<Emphasis Type="Italic">Prunus</Emphasis> microsatellite marker transferability across rosaceous crops

A total of 145 microsatellite primer pairs from Prunus DNA sequences were studied for transferability in a set of eight cultivars from nine rosaceous species (almond, peach, apricot, Japanese plum, European plum, cherry, apple, pear, and strawberry), 25 each of almond genomic, peach genomic, peach expressed sequence tags (EST), and Japanese plum genomic, 22 of almond EST, and 23 of apricot (13 EST and 10 genomic), all known to produce single-locus and polymorphic simple-sequence repeats in the species where they were developed. Most primer pairs (83.6%) amplified bands of the expected size range in other Prunus. Transferability, i.e., the proportion of microsatellites that amplified and were polymorphic, was also high in Prunus (63.9%). Almond and Japanese plum were the most variable among the diploid species (all but the hexaploid European plum) and peach the least polymorphic. Thirty-one microsatellites amplified and were polymorphic in all Prunus species studied, 12 of which, covering its whole genome, are proposed as the “universal Prunus set”. In contrast, only 16.3% were transferable in species of other Rosaceae genera (apple, pear, and strawberry). Polymorphic Prunus microsatellites also detected lower levels of variability in the non-congeneric species. No significant differences were detected in transferability and the ability to detect variability between microsatellites of EST and genomic origin.     

Characteristics and transferability of new apple EST-derived SSRs to other Rosaceae species

Genic microsatellites or simple sequence repeat markers derived from expressed sequence tags (ESTs), referred to as EST–SSRs, are inexpensive to develop, represent transcribed genes, and often have assigned putative function. The large apple (Malus × domestica) EST database (over 300,000 sequences) provides a valuable resource for developing well-characterized DNA molecular markers. In this study, we have investigated the level of transferability of 68 apple EST–SSRs in 50 individual members of the Rosaceae family, representing three genera and 14 species. These representatives included pear (Pyrus communis), apricot (Prunus armeniaca), European plum (P. domestica), Japanese plum (P. salicina), almond (P. dulcis), peach (P. persica), sour cherry (P. cerasus), sweet cherry (P. avium), strawberry (Fragaria vesca, F. moschata, F. virginiana, F. nipponica, and F. pentaphylla), and rose (Rosa hybrida). All 68 primer pairs gave an amplification product when tested on eight apple cultivars, and for most, the genomic DNA-derived amplification product matched the expected size based on EST (in silico) data. When tested across members of the Rosaceae, 75% of these primer pairs produced amplification products. Transferability of apple EST–SSRs across the Rosaceae ranged from 25% in apricot to 59% in the closely related pear. Besides pear, the highest transferability of these apple EST–SSRs, at the genus level, was observed for strawberry and peach/almond, 49 and 38%, respectively. Three markers amplified in at least one genotype within all tested species, while eight additional markers amplified in all species, except for cherry. These 11 markers are deemed good candidates for a widely transferable Rosaceae marker set provided their level of polymorphism is adequate. Overall, these findings suggest that transferability of apple EST–SSRs across Rosaceae is varied, yet valuable, thereby providing additional markers for comparative mapping and for carrying out evolutionary studies.     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.     

Detection of a 16SrXII phytoplasma associated with sugarcane yellow leaf syndrome in India

Phytoplasma strain was detected in leaves of sugarcane in India exhibiting symptoms of yellowing of midribs. A phytoplasma characteristic 1.2 kb rDNA PCR product was amplified from DNAs of all diseased samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs P1/P7 and f5U/r3U. Restriction fragment length polymorphism (RFLP) analysis of amplified 16S rDNA indicated that diseased sugarcane was infected by phytoplasma. The 16S rDNA sequence of the Indian sugarcane yellow leaf phytoplasma (SCYLP) showed the closest identity (99%) to that of SCYLP in Cuba identified as Macroptilium lathyroides (AY725233), which belongs to 16SrXII (Stolbur group). This is the first record of the detection of SCYLP, and identification of the 16SrXII group of phytoplasma associated with yellow leaf syndrome (YLS) in India.     

SSRs isolated from apple can identify polymorphism and genetic diversity in pear

Apple simple sequence repeats (SSRs) were intergenerically applied to the characterization of 36 pear accessions, including 19 Japanese pears (Pyrus pyrifolia), 7 Chinese pears (P. bretschneideri, P. ussuriensis), 5 European pears (P. communis), 3 wild relatives (P. calleryana), and 2 hybrids between P. pyrifolia and P. communis. All of the tested SSR primers derived from apple produced discrete amplified fragments in all pear accessions. Nucleotide repeats were detected in the amplified bands by both Southern blot and sequencing analysis, and nucleotide sequences of pear were compared with those of apple. The differences in fragment size among pear or between pear and apple were, in many cases, due to the differences in repeat number. Interestingly, the DNA sequence of flanking regions in apple was highly conserved in pear. Hybrids from P. pyrifolia×P. communis showed one fragment inherited from each parent in all scorable cases, which suggested that each primer pair amplified fragments originating from the same locus. A total of 79 alleles were detected from seven SSR loci in pear, and all pear varieties except for the mutants could be differentiated. In conclusion, SSRs isolated from apple are highly conserved in pear and could be utilized as DNA markers in the latter genus. Received: 17 July 2000 / Accepted: 22 September 2000     

Minor oil-producing crops of the United States

The pits and nuts of almond, Persian walnut, pecan, filbert, tung, apricot, prune, peach, cherry and plum; the fruit pulp of avocado and olive; and the seeds of citrus fruits, grape, apple, pear, cranberry and numerous other domestically cultivated plants are sources of valuable oils already in use.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

Detection of 'Candidatus Phytoplasma pini' in Pinus sylvestris trees in Poland

Abnormal shoot branching was observed in Pinus sylvestris trees in Poland. These abnormalities resulted in the formation of dense, ball‐like structures with dwarfed needles. The presence of phytoplasma in the needles of branched and surrounding symptomless shoots was demonstrated using nested‐polymerase chain reaction (PCR) with universal primer pairs that amplified phytoplasma 16S rDNA, as well as using restriction fragment length polymorphic analysis of PCR products. Comparison of nucleotide sequences of DNA samples from three P. sylvestris trees with ball‐like structures revealed that their fragments of 16S rDNA were identical. The nucleotide sequence showed more than 99% similarity with the corresponding fragments of sequence of ‘Candidatus phytoplasma pini’.     

Development of a real-time PCR assay for the detection of Cladosporium fulvum in tomato leaves

Aims: The aim of this study was to develop a sensitive real-time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves. Methods and Results: Three PCR primer pairs were designed based on the nucleotide sequences of: (i) the internal transcribed spacer regions of ribosomal RNA; (ii) a microsatellite region amplified by the microsatellite primer M13; and (iii) the β-tubulin gene of C. fulvum. Each primer pair amplified the expected target DNA fragment from geographically diverse isolates of C. fulvum. No PCR products were amplified with these primer pairs from DNA of other fungal species. Among the three pairs of primers, the primer pair CfF1/CfR1 developed based on the microsatellite region was the most sensitive. Using this sensitive primer pair, a real-time PCR assay was developed to detect early infection of C. fulvum in tomato leaves. Significance and Impact of the Study: DNA regions amplified by the microsatellite primer M13 have a high potential for developing highly sensitive species-specific PCR primers for the detection of phytopathogenic fungi. The real-time PCR assay developed in this study is useful in monitoring early infection of C. fulvum, and can help growers make timely decisions on fungicide application.     

Molecular markers derived from RAPD,SCAR, and the conserved 18S rDNA sequences for classification and identification in<Emphasis Type="Italic"> Pyrus pyrifolia</Emphasis> and<Emphasis Type="Italic"> P</Emphasis>.<Emphasis Type="Italic"> communis</Emphasis>

We generated RAPD, SCAR, and conserved 18S rDNA markers for classifying and identifying cultivars of Pyrus pyrifolia (Japanese pear) and P. communis (European pear). PCR amplification with selected specific primers—LCH327UP and LCH327DOWN—was performed using DNA extracted from 25 P. pyrifolia and P. communis cultivars. The 1,380-bp fragment was amplified from P. communis cvs. Beurre Giffard, Cascade, Conference, Clapps Favorite, Packhams Triumph, and Winter Nelis. RAPD has only a dominant single band of 1,380-bp, however, SCAR has one or more band of the same size. Amplification involving sequence-specific primer pairs LCH346UP and LCH346DOWN resulted in a loss of polymorphism. The 1,190-bp fragment was amplified from all P. pyrifolia cultivars. The conserved sequences of the 18S rDNA fragment of 25 pear cultivars were amplified and analyzed with 42 restriction enzymes. Compared with P. pyrifolia cultivars, they lacked the restriction enzyme site of KpnI and had one less RsaI site. Cultivar Gamcheonbae had a specific PstI restriction site, while cvs. Mansoo and Conference pear digested with AluI showed a different presentation than other cultivars. For the Okusankichi and Shinil pears TaqI was best marker for identification in P. pyrifolia. These results can be adopted for identifying pear cultivars; to date there is no standard marker for identifying the cultivars of fruit trees in Korean fruit tree breeding programs.Communicated by J.S. Heslop-Harrison     

A Plating‐PCR Technique for Detection and Quantification of the Biological Control Agent Agrobacterium vitis Strain E26 in Soil under Controlled Conditions

Agrobacterium vitis strain E26 is a promising biocontrol agent of grapevine crown gall, an economically important disease of grape worldwide. In this report, we developed a Plating‐PCR method that allows specific detection and quantification of E26 by combining classical microbiological techniques with molecular tools. Random amplified polymorphic DNA fingerprints were used to differentiate E26 from other A. vitis strains. A differentially amplified fragment from E26 was sequenced and characterized as a sequence characterized amplified region (SCAR) marker. Two primer pairs were then designed and evaluated for their specificity against E26. One of the two SCAR primer pairs, 740F/R, was further selected for specific detection of strain E26. A plating assay coupled to PCR with the SCAR primers 740F/R allowed the assessment of population dynamics of E26 in non‐sterile grape rhizosphere soil under controlled conditions.     

Molecular Evidence for the Presence of Aster Yellows-Related Phytoplasmas in Lilies with Leaf Scorch and Flower Virescence

Severe leaf scorch symptoms occurred on oriental lily hybrids cv. Woodriff's Memory cultivated in two commercial greenhouses in Poland. Symptoms included leaf necrosis and malformation, flower bud abscission and flower virescence, distortion and abortion. Naturally infected lily plants with severe symptoms in 1999 had retarded growth and leaf chlorosis and they failed to flower the following year. The presence of phytoplasmas in diseased lilies was demonstrated using nested polymerase chain reaction (PCR) assays with universal and 16SrI group specific primer pairs that amplified the phytoplasma 16S rDNA fragment. The PCR products (1.1 kb) of all samples used for restriction fragment length polymorphism analysis had the same restriction profiles after digestion with endonucleases Alu I and Mse I. The restriction profiles of phytoplasma DNA from these plants corresponded to those of an aster yellows phytoplasma reference strain.     

Genetically stable regeneration of apple plants from slow growth

Shoot-tips of apple cultivar `Gala' were stored in vitrousing a low temperature slow-growth culture method. All shoot-tips survived 1-year storage, with a significant height increment over that period. Eight `Gala' single-bud sibling lines were established for genetic analysis. Although cytological examination detected chromosomal variation in plants recovered from slow growth culture, the ploidy remained genetically stable relative to the before-storage cultures. An amplified fragment length polymorphism (AFLP) assay was performed to detect DNA sequence variation. No differences in the DNA fragment patterns were observed using 20 primer combinations between the before-storage and the stored samples. In addition, a methylation sensitive amplified polymorphism (MSAP) assay was performed to investigate the DNA methylation status in both the before-storage and stored samples. It was found that the slow-growth storage resulted in a significant DNA methylation change in the stored shoots compared with the before-storage samples.     

Detection and Molecular Characterization of a Phytoplasma Associated with Big Bud Disease of Tomatoes in Jordan

Tomato big bud was detected for the first time in tomato plants (Lycopersicon esculentum Mill.) in the eastern region (Al‐Mafraq) of Jordan. Infected plants showed proliferation of lateral shoots, hypertrophic calyxes and greening of flower petals. The presence of phytoplasmas in diseased tomato plants was demonstrated using polymerase chain reaction (PCR) assays. The amplified DNAs yielded products of 1.8 kb (primer pair P1/P7) and 1.2 kb (primer pair R16F2/R2) by direct and nested‐PCR, respectively. DNA from tomato isolates T1 and T2 could not be amplified in the nested‐PCR assays when the aster yellow‐specific primer pair R16(1)F1/R1 was used, suggesting that the phytoplasma in these isolates is not genetically related to the 16SrI (aster yellows) group. After restriction fragment length polymorphism (RFLP) analyses, using four endonuclease enzymes (HhaI, RsaI, AluI and Bsp143I) similar patterns were formed among the digested 1.2 kb PCR products of two tomato isolates suggesting that both isolates belonged to the same phytoplasma. Compared with the RFLP profile of the reference strains, no difference in the digestion pattern was found between the tomato isolates and that of the catharanthus phyllody agent from Sudan, indicating that the phytoplasma belongs to 16SrDNA VI (clover proliferation) group.     

GDR (<Emphasis Type="Underline">G</Emphasis> enome <Emphasis Type="Underline">D</Emphasis> atabase for <Emphasis Type="Underline">R</Emphasis> osaceae): integrated web resources for Rosaceae genomics and genetics research

Background  

Peach is being developed as a model organism for Rosaceae, an economically important family that includes fruits and ornamental plants such as apple, pear, strawberry, cherry, almond and rose. The genomics and genetics data of peach can play a significant role in the gene discovery and the genetic understanding of related species. The effective utilization of these peach resources, however, requires the development of an integrated and centralized database with associated analysis tools.     

Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato

We have cloned and sequenced six RAPD fragments tightly linked to the Tm-1 gene which confers tomato mosaic virus (ToMV) resistance in tomato. The terminal ten bases in each of these clones exactly matched the sequence of the primer for amplifying the corresponding RAPD marker, except for one in which the 5-endmost two nucleotides were different from those of the primer. These RAPD clones did not cross-hybridize with each other, suggesting that they were derived from different loci. From Southern-hybridization experiments, five out of the six RAPD clones were estimated to be derived from middle- or high-repetitive sequences, but not from any parts of the ribosomal RNA genes (rDNA), which are known to be tightly linked with the Tm-1 locus. The remaining clone appeared to be derived from a DNA family consisting of a few copies. These six RAPD fragments were converted to sequence characterized amplified region (SCAR) markers, each of which was detectable using a pair of primers having the same sequence as that at either end of the corresponding RAPD clone. All pairs of SCAR primers amplified distinct single bands whose sizes were the same as those of the RAPD clones. In four cases, the SCAR markers were present in the line with Tm-1 but absent in the line without it, as were the corresponding RAPD markers. In the two other cases, the products of the same size were amplified in both lines. When these SCAR products were digested with different restriction endonucleases which recognize 4-bp sequences, however, polymorphisms in fragment length were found between the two lines. These co-dominant markers are useful for differentiating heterozygotes from both types of homozygote.     

AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L) Batsch]: isolation, characterisation and cross-species amplification in Prunus

 We report the sequences of 17 primer pairs of microsatellite loci, which we have cloned and sequenced from two genomic libraries of peach [Prunus persica (L) Batsch] ‘Redhaven’, enriched for AC/GT and AG/CT repeats respectively. For ten of these microsatellite loci we were able to demonstrate Mendelian inheritance in a segregating back-cross population; the remainder did not segregate. The polymorphism of the microsatellites was evaluated in a panel of ten peach genotypes, including true-to-type peaches, nectarines and one canning-peach. Fifteen microsatellites (88%) were polymorphic showing 2–4 alleles each. The mean heterozygosity, averaged over all loci, was 0.32 and significantly higher than that reported in the literature for isozymes and molecular markers, such as RFLPs and RAPDs. We have also assayed the cross-species transportability and found that ten microsatellite (59%) gave apparently correct amplification in all Prunus species surveyed, namely P. domestica (European plum), P. salicina (Japanese plum), P. armeniaca (apricot), P. dulcis (almond), P. persica var. vulgaris (peach), P. persica var. laevis (nectarine), P. avium (sweet cherry) and P. cerasus (sour cherry), with three of them also being amplified in Malus (apple). The remaining microsatellites gave less-extensive amplification. Because of their appreciable polymorphism and wide cross-species transportability, most of these new markers can be integrated into the linkage maps which are currently being constructed in peach, as well as in other stone fruit crops, such as almond, apricot, cherry and plum. Received: 3 September 1998 / Accepted: 28 November 1998     

Specific Detection of Xylella fastidiosa Pierce's Disease Strains

Pierce's disease (PD, Xylella fastidiosa) of grapevine is the primary pathogen limiting vinifera grape production in Florida and other regions of the southeastern United States. Quick and accurate detection of PD strains is essential for PD studies and control. A unique random amplified polymorphic DNA (PD1-1-2) was isolated from a PD strain from Florida. Fragment PD1-1-2 was cloned, sequenced, and found to be 1005 bp in length. PCR primers were designed to utilize these sequence data for PD strain detection. One primer set (XF176f–XF954r) amplified a 779-bp DNA fragment from 34 PD strains including seven pathotypes of X. fastidiosa, but not from strains of Xanthomonas campestris pv. campestris, Xan. vesicatoria or Escherichia coli. A second primer set (XF176f and XF686r) amplified a 511-bp fragment specific to 98 PD strains, but not from strains of citrus variegated chlorosis, mulberry leaf scorch, oak leaf scorch, periwinkle wilt, phony peach, or plum leaf scald. Sequence analysis indicated that RAPD fragment PD1-1-2 contains a Ser-tRNA gene. The PD-specific region includes a TaqI restriction site (TCGA) and is 150 bp downstream of the Ser-tRNA gene. Received: 1 March 1999 / Accepted: 5 April 1999