Simple sequence repeat-based assessment of genetic relationships among Prunus rootstocks

Ten SSR loci, previously developed for Prunus, were analyzed to examine genetic relationships among 23 rootstock candidates for sweet and sour cherries, of the species P. avium, P. cerasus, P. mahaleb, and P. angustifolia. Five genotypes of P. laurocerasus, not used as rootstock, were included in the molecular analysis. The number of alleles per locus ranged from 8 to 12, with a mean of 9, while the number of microsatellite genotypes varied from 8 to 17, indicating that the SSRs were highly informative. The degree of heterozygosity (0.61) was high. Clustering analysis resulted in two main clusters. The first cluster was divided into two subclusters; the first subcluster consisted of P. avium and P. cerasus, and the second subcluster consisted of P. laurocerasus. The second cluster was divided into two subclusters. The first subcluster consisted of P. mahaleb genotypes and the second consisted of P. angustifolia genotypes. The reference rootstocks also clustered with their associated botanical species. Unweighted pair-group method with arithmetic mean analysis demonstrated that P. laurocerasus genotypes had less genetic variation and that P. avium genotypes were more closely related to P. cerasus. The SSR-based phylogeny was generally consistent with Prunus taxonomy information, suggesting the applicability of SSR analysis for genotyping and phylogenetic studies in the genus Prunus.     

Development of microsatellite markers in peach [ Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry ( Prunus avium L.)

We report the sequence of 41 primer pairs of microsatellites from a CT-enriched genomic library of the peach cultivar 'Merrill O'Henry'. Ten microsatellite-containing clones had sequences similar to plant coding sequences in databases and could be used as markers for known functions. For microsatellites segregating at least in one of the two Prunus F(2) progenies analyzed, it was possible to demonstrate Mendelian inheritance. Microsatellite polymorphism was evaluated in 27 peach and 21 sweet cherry cultivars. All primer pairs gave PCR-amplification products on peach and 33 on cherry (80.5%). Six PCR-amplifications revealed several loci (14.6%) in peach and eight (19.5%) in sweet cherry. Among the 33 single-locus microsatellites amplified in peach and sweet cherry, 13 revealed polymorphism both in peach and cherry, 19 were polymorphic only on peach and one was polymorphic only on cherry. The number of alleles per locus ranged from 1 to 9 for peach and from 1 to 6 on sweet cherry with an average of 4.2 and 2.8 in peach and sweet cherry, respectively. Cross-species amplification was tested within the Prunus species: Prunus avium L. (sweet cherry and mazzard), Prunus cerasus L. (sour cherry), Prunus domestica L. (European plum), Prunus amygdalus Batsch. (almond), Prunus armeniaca L. (apricot), Prunus cerasifera Ehrh. (Myrobalan plum). Plants from other genera of the Rosaceae were also tested: Malus (apple) and Fragaria (strawberry), as well as species not belonging to the Rosaceae: Castanea (chestnut tree), Juglans (walnut tree) and Vitis (grapevine). Six microsatellites gave amplification on all the tested species. Among them, one had an amplified region homologous to sequences encoding a MADS-box protein in Malus x domestica. Twelve microsatellites (29.3%) were amplified in all the Rosaceae species tested and 31 (75.6%) were amplified in all the six Prunus species tested. Thirty three (80.5%), 18 (43.9%) and 13 (31.7%) gave amplification on chestnut tree, grapevine and walnut tree, respectively.     

Chloroplast inheritance and DNA variation in sweet, sour, and ground cherry

Sour cherry (Prunus cerasus L.) is an allotetraploid and both sweet cherry (P avium L.) and ground cherry (P. fruticosa Pall.) are the proposed progenitor species. The study investigated the maternal species origin(s) of sour cherry using chloroplast DNA (cpDNA) markers and a diverse set of 22 sweet, 25 sour, and 7 ground cherry selections. Two cpDNA restriction fragment length polymorphisms (RFLPs) and one polymerase chain reaction (PCR) fragment length polymorphism were identified among the 54 selections. The three polymorphisms considered together resolved four haplotypes. Analysis of sour cherry progeny indicated that the chloroplast genome is maternally inherited and therefore appropriate to use in determining maternal phylogenetic relationships. Ground cherry was found more likely than sweet cherry to be the maternal progenitor species of sour cherry since 23 of 25 of the sour cherry selections had the most prevalent ground cherry haplotype. However, the other two sour cherry selections tested had the most prevalent sweet cherry haplotype and a wild French sweet cherry selection had the most prevalent ground cherry haplotype. The results underscore the importance of using diverse Prunus germplasm to investigate phylogenetic relationships.     

A pollen-expressed gene for a novel protein with an F-box motif that is very tightly linked to a gene for S-RNase in two species of cherry,Prunus cerasus and P. avium

This study describes a novel F-box protein gene in the S-locus of sour cherry (Prunus cerasus) and sweet cherry (P. avium). The gene showed an S-haplotype-specific sequence polymorphism and the expression was specific to pollen. Genomic DNA blot analysis of eight sweet cherry cultivars with the probe for the F-box protein gene under low stringency conditions yielded RFLP bands specific to the S-haplotypes of each cultivar. We discuss the possibility of the gene for the F-box protein being a candidate for the male determinant of gametophytic self-incompatibility in PRUNUS:     

2n+n Hybridization of Apomictic Paspalum dilatatum with Diploid Paspalum Species

Common dallisgrass (Paspalum dilatatum) is an apomictic pentaploid (2n=5x=50) of hybrid origin with irregular meiosis and with the genome formula IIJJX. The I and J genomes are homologous to those of diploid P. intermedium and P. jurgensii, respectively, but the source of the X genome is unknown. Members of the X genome may have genes of special biological significance, including those controlling apomixis. Common dallisgrass was crossed with several diploid Paspalum species in an attempt to identify the source of the X genome. Since common dallisgrass is apomictic, all hybrids produced will be formed by fertilization of an unreduced egg (2n+n). Any hybrid showing 30 chromosome bivalents at meiosis would indicate that the male diploid parent has a chromosome set that is homologous to the X genome of dallisgrass. Over 36,000 spikelets of dallisgrass were emasculated and dusted with pollen of 15 different diploid species (diploid species bearing I or J genomes were excluded). Only five (P. chaseanum, P. equitans, P. fasciculatum, P. notatum, and P. simplex) produced 2n+n hybrids with P. dilatatum. Meiotic chromosome behavior was similar in all hexaploid hybrids showing ca. 20 bivalents and 20 univalents. Results indicated a very low rate of 2n+n hybridization; none of the five diploid species possessed the X genome. Because several diploid species failed to hybridize with 5x dallisgrass, other methods should be attempted. Molecular markers specific for the X genome may help solve the question.     

Genetic diversity in wild sweet cherries (Prunus avium) in Turkey revealed by SSR markers

Wild sweet cherry (Prunus avium) trees are abundant in the northern part of Turkey, including the Coruh Valley. We analyzed 18 wild sweet cherry genotypes collected from diverse environments in the upper Coruh Valley in Turkey to determine genetic variation, using 10 SSR primers. These SSR primers generated 46 alleles; the number of alleles per primer ranged from 3 to 7, with a mean of 4.6. The primer PS12A02 gave the highest number of polymorphic bands (N = 7), while CPSCT010, UDAp-401 and UDAp-404 gave the lowest number (N = 3). Seven groups were separated in the dendrogram, although most of the genotypes did not cluster according to phenological and morphological traits. This level of genetic diversity in these wild sweet cherry genotypes is very high and therefore these trees would be useful as breeders for crosses between cultivated sweet cherry and wild genotypes.     

Genetic and molecular characterization of three novel S-haplotypes in sour cherry (Prunus cerasus L.)

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.     

Characterization of microsatellites in wild and sweet cherry (Prunus avium L.)--markers for individual identification and reproductive processes.

Nuclear microsatellites were characterized in Prunus avium and validated as markers for individual and cultivar identification, as well as for studies of pollen- and seed-mediated gene flow. We used 20 primer pairs from a simple sequence repeat (SSR) library of Prunus persica and identified 7 loci harboring polymorphic microsatellite sequences in P. avium. In a natural population of 75 wild cherry trees, the number of alleles per locus ranged from 4 to 9 and expected heterozygosity from 0.39 to 0.77. The variability of the SSR markers allowed an unambiguous identification of individual trees and potential root suckers. Additionally, we analyzed 13 sweet cherry cultivars and differentiated 12 of them. An exclusion probability of 0.984 was calculated, which indicates that the seven loci are suitable markers for paternity analysis. The woody endocarp was successfully used for resolution of all microsatellite loci and exhibited the same multilocus genotype as the mother tree, as shown in a single seed progeny. Hence, SSR fingerprinting of the purely maternal endocarp was also successful in this Prunus species, allowing the identification of the mother tree of the dispersed seeds. The linkage of microsatellite loci with PCR-amplified alleles of the self-incompatibility locus was tested in two full-sib families of sweet cherry cultivars. From low recombination frequencies, we inferred that two loci are linked with the S locus. The present study provides markers that will significantly facilitate studies of spatial genetic variation and gene flow in wild cherry, as well as breeding programs in sweet cherry.     

Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map

The concept of selective (or bin) mapping is used here for the first time, using as an example the Prunus reference map constructed with an almond x peach F2 population. On the basis of this map, a set of six plants that jointly defined 65 possible different genotypes for the codominant markers mapped on it was selected. Sixty-three of these joint genotypes corresponded to a single chromosomal region (a bin) of the Prunus genome, and the two remaining corresponded to two bins each. The 67 bins defined by these six plants had a 7.8-cM average length and a maximum individual length of 24.7 cM. Using a unit of analysis composed of these six plants, their F1 hybrid parent, and one of the parents of the hybrid, we mapped 264 microsatellite (or simple-sequence repeat, SSR) markers from 401 different microsatellite primer pairs. Bin mapping proved to be a fast and economic strategy that could be used for further map saturation, the addition of valuable markers (such as those based on microsatellites or ESTs), and giving a wider scope to, and a more efficient use of, reference mapping populations.     

Heterozygote excess in a self-incompatible and partially clonal forest tree species -- Prunus avium L

Wild cherry (Prunus avium L.), a partially asexual self-incompatible forest tree, shows heterozygote excess, which is a poorly studied phenomenon. In three natural populations, we found significant heterozygote excess at almost all investigated loci (eight microsatellites and markers for the self-incompatibility locus). We examined four hypotheses to account for this observed heterozygote excess. First, negative F(IS) can result from a lack of selfed progeny in small populations of outcrossing species. A second explanation for negative F(IS) is selection during the life cycle of the most heterozygous individuals. A third explanation is negative assortative mating when reproduction occurs between individuals bearing phenotypes more dissimilar than by chance. The last explanation for negative F(IS) relies on asexual reproduction. Expectations for each hypothesis were tested using empirical data. Patterns of F(IS) differed among loci. Nevertheless, our experimental results did not confirm the small sample size hypothesis. Although one locus is probably under a hitch-hiking effect from the SI locus, we rejected the effect of the self-incompatibility locus for the genome as a whole. Similarly, although one locus showed a clear pattern consistent with the selection of heterozygous individuals, the heterosis effect over the whole genome was rejected. Finally, our results revealed that clonality probably explains significant negative F(IS) in wild cherry populations when considering all individuals. More theoretical effort is needed to develop expectations and hypotheses, and test them in the case of species combining self-incompatibility and partially asexual reproduction.     

Rapd fingerprinting of diploid Lolium perenne × hexaploid Festuca arundinacea hybrid genomes

We tested the application of RAPD technology for identification of hybrid genomes originated from a maternal clone of Lolium perenne L. (2n = 2x = 14) bearing cytoplasmic male sterility, which was pollinated separately by five clones of Festuca arundinacea Schreb. cv. Barocco (2n = 6x = 42). Six classes of RAPD markers were recognized, specific to: 1) Festuca genome and inherited into F1 hybrid genomes, 2) Lolium genome inherited into F1 hybrid genomes, 3) Lolium-specific bands not found in F1 progeny, 4) Festuca-specific bands not found in F1 progeny, 5) new bands found only in F1 hybrid profiles, 6) bands common to all parental and F1 hybrid genotypes. RAPD data were shown to have full potential a) to serve as an unequivocal proof of genome recombination in perennial ryegrass × tall fescue hybrids, b) to identify hybrid genomes, c) to reveal phenetic relationships of the accessions from crossing families, d) to enhance, by fingerprinting, the selection of superior hybrid material for further breeding. RAPD data were found to be consistent with the festucoid phenotype of F1 hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

A set of simple-sequence repeat (SSR) markers covering the Prunus genome

A set of 109 microsatellite primer pairs recently developed for peach and cherry have been studied in the almond x peach F(2) progeny previously used to construct a saturated Prunus map containing mainly restriction fragment length polymorphism markers. All but one gave amplification products, and 87 (80%) segregated in the progeny and detected 96 loci. The resulting Prunus map contains a total of 342 markers covering a total distance of 522 cM. The approximate position of nine additional simple sequence repeats (SSRs) was established by comparison with other almond and peach maps. SSRs were placed in all the eight linkage groups of this map, and their distribution was relatively even, providing a genome-wide coverage with an average density of 5.4 cM/SSR. Twenty-four single-locus SSRs, highly polymorphic in peach, and each falling within 24 evenly spaced approximately 25-cM regions covering the whole Prunus genome, are proposed as a 'genotyping set' useful as a reference for fingerprinting, pedigree and genetic analysis of this species.     

Reproduction and hybrid load in all-hybrid populations of Rana esculenta water frogs in Denmark

All-hybrid populations of the water frog, Rana esculenta, are exceptional in consisting of independently and to some extent sexually reproducing interspecific hybrids. In most of its range R. esculenta reproduces hemiclonally with one of the parental species, R. lessonae or R. ridibunda, but viable populations of diploid and triploid hybrids, in which no individuals of the parental species have been found, exist in the northern part of the range. We test the hypothesis that nonhybrids arise every year in these all-hybrid populations, but die during larval development. Microsatellite markers were used to determine the genotypes of adults and abnormal and healthy offspring in three all-hybrid populations of R. esculenta in Denmark. Of all eggs and larvae, 63% developed abnormally or died, with some being nonhybrid (genomes matching one of the parental species), many being aneuploid (with noninteger chromosome sets), a few being tetraploid, and many eggs possibly being unfertilized. The 37% surviving and apparently healthy froglets were all diploid or triploid hybrids. In all three populations, gametogenesis matched the pattern previously described for all-hybrid R. esculenta populations in which most triploid adults have two R. lessonae genomes. This pattern was surprising for the one population in which triploid adults had two R. ridibunda genomes, because here it leads to a deficiency of gametes with an R. lessonae genome and should compromise the stability of this population. We conclude that faulty gametogenesis and mating between frogs with incompatible gametes induce a significant hybrid load in all-hybrid populations of R. esculenta, and we discuss compensating advantages and potential evolutionary trajectories to reduce this hybrid load.     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

Allelic diversity of S-RNase at the self-incompatibility locus in natural flowering cherry populations (Prunus lannesiana var. speciosa)

In the Rosaceae family, which includes Prunus, gametophytic self-incompatibility (GSI) is controlled by a single multiallelic locus (S-locus), and the S-locus product expressed in the pistils is a glycoprotein with ribonuclease activity (S-RNase). Two populations of flowering cherry (Prunus lannesiana var. speciosa), located on Hachijo Island in Japan's Izu Islands, were sampled, and S-allele diversity was surveyed based on the sequence polymorphism of S-RNase. A total of seven S-alleles were cloned and sequenced. The S-RNases of flowering cherry showed high homology to those of Prunus cultivars (P. avium and P. dulcis). In the phylogenetic tree, the S-RNases of flowering cherry and other Prunus cultivars formed a distinct group, but they did not form species-specific subgroups. The nucleotide substitution pattern in S-RNases of flowering cherry showed no excess of nonsynonymous substitutions relative to synonymous substitutions. However, the S-RNases of flowering cherry had a higher Ka/Ks ratio than those of other Prunus cultivars, and a subtle heterogeneity in the nucleotide substitution rates was observed among the Prunus species. The S-genotype of each individual was determined by Southern blotting of restriction enzyme-digested genomic DNA, using cDNA for S-RNase as a probe. A total of 22 S-alleles were identified. All individuals examined were heterozygous, as expected under GSI. The allele frequencies were, contrary to the expectation under GSI, significantly unequal. The two populations studied showed a high degree of overlap, with 18 shared alleles. However, the allele frequencies differed considerably between the two populations.     

The potential of Prunus davidiana for introgression into peach [Prunus persica (L.) Batsch] assessed by comparative mapping

The potential for introgression of Prunus davidiana, a wild species related to peach, was evaluated with respect to problems of non-Mendelian segregation or suppressed recombination which often hamper breeding processes based on interspecific crosses. Three connected (F1, F2 and BC2) populations, derived from a cross between P. davidiana clone P1908 and the peach cultivar Summergrand were used. The intraspecific map of P. davidiana already established using the F1 progeny was complemented, and two interspecific maps, for the F2 and BC2 progenies, were built with a set of markers selected from the Prunus reference map. With the molecular data collected for the F2 map construction, regions with distorted marker segregation were detected on the genome; one third of all loci deviated significantly from the expected Mendelian ratios. However, some of these distorted segregations were probably not due to the interspecific cross. On linkage group 6, a skewed area under gametic selection was most likely influenced by the self-incompatibility gene of P. davidiana. Using anchor loci, a good colinearity between the three maps built and the Prunus reference map was demonstrated. Comparative mapping also revealed that homologous recombination occurred normally between P. davidiana and the Prunus persica genome. This confirmed the closeness of the two species. Higher recombination rates were generally observed between P. davidiana and P. persica than between Prunus amygdalus and P. persica. The consequences for plant breeding strategy are discussed. The three maps of the F1, F2 and BC2 progenies provide useful tools for QTL detection and marker-assisted selection, as well as for assessing the efficiency of the peach breeding scheme applied to introgress P. davidiana genes into peach cultivated varieties.     

Polymorphism of heterochromatin C-blocks of rye genome chromosomes in rye-wheat amphidiploids and their chromosome substitution lines

Polymorphism of heterochromatin C-blocks in chromosomes of rye genome has been studied in the F1BC1 hybrids and the D/A substitution lines of rye-wheat amphidiploids (Verasen x L374, Novosibirskaya x x L246)--secalotriticum for revealing cytogenetic markers of rye chromosomes. An increase in polymorphism for the presence and value of heterochromatin blocks in chromosomes of rye genome was shown in the F1BC1 hybrids ((Verasen x L374) x L145 x L145) and unstable genotypes of the F6BC1 chromosome substitution lines (line 118 (Novosibirskaya x L246) x Reso x Reso), line 104 ((Verasen x x L374) x Garmoniya x Garmoniya) that was related to activation of mobile genetic elements present in cereals. Heterochromatin markers of all seven chromosomes in rye genome were revealed. The polymorphism system of heterochromatin blocks may serve as a marker for specificity of the linear chromosome structure during reconstruction of synthetic cereal genomes, and as a test for cytological and morphogenetic stability of hybrid polygenomes in a series of generations.     

The development of a bin mapping population and the selective mapping of 103 markers in the diploid Fragaria reference map.

We have identified a set of plants (the bin set) to permit "selective" or "bin" mapping using the diploid strawberry mapping population FV x FN, derived from the F2 cross F. vesca 815 x F. nubicola 601, which has been used to develop the Fragaria reference map. The bin set consists of 8 plants: the F. vesca 815 parent, the F1 hybrid individual, and 6 seedlings of the F2 population. This bin set divides the 578 cM of the diploid Fragaria genome into 46 bins, the largest mapping bin being 26 cM in length and the average bin size being 12.6 cM. To validate the FV x FN bin set, we used it to locate 103 loci into bins on the FV x FN map. These loci comprised 61 previously described SSRs, 38 new SSRs developed in this investigation from Fragaria x ananassa genomic DNA, EST and gene sequences, and 4 ripening-related genes developed for Prunus. The 103 markers were located to bins on all 7 linkage groups of the Fragaria map and a new mapping bin was identified with the novel markers, demonstrating that the map covers the majority of the diploid Fragaria genome and that the 6 bin-set seedlings selected were appropriate for bin mapping using this progeny.     

Does the bird cherry have its ‘fair share’ of insect pests? An appraisal of the species—area relationships of the phytophagous insects associated with British Prunus species

Abstract. 1. Field and published data reveal that there are at least twenty-three species of phytophagous insects associated with Prunus padus L. in Britain. Most of these species occur on the tree between May and September. Interspecific competition does not appear to be severe.
2. There are a total of 196 species of phytophagous insects feeding upon the various tissues of P. avium, P. cerasifera, P. cerasus, P. domestica, P. laurocerasus, P. padus and P. spinosa.
3. A highly significant species (5)-distribution (A) relationship of the form In (S+l)=0.92 In A-2.19, was obtained for the Prunus species examined within Britain (excluding Ireland), i.e. the wider the distribution of the plant the greater the number of insect species associated with it.
4. However, critical analysis of the data indicate that P. padus does not support as many different insect species as expected.
5. Neither plant form nor age of establishment within the British Isles accounted for this discrepancy.
6. The possibility of using a plant's insect fauna to describe its relationship to other plants in the same genus is discussed.