Journal of Heredity Advance Access originally published online on September 19, 2006
Journal of Heredity 2006 97(5):514-520; doi:10.1093/jhered/esl029
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The Mutated S1-Haplotype in Sour Cherry Has an Altered S-Haplotype–Specific F-Box Protein Gene
From the Department of Horticulture, Michigan State University, East Lansing, MI 48824 (Hauck and Iezzoni); and the Laboratory of Pomology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan (Ikeda and Tao)
Address correspondence to A. F. Iezzoni at the address above, e-mail: iezzoni{at}msu.edu.
Gametophytic self-incompatibility (GSI) is an outcrossing mechanism in flowering plants that is genetically controlled by 2 separate genes located at the highly polymorphic S-locus, termed S-haplotype. This study characterizes a pollen part mutant of the S1-haplotype present in sour cherry (Rosaceae, Prunus cerasus L.) that contributes to the loss of GSI. Inheritance of S-haplotypes from reciprocal interspecific crosses between the self-compatible sour cherry cultivar Újfehértói Fürtös carrying the mutated S1-haplotype (S1'S4SdSnull) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type S1-haplotype revealed that the mutated S1-haplotype confers unilateral incompatibility with a functional pistil component and a nonfunctional pollen component. The altered sour cherry S1-haplotype pollen part mutant, termed S1', contains a 615-bp Ds-like element within the S1-haplotype–specific F-box protein gene (SFB1'). This insertion generates a premature in-frame stop codon that would result in a putative truncated SFB1 containing only 75 of the 375 amino acids present in the wild-type SFB1. S1' along with 2 other previously characterized Prunus S-haplotype mutants, Sf and S6m, illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of GSI.
Sour cherry (Prunus cerasus L.) is a segmental allotetraploid produced by hybridization of the diploid sweet cherry (Prunus avium L.) and the tetraploid ground cherry (Prunus fruticosa Pall.) (Olden and Nybom 1968; Beaver and Iezzoni 1993). Like sweet cherry, sour cherry exhibits an S-RNase–based gametophytic self-incompatibility (GSI) system (Yamane et al. 2001). However, in contrast to sweet cherry where natural types are self-incompatible (SI), sour cherry includes both SI and self-compatible (SC) types (Lansari and Iezzoni 1990).
In sweet cherry, as in other diploid GSI systems, matching S-haplotypes in the pollen and style will result in an incompatible reaction, and the growth of this "self"-pollen tube will be inhibited (de Nettancourt 2001). In sour cherry, the genetic control of self-pollen recognition is more complicated because a pollen grain contains 2 S-haplotypes. Sour cherry pollen is incompatible if 1 or 2 of the S-haplotypes in the pollen matches an S-haplotype in the style (Hauck et al. 2006). In contrast, SC sour cherry pollen must contain 2 S-haplotypes that have lost pollen-S and/or pistil-S function, termed nonfunctional S-haplotypes. Therefore, the genotype-dependent loss of SI in sour cherry is due to the accumulation of at least 2 nonfunctional S-haplotypes (Hauck et al. 2006).
In sour cherry, 4 common sweet cherry S-haplotypes, S1, S4, S6, and S9, have been identified and determined to retain their SI function. In addition, 7 nonfunctional S-haplotypes have been identified in sour cherry based on genetic inheritance studies (Hauck et al. 2006). This includes a nonfunctional S-haplotype, termed Snull, that is hypothesized to be a deletion containing the S-haplotype because no restriction fragment length polymorphism (RFLP) fragment associated with Snull was visualized with either an S-RNase or an SFB probe (Hauck et al. 2006). To date, the molecular changes resulting in nonfunctional S-haplotypes have been reported for 4 of the 6 other mutants, including the stylar part mutant, S6m (Yamane et al. 2003; Tsukamoto et al. 2006). This mutant has a 2.6-kbp insert approximately 800 bp upstream of the S6-RNase. S6m was originally identified as a novel S-haplotype as it exhibited a unique RFLP fragment that resulted from an altered HindIII cut site compared with the wild-type S6-haplotype (Yamane et al. 2001).
The S-haplotype mutant described in this work was also initially identified due to a unique RFLP fragment (Yamane et al. 2001). In a survey of the S-RNases in sour cherry, only one selection, Újfehértói Fürtös (UF), had a unique HindIII S-RNase fragment of 9.6 kb (Yamane et al. 2001). This fragment was tentatively named the Se-haplotype, and the UF S-haplotype phenotype was determined to be S4SdSe. Segregation analysis subsequently determined that the fourth S-haplotype in UF was Snull (Hauck et al. 2006). In self-pollinated progeny of UF (S4SdSeSnull), the S4-haplotype segregated 1:1 (presence:absence) as would be expected for a fully functional S-haplotype. In contrast, both the Sd- and Se-haplotypes were present in all the self-pollinated progeny, indicating that the Sd- and Se-haplotypes have impaired functions (Hauck et al. 2006).
UF is a SC selection from the SI sour cherry landrace variety Pándy (syn. Cri
ana, Köröser) that is native to Hungary and Romania (Iezzoni et al. 1990). The S-haplotype phenotype for Pándy was determined to be S1S4Sd, and the functionality of the S1- and S4-haplotypes were confirmed using reciprocal crosses with sweet cherry (Rainier, S1S4) (Hauck et al. 2002). The origin of UF as an SC selection from the SI Pándy landrace, and the similar S-haplotype phenotypes between these selections, led to the hypothesis that Se is a mutated nonfunctional S1-haplotype that contributes to the SC of UF. Herein, we confirm this hypothesis and report that UF has an altered S1-haplotype, termed S1', in which the S-haplotype–specific F-box protein gene (SFB) (Ushijima et al. 2003; SLF, Entani et al. 2003) is nonfunctional due to the insertion of a Ds-like element.
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Plant Material and DNA Extraction
Three sour cherry cultivars, UF, Pándy 114 (P114), and Montmorency (Mont; S-phenotype S6S13mSa) (Hauck et al. 2006; Tsukamoto et al. 2006), and 3 sweet cherry cultivars, Emperor Francis (EF), Regina, and Summit, were used. S-alleles have been previously reported for the sour cherry cultivars (Yamane et al. 2001; Hauck et al. 2006) and sweet cherry cultivars (Schmidt and Timmann 1997;
and Tobutt 2001). The cultivars were grown at the Michigan State University Experimental Station, Clarksville, MI. Triploid progenies were generated from fully fertile reciprocal interspecific crosses between UF and Regina, EF, and Summit. Progeny genotyping was done using DNA extracted from mature seed from which the testa was removed using the procedure of Hauck et al. (2006). DNA extraction from leaves was done as previously described (Hauck et al. 2001).
S-RNase and SFB Genotyping
The S-RNase gene–specific primer set, Pru-C2 and PCE-R (Tao et al. 1999; Yamane et al. 2001), was used for S-haplotype determination of the progeny/seed from the interspecific crosses. This primer pair can differentiate between most S-RNase alleles based on polymorphisms in the length of the second intron in the Prunus S-RNase. However, the S2-RNase allele could not be reliably amplified using this primer pair. Instead, the S2-allele–specific primer pair PaS2-Fnew/PaS2-R (Sonneveld et al. 2003) was used. The SFB gene–specific primer set, SFB-C1F and FB3R (Ushijima et al. 2003), was used to amplify all SFB alleles and for the initial discovery of an insertion in SFB1'. In addition, the SFB1 allele was amplified with the allele-specific primer set PaSFB1-F and PaSFB1-R (Ikeda et al. 2005). A newly designed primer PcSFB1'-R (5'-GTTCGGTCCGGTTCTGAC-3') was used in conjunction with PaSFB1-F (Ikeda et al. 2005) for differentiating between the S1- and S1'-haplotypes. Polymerase chain reaction (PCR) conditions were identical to Ikeda et al. (2005) but with an annealing temperature of 62 °C.
S-RNase and SFB Reverse Transcriptase–PCR
Total RNA was isolated from styles with stigma or from pollen grains at the balloon stage of flower development as described elsewhere (Tao et al. 1999). One microgram of DNase (Roche Applied Science, Indianapolis, IN)-treated RNA was used for first strand cDNA synthesis by SuperScript II reverse transcriptase (RT) (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. For S1-RNase expression, PaS1-F and PaS1-R (Sonneveld et al. 2001) were used on stylar cDNA. For SFB1 and SFB1' expression, PaSFB1-F and PcSFB1'-R were used on pollen cDNA. Expression of the actin gene was used as a reference with the primers ActF1 and ActR1 (Yamane et al. 2003). PCR conditions were identical to those described by Yamane et al. (2003).
Genomic Library Construction and Screening
DNA from UF and P114 was used to create fosmid libraries using the CopyControl Fosmid Library Production Kit (Epicentre Technologies, Madison, WI). Clones containing either SFB1' or SFB1 were obtained using a digoxigenin–labeled SFB1 probe as previously described (Ushijima et al. 2001). Plasmid DNA was prepared using Wizard Plus Minipreps DNA Purification Kit (Promega, Madison, WI).
SFB1 and SFB1' Sequencing
Two micrograms of plasmid DNA was used as templates to sequence the SFB1' and SFB1 by primer walking using the primers SFB-C1F, SFB-C2R, SFB-C5F, and FB3R (Ushijima et al. 2003). In addition, PcSFB1'-R was used to obtain the sequence of the 5' end of the SFB1' gene. Sequencing was done by the Michigan State University Research Technology Support Facility using an ABI PRISM® 3100 Genetic Analyzer.
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The ability of UF (S1'S4SdSnull) styles to arrest S4 and S1 pollen was tested by pollinating UF with pollen from the sweet cherry cultivars EF (S3S4) and Regina (S1S3). All the progenies from these 2 crosses contained the S3-haplotype (Table 1). This indicates that EF and Regina S3-pollen were compatible in the UF styles, whereas Regina S1-pollen and the EF S4-pollen were selectively arrested by the presence of functional S1- and S4-RNases. Therefore, the UF S1- and S4-haplotypes exhibit functional stylar-S components. Because the UF styles were able to carry out this SI reaction in an allele-specific manner, the genetic changes causing self-compatibility in UF must disrupt specific GSI interactions through disruption of pollen-S function.
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Previously we demonstrated that UF has a functional S4-SFB as UF S4-containing pollen was unsuccessful when placed on EF (S3S4) pistils (Hauck et al. 2006). We tested the compatibility of S1'-containing UF (S1'S4SdSnull) pollen by placing UF pollen on pistils of Summit (S1S2) and Regina (S1S3). The presence of progeny resulting from the UF gametes S1' Sd and S1'Snull indicates that the UF S1'-haplotype has a nonfunctional pollen component (Table 2). If the S1 pollen component had been functional, S1Sd and S1Snull pollen would have been incompatible because the match of one-functional S-haplotype between the pollen and style results in incompatibility in Prunus (Hauck et al. 2006). This mutated SFB1 S-haplotype is termed S1' according to the Prunus nomenclature for a pollen part mutant (Lewis and Crowe 1954).
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The S1'-haplotype was also present in a progeny from the cross UF x Mont. The S-genotype of this individual was S1'S4S6Sa, and the individual was determined to be SC (Hauck et al. 2006). Because S4 and S6 are fully functional S-haplotypes, both Sa and S1' would need to be nonfunctional for this individual to be SC.
To determine the molecular basis of the nonfunctional SFB1', 2 fosmid clones each from UF and P114 that contained the S1-RNase were identified. PCR analysis using S-RNase primers amplified an S1-RNase fragment of identical size from the fosmid clones, UF and P114 (Figure 1A). As expected, S-RNase fragments representing the S4- and Sd-haplotypes were also amplified from UF and P114 (Hauck et al. 2006). The nucleotide and amino acid sequences for 280 bp of the Pru-C2/PCE-R S1 fragment from the UF fosmid clone were identical to those of the reported S1-RNase (GenBank number AB028153, data not presented).
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The UF and P114 fosmid clones containing the S1-RNase also contained SFB1' and SFB1, respectively. This was expected as the intergenic distance between the S-RNase and SFB in the S1-haplotype is only approximately 600 bp (Ikeda et al. 2005). The SFB1' fragment amplified with the universal SFB primers SFB-C1F and FB3R was approximately 600 bp longer than expected for wild-type SFB alleles, including the wild-type SFB1 from P114, SFB26, and SFB13 (Figure 1B). The increased length of SFB1' compared with SFB1 indicates the presence of an insertion.
The nucleotide alignment of SFB1 from P114 and sweet cherry from which it was originally sequenced was identical (GenBank number AB111518, data not presented). In contrast, SFB1' from UF contained a 615-bp insertion located +225 bp from the translation start site (Figure 2). The first 3 nucleotides of the insert encode an in-frame stop codon. The insert has 11-bp inverted repeats at the termini that are flanked by a 8-bp direct repeat. Both the terminal inverted repeat and the direct repeat are characteristic of Ds transposable elements (Kunze and Weil 2002).
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The premature in-frame stop codon in SFB1' is located in the V1 region of SFB, upstream from the 3 other SFB variable regions, V2, HVa, and HVb (Figure 3) (Ikeda et al. 2004). Therefore, SFB1' lacks 3 of the 4 variable regions and has only 75 amino acids from the N-terminus of the protein compared with wild-type SFB1 protein that has 375 amino acids.
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RT–PCR using the SFB1 forward primer, PaSFB1-F, and a newly designed reverse primer using sequence from the SFB1' insert, named PcSFB1'-R, amplified a fragment from UF pollen cDNA, indicating that SFB1' is transcribed (Figure 4). Therefore, it is possible that a truncated variant of the SFB1 protein is encoded from the S1'-haplotype.
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S1' is the fourth pollen part mutant identified in Prunus for which the molecular basis of the mutation has been characterized. The other 3 pollen part mutants are the naturally occurring Sf in Prunus mume and the irradiation-induced pollen part mutants in sweet cherry, S3' and S4'. All 4 of these pollen part mutants contain structural alternations in SFB (Ushijima et al. 2004; Sonneveld et al. 2005). The most severe structural change is present in the sweet cherry irradiation-induced S3' (Lewis and Crowe 1954), as the SFB3-coding region is completely deleted (Sonneveld et al. 2005). In contrast, the other 3 altered SFBs, Sf, S1', and S4', are at least partially transcribed, suggesting that they encode putative truncated proteins (Ushijima et al. 2004; Sonneveld et al. 2005). The F-box domain and the N-terminus of these proteins are unaffected; yet, the variable and hypervariable domains at the C-terminus, which are thought to be involved in specific interactions (Ikeda et al. 2004), would be changed or lost. The altered putative proteins are therefore unlikely to perform their normal specificity function. Compared with Sf and S4', S1' is noteworthy in that it would encode the shortest protein, completely lacking the variable region V2 and the hypervariable regions HVa and HVb.
The finding that alterations disrupting SFB function lead to pollen part mutants suggests that in Prunus, S-allele–specific S-RNase degradation follows the modified inhibitor model (Luu et al. 2001; see review by Wang et al. 2003). In this model, the pollen-S determines specificity and a separate inhibitor prevents the cytotoxic activity of the S-RNases. Therefore, SFB would act to block the binding of a general inhibitor to its cognate S-RNase, and the absence of SFB would result in the binding of a general inhibitor to the S-RNases and compatibility. This mechanistic model is also consistent with the "one-allele match" genetic model for the control of SC and SI in sour cherry (Hauck et al. 2006). If 1 or 2 of the SFBs in the 2x sour cherry pollen match a stylar S-RNase, the pollen would be incompatible because the SFBs would protect their cognate S-RNases from general inhibitor binding. Therefore, these S-RNases would maintain their cytotoxic activity.
SFB1' is the third nonfunctional S-haplotype mutation that is caused by the insertion of a transposable element. The sour cherry stylar part mutant S6m-haplotype has a 2.7-kb Mutator-like element insertion in the putative promoter region of the S6-RNase (Yamane et al. 2003). The S6-RNase failed to express in the mutant plant. SFBf has a 6.8-kb insertion that is a nonautonomous long terminal repeat element (Ushijima et al. 2004). Like the Ds insertion in SFB1', this insertion also results in a premature stop codon. The above examples illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of SI in Prunus, either by suppressing the expression or by interrupting the coding region of the relevant genes.
For centuries, the SI sour cherry landrace variety Pándy was grown in backyards and lined village streets in its native Hungary and Romania due to its excellent fruit quality (Iezzoni et al. 1990). In the village of Újfehértó, Hungary, the local residents identified and vegetatively propagated a particular selection of Pándy that was SC and therefore more productive. This selection, named Újfehértói Fürtös (translated "Bunched of Újfehértó") subsequently became the most widely planted sour cherry cultivar grown in Hungary. Pándy has the S-haplotype phenotype S1S4Sd. This lineage suggests that the SC in UF is at least partially due to the loss of function of the S1'-haplotype in the Újfehértó selection from the Pándy landrace. Therefore, SFB1' represents a novel allele resulting from Ds-element insertion that was selected and maintained in the population by local people due to the increased yield accompanying self-compatibility.
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Acknowledgments |
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We thank Audrey Sebolt for technical assistance and Ning Jiang for her critical review of the manuscript. This project was supported by the USDA Cooperative State Research, Education and Extension Service, National Research Initiative—Plant Genome Program.
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Footnotes |
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Corresponding Editor: Reid Palmer
Received January 16, 2006
Accepted July 16, 2006
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