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Gametophytic self-incompatibility (GSI) in sweet cherry ( Prunus avium, Rosaceae) is controlled by the multi-allelic S-locus. This complex locus contains two linked genes, an S- RNase that controls pistil specificity, and an F-box gene named SFB ( S-haplotype-specific F-box protein gene) that is the putative determinant of pollen specificity in Prunus species. The S-locus is considered to be a region of repressed recombination, as recombination has not been observed. Recombination between the pistil- S and pollen- S determinant genes would result in non-functional S-haplotypes and loss of self-incompatibility. With the recent identification of multiple SFB alleles in sweet cherry, along with the existing S- RNase alleles, it is now possible to estimate the linkage distance and quantify the physical distance between the two GSI specificity genes in multiple sweet cherry S-haplotypes. A recombinational analysis between S- RNase and SFB was performed for four sweet cherry S-haplotypes ( S2, S3, S4, S6) using F1 progeny from reciprocal crosses between ‘Emperor Francis’ ( S3 S4) and ‘NY 54’ ( S2 S6). For SFB genotyping, allele-specific primer sets were designed from the sequence of the SFB coding region. The S- RNase and SFB genotypes of 511 progeny, representing the outcomes of 1,022 meioses, were determined. All four S-haplotypes individually segregated according to the expected Mendelian ratio of 1:1. The S- RNase and SFB loci were completely linked as no recombinant individuals were identified, thus maintaining the co-evolved allele specificities for the pistil and putative pollen determinant. The physical distance between S- RNase and SFB in six sweet cherry S-haplotypes ( S1- S6) was determined using PCR with genomic clones as the template. The relative order and transcriptional orientation of S- RNase and SFB were conserved across the six S-haplotypes. However, the physical distance between these two genes varied widely, ranging from 380 bp to approximately 40 kb. This study represents the first large-scale recombinational analysis of the S-locus region in the Rosaceae, serving as the starting point for future comparative analyses of physical distances, linkage distances, and sequence diversity among Prunus S-haplotypes. [ABSTRACT FROM AUTHOR]

The gene SFB encodes an F-box protein that has appropriate S-haplotype-specific variation to be the pollen determinant in the S-RNase-based gametophytic self-incompatibility (GSI) reaction in Prunus (Rosaceae). To further characterize Prunus SFB, we cloned and sequenced four additional alleles from sweet cherry (P. avium), SFB1, SFB2, SFB4, and SFB5. These four alleles showed haplotype-specific sequence diversity similar to the other nine SFB alleles that have been cloned. In an amino acid alignment of Prunus SFBs, including the four newly cloned alleles, 121 out of the 384 sites were conserved and an additional 65 sites had only conservative replacements. Amino acid identity among the SFBs ranged from 66.0% to 82.5%. Based on normed variability indices (NVI), 34 of the non-conserved sites were considered to be highly variable. Most of the variable sites were located at the C-terminal region. A window-averaged plot of NVI indicated that there were two variable and two hypervariable regions. These variable and hypervariable regions appeared to be hydrophilic or at least not strongly hydrophobic, which suggests that these regions may be exposed on the surface and function in the allele specificity of the GSI reaction. Evidence of positive selection was detected using maximum likelihood methods with sites under positive selection concentrated in the variable and hypervariable regions. [ABSTRACT FROM AUTHOR]

The Spanish local cultivar 'Ambrunés' stands out due to its high organoleptic quality and fruit firmness. These characteristics make it an important parent for breeding cherries with excellent fresh and post-harvest quality. In this work, an F1 sweet cherry population (n = 140) from 'Ambrunés' × 'Sweetheart' was phenotyped for 2 years for fruit diameter, weight and firmness and genotyped with the RosBREED cherry Illumina Infinium® 6K SNP array v1. These data were used to construct a linkage map and to carry out quantitative trait locus (QTL) mapping of these fruit quality traits. Genotyping of the parental cultivars revealed that 'Ambrunés' is highly heterozygous, and its genetic map is the longest reported in the species using the same SNP array. Phenotypic data analyses confirmed a high heritability of fruit size and firmness and a distorted segregation towards softer and smaller fruits. However, individuals with larger and firmer fruits than the parental cultivars were observed, revealing the presence of alleles of breeding interest. In contrast to other genetic backgrounds in which a negative correlation was observed between firmness and size, in this work, no correlation or low positive correlation was detected between both traits. Firmness, diameter and weight QTLs detected validated QTLs previously found for the same traits in the species, and major QTLs for the three traits were located on a narrow region of LG1 of 'Ambrunés'. Haplotype analyses of these QTLs revealed haplotypes of breeding interest in coupling phase in 'Ambrunés', which can be used for the selection of progeny with larger and firmer fruits. [ABSTRACT FROM AUTHOR]

Background: Sweet cherry is consumed widely across the world and provides substantial economic benefits in regions where it is grown. While cherry breeding has been conducted in the Pacific Northwest for over half a century, little is known about the genetic architecture of important traits. We used a genome-enabled mixed model to predict the genetic performance of 505 individuals for 32 phenological, disease response and fruit quality traits evaluated in the RosBREED sweet cherry crop data set. Genome-wide predictions were estimated using a repeated measures model for phenotypic data across 3 years, incorporating additive, dominance and epistatic variance components. Genomic relationship matrices were constructed with high-density SNP data and were used to estimate relatedness and account for incomplete replication across years. Results: High broad-sense heritabilities of 0.83, 0.77, and 0.76 were observed for days to maturity, firmness, and fruit weight, respectively. Epistatic variance exceeded 40% of the total genetic variance for maturing timing, firmness and powdery mildew response. Dominance variance was the largest for fruit weight and fruit size at 34% and 27%, respectively. Omission of non-additive sources of genetic variance from the genetic model resulted in inflation of narrow-sense heritability but minimally influenced prediction accuracy of genetic values in validation. Predicted genetic rankings of individuals from single-year models were inconsistent across years, likely due to incomplete sampling of the population genetic variance. Conclusions: Predicted breeding values and genetic values revealed many high-performing individuals for use as parents and the most promising selections to advance for cultivar release consideration, respectively. This study highlights the importance of using the appropriate genetic model for calculating breeding values to avoid inflation of expected parental contribution to genetic gain. The genomic predictions obtained will enable breeders to efficiently leverage the genetic potential of North American sweet cherry germplasm by identifying high quality individuals more rapidly than with phenotypic data alone. [ABSTRACT FROM AUTHOR]

Understanding the process of Prunus species floral development is crucial for developing strategies to manipulate bloom time and prevent crop loss due to climate change. Here, we present a detailed examination of flower development from initiation until bloom for early- and late-blooming sour cherries (Prunus cerasus) from a population segregating for a major bloom time QTL on chromosome 4. Using a new staging system, we show floral buds from early-blooming trees were persistently more advanced than those from late-blooming siblings. A genomic DNA coverage analysis revealed the late-blooming haplotype of this QTL, k, is located on a subgenome originating from the late-blooming P. fruticosa progenitor. Transcriptome analyses identified many genes within this QTL as differentially expressed between early- and late-blooming trees during the vegetative-to-floral transition. From these, we identified candidate genes for the late bloom phenotype, including multiple transcription factors homologous to Reproductive Meristem B3 domain-containing proteins. Additionally, we determined that the basis of k in sour cherry is likely separate from candidate genes found in sweet cherry-suggesting several major regulators of bloom time are located on Prunus chromosome 4., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Sour cherry ( Prunus cerasus L.) is a valuable fruit crop in the Rosaceae family and a hybrid between progenitors closely related to extant Prunus fruticosa (ground cherry) and Prunus avium (sweet cherry). Here we report a chromosome-scale genome assembly for sour cherry cultivar Montmorency, the predominant cultivar grown in the USA. We also generated a draft assembly of P. fruticosa to use alongside a published P. avium sequence for syntelog-based subgenome assignments for 'Montmorency' and provide compelling evidence P. fruticosa is also an allotetraploid. Using hierarchal k-mer clustering and phylogenomics, we show 'Montmorency' is trigenomic, containing two distinct subgenomes inherited from a P. fruticosa -like ancestor (A and A') and two copies of the same subgenome inherited from a P. avium -like ancestor (BB). The genome composition of 'Montmorency' is AA'BB and little-to-no recombination has occurred between progenitor subgenomes (A/A' and B). In Prunus , two known classes of genes are important to breeding strategies: the self-incompatibility loci ( S- alleles), which determine compatible crosses, successful fertilization, and fruit set, and the Dormancy Associated MADS-box genes ( DAMs ), which strongly affect dormancy transitions and flowering time. The S -alleles and DAMs in 'Montmorency' and P. fruticosa were manually annotated and support subgenome assignments. Lastly, the hybridization event 'Montmorency' is descended from was estimated to have occurred less than 1.61 million years ago, making sour cherry a relatively recent allotetraploid. The 'Montmorency' genome highlights the evolutionary complexity of the genus Prunus and will inform future breeding strategies for sour cherry, comparative genomics in the Rosaceae, and questions regarding neopolyploidy., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

The Rosaceae crop family (including almond, apple, apricot, blackberry, peach, pear, plum, raspberry, rose, strawberry, sweet cherry, and sour cherry) provides vital contributions to human well-being and is economically significant across the U.S. In 2003, industry stakeholder initiatives prioritized the utilization of genomics, genetics, and breeding to develop new cultivars exhibiting both disease resistance and superior horticultural quality. However, rosaceous crop breeders lacked certain knowledge and tools to fully implement DNA-informed breeding-a "chasm" existed between existing genomics and genetic information and the application of this knowledge in breeding. The RosBREED project ("Ros" signifying a Rosaceae genomics, genetics, and breeding community initiative, and "BREED", indicating the core focus on breeding programs), addressed this challenge through a comprehensive and coordinated 10-year effort funded by the USDA-NIFA Specialty Crop Research Initiative. RosBREED was designed to enable the routine application of modern genomics and genetics technologies in U.S. rosaceous crop breeding programs, thereby enhancing their efficiency and effectiveness in delivering cultivars with producer-required disease resistances and market-essential horticultural quality. This review presents a synopsis of the approach, deliverables, and impacts of RosBREED, highlighting synergistic global collaborations and future needs. Enabling technologies and tools developed are described, including genome-wide scanning platforms and DNA diagnostic tests. Examples of DNA-informed breeding use by project participants are presented for all breeding stages, including pre-breeding for disease resistance, parental and seedling selection, and elite selection advancement. The chasm is now bridged, accelerating rosaceous crop genetic improvement.

A Rosaceae family-level candidate gene approach was used to identify genes associated with sugar content in blackberry ( Rubus subgenus Rubus ). Three regions conserved among apple ( Malus × domestica ), peach ( Prunus persica ), and alpine strawberry ( Fragaria vesca ) were identified that contained previously detected sweetness-related quantitative trait loci (QTL) in at least two of the crops. Sugar related genes from these conserved regions and 789 sugar-associated apple genes were used to identify 279 Rubus candidate transcripts. A Hyb-Seq approach was used in conjunction with PacBio sequencing to generate haplotype level sequence information of sugar-related genes for 40 cultivars with high and low soluble solids content from the University of Arkansas and USDA blackberry breeding programs. Polymorphisms were identified relative to the 'Hillquist' blackberry ( R. argutus ) and ORUS 4115-3 black raspberry ( R. occidentalis ) genomes and tested for their association with soluble solids content (SSC). A total of 173 alleles were identified that were significantly (α = 0.05) associated with SSC. KASP genotyping was conducted for 92 of these alleles on a validation set of blackberries from each breeding program and 48 markers were identified that were significantly associated with SSC. One QTL, qSSC-Ruh-ch1.1, identified in both breeding programs accounted for an increase of 1.5 °Brix and the polymorphisms were detected in the intron space of a sucrose synthase gene. This discovery represents the first environmentally stable sweetness QTL identified in blackberry. The approach demonstrated in this study can be used to develop breeding tools for other crops that have not yet benefited directly from the genomics revolution., (Copyright © 2020 Zurn et al.)

Sweet cherry (Prunus avium L.) trees are both economically important fruit crops but also important components of natural forest ecosystems in Europe, Asia and Africa. Wild and domesticated trees currently coexist in the same geographic areas with important questions arising on their historical relationships. Little is known about the effects of the domestication process on the evolution of the sweet cherry genome. We assembled and annotated the genome of the cultivated variety "Big Star*" and assessed the genetic diversity among 97 sweet cherry accessions representing three different stages in the domestication and breeding process (wild trees, landraces and modern varieties). The genetic diversity analysis revealed significant genome-wide losses of variation among the three stages and supports a clear distinction between wild and domesticated trees, with only limited gene flow being detected between wild trees and domesticated landraces. We identified 11 domestication sweeps and five breeding sweeps covering, respectively, 11.0 and 2.4 Mb of the P. avium genome. A considerable fraction of the domestication sweeps overlaps with those detected in the related species, Prunus persica (peach), indicating that artificial selection during domestication may have acted independently on the same regions and genes in the two species. We detected 104 candidate genes in sweep regions involved in different processes, such as the determination of fruit texture, the regulation of flowering and fruit ripening and the resistance to pathogens. The signatures of selection identified will enable future evolutionary studies and provide a valuable resource for genetic improvement and conservation programs in sweet cherry., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Cherry breeding and genetic studies can benefit from genome-wide genetic marker assays. Currently, a 6K SNP array enables genome scans in cherry; however, only a third of these SNPs are informative, with low coverage in many genomic regions. Adding previously detected SNPs to this array could provide a cost-efficient upgrade with increased genomic coverage across the 670 cM/352.9 Mb cherry whole genome sequence. For sweet cherry, new SNPs were chosen following a focal point strategy, grouping six to eight SNPs within 10-kb windows with an average of 0.6 cM (627 kb) between focal points. Additional SNPs were chosen to represent important regions. Sweet cherry, the fruticosa subgenome of sour cherry, and cherry organellar genomes were targeted with 6942, 2020, and 38 new SNPs, respectively. The +9K add-on provided 2128, 1091, and 70 new reliable, polymorphic SNPs for sweet cherry and the avium and the fruticosa subgenomes of sour cherry, respectively. For sweet cherry, 1241 reliable polymorphic SNPs formed 237 informative focal points, with another 2504 SNPs in-between. The +9K SNPs increased genetic resolution and genome coverage of the original cherry SNP array and will help increase understanding of the genetic control of key traits and relationships among individuals in cherry.

Bloom time in sweet cherry ( Prunus avium L.) is a highly heritable trait that varies between genotypes and depends on the environmental conditions. Bud-break occurs after chill and heat requirements of each genotype are fulfilled, and dormancy is released. Bloom time is a critical trait for fruit production as matching cultivar adaptation to the growing area is essential for adequate fruit set. Additionally, low chilling cultivars are of interest to extend sweet cherry production to warmer regions, and for the crop adaptation to increasing winter and spring temperatures. The aim of this work is to investigate the genetic control of this trait by analyzing multiple families derived from the low chilling and extra-early flowering local Spanish cultivar 'Cristobalina' and other cultivars with higher chilling requirements and medium to late bloom times. Bloom time evaluation in six related sweet cherry populations confirmed a high heritability of this trait, and skewed distribution toward late flowering, revealing possible dominance of the late bloom alleles. SNP genotyping of the six populations (n = 406) resulted in a consensus map of 1269 SNPs. Quantitative trait loci (QTL) analysis using the Bayesian approach implemented by FlexQTL™ software revealed two major QTLs on linkage groups 1 and 2 (qP-BT1.1m and qP-BT2.1m) that explained 47.6% of the phenotypic variation. The QTL on linkage group 1 was mapped to a 0.26 Mbp region that overlaps with the DORMANCY ASSOCIATED MADS-BOX (DAM) genes. This finding is consistent with peach results that indicate that these genes are major determinants of chilling requirement in Prunus . Haplotype analysis of the linkage group 1 and 2 QTL regions showed that 'Cristobalina' was the only cultivar tested that contributed early bloom time alleles for these two QTLs. This work contributes to knowledge of the genetic control of chilling requirement and bloom date and will enable marker-assisted selection for low chilling in sweet cherry breeding programs., (Copyright © 2020 Calle, Cai, Iezzoni and Wünsch.)

Prior to the availability of whole-genome sequences, our understanding of the structural and functional aspects of Prunus tree genomes was limited mostly to molecular genetic mapping of important traits and development of EST resources. With public release of the peach genome and others that followed, significant advances in our knowledge of Prunus genomes and the genetic underpinnings of important traits ensued. In this review, we highlight key achievements in Prunus genetics and breeding driven by the availability of these whole-genome sequences. Within the structural and evolutionary contexts, we summarize: (1) the current status of Prunus whole-genome sequences; (2) preliminary and ongoing work on the sequence structure and diversity of the genomes; (3) the analyses of Prunus genome evolution driven by natural and man-made selection; and (4) provide insight into haploblocking genomes as a means to define genome-scale patterns of evolution that can be leveraged for trait selection in pedigree-based Prunus tree breeding programs worldwide. Functionally, we summarize recent and ongoing work that leverages whole-genome sequences to identify and characterize genes controlling 22 agronomically important Prunus traits. These include phenology, fruit quality, allergens, disease resistance, tree architecture, and self-incompatibility. Translationally, we explore the application of sequence-based marker-assisted breeding technologies and other sequence-guided biotechnological approaches for Prunus crop improvement. Finally, we present the current status of publically available Prunus genomics and genetics data housed mainly in the Genome Database for Rosaceae (GDR) and its updated functionalities for future bioinformatics-based Prunus genetics and genomics inquiry., Competing Interests: The authors declare that they have no conflict of interest.

Fruit firmness is an important market driven trait in sweet cherry (Prunus avium L.) where the desirable increase in fruit firmness is associated with landrace and bred cultivars. The aim of this work was to investigate the genetic basis of fruit firmness using plant materials that include wild cherry (syn. mazzard), landrace and bred sweet cherry germplasm. A major QTL for fruit firmness, named qP-FF4.1, that had not previously been reported, was identified in three sweet cherry populations. Thirteen haplotypes (alleles) associated with either soft or firm fruit were identified for qP-FF4.1 in the sweet cherry germplasm, and the "soft" alleles were dominant over the "firm" alleles. The finding that sweet cherry individuals that are homozygous for the "soft" alleles for qP-FF4.1 are exclusively mazzards and that the vast majority of the bred cultivars are homozygous for "firm" alleles suggests that this locus is a signature of selection. Candidate genes related to plant cell wall modification and various plant hormone signaling pathways were identified, with an expansin gene being the most promising candidate. These results advance our understanding of the genetic basis of fruit firmness and will help to enable the use of DNA informed breeding for this trait in sweet cherry breeding programs.

Tetraploid sour cherry (Prunus cerasus) has an S-RNase-based gametophytic self-incompatibility (GSI) system; however, individuals can be either self-incompatible (SI) or self-compatible (SC). Unlike the situation in the Solanaceae, where self-compatibility accompanying polyploidization is often due to the compatibility of heteroallelic pollen, the genotype-dependent loss of SI in sour cherry is due to the compatibility of pollen containing two nonfunctional S haplotypes. Sour cherry individuals with the S(4)S(6)S(36a)S(36b) genotype are predicted to be SC, as only pollen containing both nonfunctional S(36a) and S(36b) haplotypes would be SC. However, we previously found that individuals of this genotype were SI. Here we describe four nonfunctional S(36) variants. Our molecular analyses identified a mutation that would confer loss of stylar S function for one of the variants, and two alterations that might cause loss of pollen S function for all four variants. Genetic crosses showed that individuals possessing two nonfunctional S(36) haplotypes and two functional S haplotypes have reduced self-fertilization due to a very low frequency of transmission of the one pollen type that would be SC. Our finding that the underlying mechanism limiting successful transmission of genetically compatible gametes does not involve GSI is consistent with our previous genetic model for Prunus in which heteroallelic pollen is incompatible. This provides a unique case in which breakdown of SI does not occur despite the potential to generate SC pollen genotypes.

Use of dwarfing rootstocks has dramatically increased the profitability of fruit production by reducing production costs, reduced chemical use and higher density plantings. Despite the importance of rootstock-induced dwarfing, the cause of this phenomenon is not known. Using two commercially available graft combinations consisting of a sweet cherry scion, 'Bing', on a dwarfing rootstock (Gi5) or a semi-vigorous rootstock (Gi6), we discovered that the difference in grafted tree height was due to a significantly earlier cessation of terminal meristem growth of the scion on Gi5 compared to Gi6 rootstock, rather than shorter metamer length. We then carried out cDNA-AFLP analysis to investigate differential gene expression between the two graft combinations. Transcript-derived fragments (TDFs) identified as differentially expressed were cloned and printed on microarrays for further confirmation of the differential expression. A total of 99 TDFs were identified as differentially expressed between the 'Bing'/Gi5 and 'Bing'/Gi6 samples, including genes involved in transcription regulation, brassinosteroid signaling, flavonoid metabolism and cell wall biosynthesis or modification. Rootstock vigor has a significant effect on gene expression at the scion and the graft union. Timing of the differential gene expression in the dwarf trees coincides with the earlier cessation of terminal shoot growth, suggesting that these differentially expressed genes may be involved in the dwarfing phenomenon.

The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and wood crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. Most rosaceous crops have been enhanced by human intervention through sexual hybridization, asexual propagation, and genetic improvement since ancient times, 4,000 to 5,000 B.C. Modern breeding programs have contributed to the selection and release of numerous cultivars having significant economic impact on the U.S. and world markets. In recent years, the Rosaceae community, both in the United States and internationally, has benefited from newfound organization and collaboration that have hastened progress in developing genetic and genomic resources for representative crops such as apple (Malus spp.), peach (Prunus spp.), and strawberry (Fragaria spp.). These resources, including expressed sequence tags, bacterial artificial chromosome libraries, physical and genetic maps, and molecular markers, combined with genetic transformation protocols and bioinformatics tools, have rendered various rosaceous crops highly amenable to comparative and functional genomics studies. This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.

Tetraploid sour cherry (Prunus cerasus L.) exhibits gametophytic self-incompatibility (GSI) whereby the specificity of self-pollen rejection is controlled by alleles of the stylar and pollen specificity genes, S-RNase and SFB (S haplotype-specific F-box protein gene), respectively. As sour cherry selections can be either self-compatible (SC) or self-incompatible (SI), polyploidy per se does not result in SC. Instead the genotype-dependent loss of SI in sour cherry is due to the accumulation of non-functional S-haplotypes. The presence of two or more non-functional S-haplotypes within sour cherry 2x pollen renders that pollen SC. Two new S-haplotypes from sour cherry, S(33) and S(34), that are presumed to be contributed by the P. fruticosa species parent, the complete S-RNase and SFB sequences of a third S-haplotype, S(35), plus the presence of two previously identified sweet cherry S-haplotypes, S(14) and S(16) are described here. Genetic segregation data demonstrated that the S(16)-, S(33)-, S(34)-, and S(35)-haplotypes present in sour cherry are fully functional. This result is consistent with our previous finding that 'hetero-allelic' pollen is incompatible in sour cherry. Phylogenetic analyses of the SFB and S-RNase sequences from available Prunus species reveal that the relationships among S-haplotypes show no correspondence to known organismal relationships at any taxonomic level within Prunus, indicating that polymorphisms at the S-locus have been maintained throughout the evolution of the genus. Furthermore, the phylogenetic relationships among SFB sequences are generally incongruent with those among S-RNase sequences for the same S-haplotypes. Hypotheses compatible with these results are discussed.

Tetraploid sour cherry (Prunus cerasus) exhibits a genotype-dependent loss of gametophytic self-incompatibility that is caused by the accumulation of non-functional S-haplotypes with disrupted pistil component (stylar-S) and/or pollen component (pollen-S) function. Genetic studies using diverse sour cherry germplasm identified non-functional S-haplotypes for which an equivalent wild-type S-haplotype was present in sweet cherry (Prunus avium), a diploid progenitor of sour cherry. In all cases, the non-functional S-haplotype resulted from mutations affecting the stylar component S-RNase or Prunus pollen component S-haplotype-specific F-box protein (SFB). This study determines the molecular bases of three of these S-haplotypes that confer unilateral incompatibility, two stylar-part mutants (S(6m2) and S(13m)) and one pollen-part mutant (S(13)'). Compared to their wild-type alleles, S(6m2)-RNase has a 1 bp deletion, S(13m) -RNase has a 23 bp deletion and SFB(13)' has a 1 bp substitution that lead to premature stop codons. Transcripts were identified for these three alleles, S(6m2)-RNase, S(13m)-RNase, and SFB(13)', however, these transcripts presumably result in altered proteins with a resulting loss of activity. Our characterization of natural pollen-part and stylar-part mutants in sour cherry along with other natural S-haplotype mutants identified in Prunus supports the view that loss of pollen specificity and stylar rejection evolve independently and are caused by structural alterations affecting the S-haplotype. The prevalence of non-functional S-haplotypes in sour cherry but not in sweet cherry (a diploid) suggests that polyploidization and gene duplication were indirectly responsible for the dysfunction of some S-haplotypes and the emergence of self-compatibility in sour cherry. This resembles the specific mode of evolution in yeast where accelerated evolution occurred to one member of the duplicated gene pair.

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 S(1)-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 Ujfehértói Fürtös carrying the mutated S(1)-haplotype (S(1)'S(4)S(d)S(null)) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type S(1)-haplotype revealed that the mutated S(1)-haplotype confers unilateral incompatibility with a functional pistil component and a nonfunctional pollen component. The altered sour cherry S(1)-haplotype pollen part mutant, termed S(1)', contains a 615-bp Ds-like element within the S(1)-haplotype-specific F-box protein gene (SFB(1)'). This insertion generates a premature in-frame stop codon that would result in a putative truncated SFB(1) containing only 75 of the 375 amino acids present in the wild-type SFB(1). S(1)' along with 2 other previously characterized Prunus S-haplotype mutants, S(f) and S(6m), illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of GSI.

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.