S7, with a segregation ratio of 13 : 14 (Table 2). S-RNase genotypes in the selfed

progeny of ‘Koshinoume’ (S3'Sf) segregated into all three possible classes of S3'S3', S3'Sf, and SfSf with a segregation ratio of 10 : 16 : 5 (Table 2).

Cloning of S locus genes

Four and seven positive clones that contained S3- RNase were obtained from the fosmid libraries of 1K0- 26 (S3'S7) and 1C1-10 (S3S7), respectively. The positive clones were further screened by PCR with SFB gene- specific primers to obtain clones that contained both S- RNase and SFB. It appeared that two of the four positive clones from 1K0-26 contained both S-RNase and SFB, while the remaining two contained only S-RNase. As for 1C1-10 positive clones, we only checked five of the seven positive clones and found that all five clones contained both S-RNase and SFB. DNA sequencing of the clones showed that S3-RNase sequences of 1K0-26 and 1C1-10 are exactly the same and showed 100%

identity to the previously reported partial sequence of S3-RNase of Japanese apricot (Habu et al., 2008; Yaegaki et al., 2001). Although the previously reported sequence

Table 2. Segregation of S-RNase alleles in selfed progenies of 1K0-26 and ‘Koshinoume’.

Cross Expected ratio Observed ratio χ2 p

1K0-26 self S3'S3' : S3'S7 = 1 : 1 S3'S3' : S3'S7 = 13 : 14 0.037 0.848 Koshinoume self S3'S3' : S3'Sf : SfSf = 1 : 1 : 1 S3'S3' : S3'Sf : SfSf = 10 : 16 : 5 1.64 0.439

Fig. 1. Pollen tube growth after different combinations of crosses. Left and right photos in each panel show the stigma and upper style of pistil and the base of the style of pistil, respectively.

(6)

Fig. 2. Structure of SFB3'. (A) Schematic representation of SFB3' as compared with SFBf (Ushijima et al., 2004). The nucleotide sequences of the open box are similar to the coding region of a functional SFB. In the case of SFB3', nucleotide sequences of the open box are exactly the same as the coding region of the functional SFB3. ‘A’ of the start codon corresponding to that of the functional SFB is positioned as +1. The sequences of both ends (gray box) of the inserted DNA fragments are identical in SFB3' as well as in SFBf. Black and wavy thick lines in the inserted sequences indicate sequences that show high identities between SFB3' and SFBf. The positions of the primers for the PCR amplification of SFB3' and SFBf are indicated by arrows. (B) Amino acid sequence comparison between SFB3, predicted intact SFB3' (OriSFB3'), and SFB3. The F-box motif and HVa and HVb regions are boxed.

Table 3. Identities of amino acid sequences between Prunus S locus genes, S-RNase and SFB, of several S haplotypesz.

z The upper half proves amino acid sequence identities (%) among Prunus SFBs and the lower half shows identities among the S-RNases.

EMBL/GenBank/DDBJ accession numbers are as follows: PmSFB1 (AB101440), PmSFB3 (AB376968), PmSFB7 (AB101441), ParSFB1 (AY587563), ParSFB2 (AY587562), PavSFB3 (AB096857), PavSFB6 (AB096858), PdSFBa (AB092966), PdSFBc (AB079766), PsSFBa (AB252411), PsSFBb (AB252413), PmS1-RNase (AB101438), Pm-S3-RNase (AB376969), PmS7-RNase (AB101439), ParS1-RNase (AY587561), ParS2-RNase (AY587562), PavS3-RNase (AB010306), PavS6-RNase (AB010305), PdSa-RNase (AB026836), PdSc-RNase (AB011470), PsSa-RNase (AB252411), and PsSb-RNase (AB252413).

P. mume P. armeniaca P. avium P. dulcis P. salicina

PmS1 PmS3 PmS7 PmSf ParS1 ParS2 PavS3 PavS6 PdSa PdSc PsSa PsSb

PmS1 83.0 81.5 80.3 81.7 82.4 82.5 80.9 71.0 80.0 79.0 81.1

PmS3 75.6 82.4 79.5 79.8 80.8 78.5 81.9 70.7 79.2 77.1 79.8

PmS7 74.9 71.2 80.2 79.5 80.2 78.4 80.4 70.9 80.2 76.8 80.3

PmSf 74.6 82.5 71.7 78.5 77.9 76.9 78.9 71.1 81.5 76.1 78.2

ParS1 77.5 80.9 72.3 77.3 80.4 77.7 80.9 69.5 77.2 78.8 79.0

ParS2 71.1 77.6 71.1 75.9 77.6 78.2 79.7 69.0 79.5 77.1 82.2

PavS3 74.6 71.6 67.7 71.1 73.5 76.0 80.0 68.8 78.5 76.9 82.2

PavS6 75.2 80.8 74.0 78.1 77.5 73.8 73.0 69.9 77.1 78.5 78.5

PdSa 59.5 55.0 56.9 56.7 59.1 56.7 58.0 56.0 70.0 68.6 68.9

PdSc 72.6 79.9 67.8 79.5 77.9 76.4 73.0 77.2 56.3 76.6 79.0

PsSa 70.3 79.3 68.9 74.0 76.6 72.5 72.6 78.8 57.1 78.8 78.0

PsSb 73.8 79.8 74.8 74.9 77.0 77.2 76.9 79.4 56.3 75.0 73.6

(7)

is a partial sequence of S3-RNase cDNA (AB047102) and the S3-RNase gene (AB364464), the complete sequence of the S3-RNase gene (AB376969) is shown here. The deduced amino acid sequence from S3-RNase exhibited characteristic variability and conservation patterns of Prunus S-RNase and showed 55% to 82.5%

identity to other Prunus S-RNases (Table 3). When we compared SFB3' of 1K0-26 with SFB3 of 1C1-10, we found a 7144 bp insertion in SFB3' at position +821 from the start codon (Fig. 2). A duplicated 5 bp segment of the putative coding region flanked the inserted sequence.

The 517 bp segments of both ends of the inserted sequence were identical as in the case of the long terminal repeats (LTR) of retrotransposons. Although the inserted sequence displayed no significant homology with any sequence in the public database, part of the inserted sequence, mainly in the LTR region, is identical to part of the inserted sequence to SFBf (Ushijima et al., 2004).

The inserted sequence makes a premature stop codon at position +823 to +825; therefore, SFB3' transcripts encode putative SFB3' that lacks 105 amino acid residues in the C-terminal half and contains an additional cysteine residue encoded by the inserted sequence. The original intact SFB3' sequence predicted from the structure of SFB3' is identical to the SFB3 of 1C1-10, which encodes a typical SFB with 70.7% to 83.0% amino acid identity to other Prunus SFBs (Table 3).

Development of a universal molecular marker for SC in Japanese apricot

Taking advantage of sequence similarities between the sequences inserted into SFB3' and SFBf, we designed a universal PCR primer set, PM-SFB-C2F and S3'-ins- R1, for the amplification of SFB3' and SFBf. PCR with this primer set successfully amplified the expected fragments of 995 bp and 587 bp from SFB3' and SFBf, respectively, from the genomic DNA of the cultivars whose S haplotypes were described previously (Fig. 3).

PCR for the 86 diverse Japanese apricot cultivars and lines with this primer set revealed that 38, 1, and 3 had only Sf, only S3', and both Sf and S3', respectively (Table 1). Although six of the 18 self-unfruitful (SU) cultivars and lines with S3' and/or Sf haplotype show

male sterility, the remaining 12 cultivars scored SU without male sterility. In contrast, all cultivars and lines that were estimated to be self-fruitful (SF) or SC had S3' and/or Sf. Since ‘Koshinoume’, a pollen parent of 1K0- 26, yielded S3' and Sf bands (Fig. 3), ‘Koshinoume’ is supposed to be S3'Sf rather than the previously scored S3Sf (Tao et al., 2002a, 2003).

Discussion

This study elucidated the molecular basis of SC observed in 1K0-26, which is the only SC plant that does not have the Sf haplotype among Japanese apricot plants for which the S haplotype has been characterized (Tao et al., 2002a). SC mutants in Prunus usually show unilateral incompatibility (UI) with either the pollen-part or stylar-part function being disrupted (Hauck et al., 2006a, 2006b; Sonneveld et al., 2005; Tsukamoto et al., 2006, 2008; Ushijima et al., 2004). In order to determine if 1K0-26 shows UI, as with other SC mutants in Prunus, we made a reciprocal cross of SC 1K0-26 and SI 1C1- 10, both of which have the same S-RNase genotypes that usually represent S haplotypes. It appeared that 1K0- 26 is SC but at the same time it showed UI. Namely, 1K0-26 pollen tubes grew down the full length of the pistil of SI 1C1-10 but 1K0-26 pistils arrested pollen tube growth of SI 1C1-10. This information is important not only for breeders but also for growers when 1K0- 26 is released as a cultivar.

Mutation in the pollen determinant, SFB, in the S locus (Hauck et al., 2006a, 2006b; Sonneveld et al., 2005; Tsukamoto et al., 2006, 2008; Ushijima et al., 2004) or the gene in the modifier locus (Vianova et al., 2006; Wűnsch and Hormaza, 2004) has been reported to result in pollen-part mutation in Prunus. The former and latter usually show S haplotype-dependent and S haplotype-independent loss of pollen function of SI, respectively. Genetic segregation data for S-RNase genotypes of the selfed population of 1K0-26 indicated that S3 haplotype is mutated in 1K0-26 while S7 haplotype is intact because only the S-RNase genotypes found in the progeny were S3S3 and S3S7. If S7 haplotype had also been nonfunctional, S7S7 progeny would have been obtained. The mutated S3 haplotype found in 1K0-26 is termed S3' according to the Prunus nomenclature for a pollen-part mutant (Lewis and Crowe, 1954). Segrega- tion data of the selfed population of ‘Koshinoume’, a pollen parent of 1K0-26, revealed that ‘Koshinoume’

also has the S3' haplotype, and thus, it appeared that 1K0- 26 inherited the S3' haplotype from ‘Koshinoume’. We previously overlooked the presence of the SC S3' haplotype in ‘Koshinoume’ because this cultivar also has the SC Sf haplotype (Tao et al., 2002a).

In order to elucidate the molecular basis of the pollen- part mutation in the S3' haplotype, we have cloned and analyzed the S locus region of the S3' haplotype. As expected, there is a structural alternation in SFB in the S3' haplotype, while there is no such mutation in S-RNase

Fig. 3. S3' and Sf haplotype-specific PCR. Lanes: M, 100 bp ladder;

1, 1K0-26 (S3'S7); 2, ‘Koshinoume’ (S3'Sf); 3, ‘Kensaki’ (SfSf);

4, ‘Benisashi’ (S7Sf); 5, ‘Hachirou’ (S8Sf); 6, ‘Ryukyou Koume’

(S8Sf); 7, ‘Rinshuu’ (S9Sf); 8, ‘Jizouume’ (S10Sf); 9, ‘Orihime’

(S6Sf); 10, ‘Nankou’ (S1S7); 11, ‘Oushuku’ (S1S5); 12, ‘Gessekai’

(S1S6); 13, ‘Gyokuei’ (S2S6); 14, ‘Kairyou Uchidaume’ (S3S4);

15, 1C1-10 (S3S7); and N, negative control (PCR with no template DNA).

(8)

in the S3' haplotype. The mutated SFB3, termed SFB3', contains a non-autonomous transposable element with the sequence resembling LTR of retrotransposons.

Interestingly, most LTR regions and their adjacent regions in the inserted sequence to SFB3' showed very high sequence identity to the inserted sequence to SFBf of Japanese apricot (Ushijima et al., 2004). Not only the mode of mutation but also the sequences inserted into SFB are similar in S3' and Sf haplotypes. Because both segments of the LTR in the sequence inserted into SFB3' and into SFBf are identical, it is supposed that the insertion event occurred quite recently. It is possible that the non-autonomous transposable elements inserted into SFB3' and SFBf could be active retrotransposons in the same family.

Sequence similarity in the inserted sequence could be used to develop a universal PCR marker to detect S3' and Sf. Since S3' and Sf are the only SC S haplotypes that have been found in Japanese apricot, the PCR marker would serve as a universal SC marker for marker-assisted selection in Japanese apricot. More than half of the diverse Japanese apricot cultivars and lines surveyed had the Sf haplotype, while only four of 86 cultivars and lines had the S3' haplotype. It is supposed that the mutated SC S haplotype could rapidly prevail over SI S haplotypes because SC has pomological advantage over SI. It is possible, therefore, that the limited number of S3' haplotypes could indicate that S3' was generated recently.

An alternative explanation for the limited number of S3' could also be drawn from the limited number of S3 in the cultivars and lines tested. The original SI S3 was only present in ‘Kairyou Uchidaume’ and its progeny 1C1-10 (Habu et al., 2008; Hideaki Yaegaki and Ryutaro Tao, unpublished data). It is possible, therefore, that S3 and its mutated version, S3', may be linked to undesirable characters that affect tree performance or fruit quality, and S3 and S3' haplotypes may have been selected out under artificial or natural selection pressure.

Yaegaki et al. (2002b) scored the self-unfruitfulness (SU) and self-fruitfulness (SF) of Japanese apricot cultivars and lines, while we used a controlled pollination and pollen tube growth test to determine GSI characters (SI/SC). Although we confirmed that 1K0-26 is SC as well as SF, some cultivars and lines were estimated to be SU but had Sf and/or S3'. Since the criterion for SF in Yaegaki et al. (2002b) was >10% fruit set in late April after completion of the physiological fruit drop period, cultivars and lines could be scored SU not only because of their GSI characters but also because of male sterility (Yaegaki et al., 2002a, 2003) or other factors that affect fruit set. Interestingly, all ornamental cultivars are SU, regardless of the presence or absence of Sf and/or S3'. In contrast to cultivars for fruit production, SU is preferable to SF in ornamental cultivars as an excessive fruit load may weaken the tree. Furthermore, the doubled flower character that is often preferable in ornamental Japanese apricot could lead to sterility or low fruit set. SC that is

determined by PCR markers for the S haplotype, does not necessarily mean SF, because plants with Sf and/or S3' can be SU from other factors, such as pollen sterility.

In conclusion, although it is still possible that there could be other SC S haplotypes or SC mechanisms than Sf and S3', which are masked by Sf and S3', all cultivars that were estimated to be SF by pollination study had either Sf and/or S3', and thus, the PCR marker for Sf and/

or S3' developed in this study could be used effectively for marker-assisted selection for SC in Japanese apricot.

However, since there is some possibility that S3' is linked to undesirable characters, implementation of the early detection and elimination of SC seedlings should consider that this selection would have effects on linked traits.

Literature Cited

Burgos, L., O. Perez-Tornero, J. Ballester and E. Olmos. 1998.

Detection and inheritance of stylar ribonucleases associated with incompatibility alleles in apricot. Sex. Plant Reprod. 11:

153–158.

Entani, T., M. Iwano, H. Shiba, F.-S. Che, A. Isogai and S.

Takayama. 2003. Comparative analysis of the self- incompatibility (S-) locus region of Prunus mume:

identification of a pollen-expressed F-box gene with allelic diversity. Genes to Cells 8: 203–213.

Habu, T., F. Kishida, M. Morikita, A. Kitajima, T. Yamada and R. Tao. 2006. A simple and rapid procedure for the detection of self-compatible individuals in Japanese apricot (Prunus mume Sieb. et Zucc.) using the loop-mediated isothermal amplification (LAMP) method. HortScience 41: 1156–1158.

Habu, T., D. Matsumoto, K. Fukuta, T. Esumi, R. Tao, H. Yaegaki, M. Yamaguchi, M. Matsuda, T. Konishi, A. Kitajima and T.

Yamada. 2008. Cloning and characterization of twelve S- RNase alleles in Japanese apricot (Prunus mume Sieb. et Zucc.). J. Japan. Soc. Hort. Sci. 77: 374–381.

Hauck, N. R., K. Ikeda, R. Tao and A. F. Iezzoni. 2006a. The mutated S1-haplotype-specific F-box protein gene. J. Hered.

97: 514–520.

Hauck, N. R., H. Yamane, R. Tao and A. F. Iezzoni. 2006b.

Accumulation of nonfunctional S-haplotypes results in the breakdown of gametophytic self-incompatibility in tetraploid Prunus. Genetics 172: 1191–1198.

Ikeda, K., B. Igic, K. Ushijima, H. Yamane, N. R. Hauck, R.

Nakano, H. Sassa, A. F. Iezzoni, J. R. Kohn and R. Tao.

2004. Primary structural features of the S haplotype-specific F-box protein, SFB, in Prunus. Sex. Plant Reprod. 16: 235–

243.

Lewis, D. and L. K. Crowe. 1954. Structure of the incompatibility gene. IV. Types of mutation in Prunus avium L. Heredity 8:

357–363.

Romero, C., S. Vilanova, L. Burgos, J. Martínez-Carvo, M.

Vicente, G. Llácer and M. L. Badenes. 2004. Analysis of the S-locus structure in Prunus armeniaca L. Identification of S- haplotype specific S-RNase and F-box genes. Plant Mol. Biol.

56: 145–157.

Sonneveld, T., T. R. Tobutt, S. P. Baughan and T. P. Robbins.

2005. Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/

mutation of an S haplotype-specific F-box gene. Plant Cell 17: 37–51.

Sugiura, A. and R. Tao. 2002. Self-incompatibility and fruit

Figure

Updating...

References

Updating...

Related subjects :