New findings in apple S-genotype analysis resolve previous confusion and request the re-numbering of some S-alleles

Apple trees display gametophytic self-incompatibility which is controlled by a series of polymorphic S-alleles. To resolve the discrepancies in S-allele assignment that appeared in the literature, we have re-examined the identity of S-alleles known from domestic apple cultivars. Upon an alignment of S-allele nucleotide sequences, we designed allele-specific primer pairs to selectively amplify a single S-allele per reaction. Alternatively, highly similar S-alleles that were co-amplified with the same primer pair were discriminated through their distinct restriction digestion pattern. This is an extension of our previously developed allele-specific PCR amplification approach to reveal the S-genotypes in apple cultivars. Amplification parameters were optimised for the unique detection of the 15 apple S-alleles of which the nucleotide sequences are known. Both the old cultivars with a known S-genotype and a number of more common cultivars were assayed with this method. In most cases, our data coincided with those obtained through phenotypic and S-RNase analysis. However, three S-alleles were shown to relate to RNases that were previously proposed as being encoded by distinct S-alleles. For another S-allele the corresponding gene product has not been discriminated. Consequently, we propose the re-numbering of these four S-alleles. Furthermore, two alleles that were previously identified as S(27a) and S(27b) now received a distinct number, despite their identical S-specificity. To ease widespread future analysis of S-genotypes, we identified common cultivars that may function as a witness for bearing a particular S-allele. We discuss the assignment of new S-alleles which should help to avoid further confusion.     

Thidiazuron substitution for chilling requirement in three apple cultivars

Thidiazuron [(TDZ)N-phenyl-N-1,2,3-thidiazol-5-ylurea] at 750 M was applied to buds of apple trees to determine if it could substitute for the chilling requirement to induce bud break. Shoots of cv. Anna (low chill), Delicious cv. Redchief (medium chill), and Northern Spy (high chill) were untreated, treated with TDZ prior to chilling (before-chill), and treated with TDZ at various intervals after the accumulation of specific amounts of chilling (after-chill). Shoots were stored in a cold room at 4°C. TDZ applied prior to chilling reduced the chill unit (CU) requirement (1 CU = 1 h at 4°C) for the promotion of bud break on 1-year-old shoots of Anna and Northern Spy and 1- and 2-year-old wood of Delicious. TDZ applied after-chill promoted bud break only for Anna and buds on 2-year-old wood of Delicious. While accumulating CUs, untreated buds or buds treated with TDZ on 1-year Delicious and Northern Spy did not respond to the cold treatment even after 1848 h of CU accumulation. For all three cultivars, TDZ treatment was more effective in promoting bud break when applied before the initiation of chilling.The use of a company or product name does not constitute an endorsement by USDA or the University of Maryland nor imply approval to the exclusion of other suitable products.     

Reduced major minerals and increased minor nutrients improve micropropagation in three apple cultivars

Mineral nutrient medium requirements for propagation of in vitro shoots of apple (Malus domestica Borkh) ‘Golden Delicious’, ‘Maksat’, and ‘Voskhod’ were studied using response surface methodology (RSM). The mineral nutritional factors evaluated were based on Murashige and Skoog (MS) mineral nutrients (NH₄NO₃, KNO₃, CaCl₂, KH₂PO₄, MgSO₄, and minor nutrients), with concentrations ranging from 0.5 to 3.0× the MS concentrations. Nine plant growth qualities were evaluated. The most significant factors were NH₄NO₃ at 0.5 to 1.0× MS, and minor nutrients at 2.0× MS. Most of the other factors were optimal at 0.5×. The quality rating was highest when minor nutrients were 2.0× MS, and most other nutrients were standard concentrations or lower. Increased KH₂PO₄ and minor nutrients were the most significant for improved multiplication, and higher KNO₃ for shoot length. Optimized media were developed for each cultivar based on these models. The cultivars were grown on the three individual optimized media, a general medium based on the three optimizations, and MS. The optimized medium for each cultivar was significantly better for shoot quality and shoot length of each cultivar than MS, but the generalized medium of minors at 2.0× and NH₄NO₃, CaCl₂, and MgSO₄ at 0.5× MS, was significantly better for two of the three cultivars and not significantly different for the third. The next step to develop a final optimized medium will require the evaluation of the minor nutrients, determination of optimal concentrations of each, and screening a wide range of Malus germplasm on the finalized medium.

     

A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars

 Commercial scale fingerprinting of potato cultivars is made difficult by the need for speed, reliability and the ability to distinguish between large numbers of genotypes. There are also problems in extrapolating the results of small experimental studies to predict the performance of techniques or primers for larger applications. The potential of ISSR-PCR for fingerprinting purposes was evaluated using four primers on 34 potato cultivars. The complex band profiles generated were reproducible between repeat PCRs, DNA extractions, electrophoreses and gel scorings. Two primers were each able to distinguish all cultivars. The combined use of any two of the four primers also allowed complete diagnosis. It is concluded that ISSR-PCR provides a quick, reliable and highly informative system for DNA fingerprinting that is amenable for routine applications. Two possible correlates of the ability of primers to distinguish between genotypes were then examined. Marker Index failed to correlate significantly with genotype diagnosis, but a strong and seemingly linear relationship was observed between Resolving Power of a primer and its ability to distinguish genotypes (r²=0.98). Resolving Power of one or a pair of primers was found to provide a moderately accurate estimate of the number of genotypes identified. Possible implications for future studies on DNA fingerprinting are discussed. Received: 7 May 1998 / Accepted: 15 July 1998     

Characterization and density of trichomes on three common bean cultivars

Trichomes have been implicated as a mechanism which can confer resistance to both plant pests and drought. A study was conducted to provide information regarding genetic variability for trichome distribution and density among three diverse dry bean (Phaseolus vulgaris L.) cultivars, and to characterize the types of trichomes present among the cultivars. Trichomes on the leaf surfaces were micrographed with a scanning electron microscope (SEM) and counted using a stereomicroscope on both the abaxial and adaxial leaf surfaces of the cultivars ‘Bill Z’, ‘Pompadour Checa’ and ‘Diacol Calima’. Straight, hooked, and glandular trichomes were observed on the leaf surfaces of each cultivar. SEM micrographs are presented for the leaf surfaces of each cultivar and trichome type. The abaxial leaf surface had more straight trichomes than the adaxial leaf surface for ‘Pompadour Checa’ and ‘Diacol Calima’, however ‘Bill Z’ had more on the adaxial surface. The opposite relationship existed among the cultivars and leaf surfaces for the hooked trichomes.     

Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in apple

Although cultivation of genetic modified (GM) annual crops has been steadily increasing in the recent 10 years, the commercial cultivation of GM fruit tree is still very limited and reports of field trials on GM fruit trees are rare. This is probably because development and evaluation of GM fruit trees require a long period of time due to long life cycles of trees. In this study, we report results from a field trial on three rolB transgenic dwarfing apple rootstocks of M26 and M9 together with non-transgenic controls grafted with five non-transgenic scion cultivars. We intended to investigate the effects of transgenic rootstock on non-transgenic scion cultivars under natural conditions as well as to evaluate the potential value of using the rolB gene to modify difficult-to-root rootstocks of fruit trees. The results showed that all rolB transgenic rootstocks significantly reduced vegetative growth including tree height regardless of scion cultivar, compared with the non-transgenic rootstocks. Flowering and fruiting were also decreased for cultivars grown on the transgenic rootstocks in most cases, but the fruit quality was not clearly affected by the transgenic rootstocks. Cutting experiment and RT-PCR analysis showed that the rolB gene was stably expressed under field conditions. PCR and RT-PCR analyses displayed that the rolB gene or its mRNA were not detectable in the scion cultivars, indicating no translocation of the transgene or its mRNA from rootstock to scion. Our results suggest that rolB modified rootstocks should be used in combination with vigorous scion cultivars in order to obtain sufficient vegetative growth and good yield. Alternatively, the rolB gene could be used to dwarf vigorous rootstocks of fruit trees or produce bonzai plants as it can significantly reduce the vegetative growth of plants.     

PCR-based method for identifying the S-genotypes of Japanese pear cultivars

 Japanese pear (Pyrus pyrifolia Nakai), a member of the Rosaceae, shows gametophytic self-incompatibility that is controlled by the S-locus. The S-genotype of Japanese pear cultivars is an important factor for crossing and breeding. We report a rapid reliable method to identify these S-genotypes. It consists of PCR amplification of the S-RNase gene from genomic DNA and subsequent digestion of the PCR fragments with S-allele-specific restriction endonucleases. Using this method, we determined the unknown S-genotypes of nine Japanese pear cultivars and selected self-compatible varieties from the offspring of the self-compatible cultivar, ‘Osa-Nijisseiki’. Received: 8 June 1998 / Accepted: 19 October 1998     

The effect of some macronutrients on adventitious root development on scion apple cultivars in vitro

The influence of some macronutrients, especially NH₄NO₃ and KNO₃, on root development of microcuttings from 3 apple scion cultivars is discussed. A reduction of the level of NH₄NO₃ in the medium from full strength to 1/4 strength significantly increased the percentage rooting of Gala and Royal Gala, but not Jonagold. Further reduction of NH₄NO₃ level from 1/4 strength to zero significantly reduced the percentage of rooting in Gala but not Royal Gala. Jonagold rooted best at zero concentration NH₄NO₃. Without NH₄NO₃, rooting percentages were as high as 100% for all 3 cultivars when KNO₃ was provided at full strength. The results show that adventitious roots can be induced on apple scion cultivars by media manipulation.     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000     

Characterization and mapping of three new mammalian ATP-binding transporter genes from an EST database

Analysis of the human expressed sequence tag (EST) database identified four clones that contain sequences of previously uncharacterized genes, members of the ATP-binding cassette (ABC) superfamily. Two new ABC genes (EST20237, 31252) are located at Chromosome (Chr) 1q42 and 1q25 respectively in humans, as determined by FISH; at locations distinct from previously mapped genes of this superfamily. Two additional clones, EST 600 and EST 1596, were found to represent different ATP-binding domains of the same gene, ABC2. This gene was localized to 9q34 in humans by FISH and to the proximal region of Chr 2 in mice by linkage analysis. All genes display extensive diversity in sequence and expression pattern. We present several approaches to characterizing EST clones and demonstrate that the analysis of EST clones from different tissues is a powerful approach to identify new members of important gene families. Some drawbacks of using EST databases, including chimerism of cDNA clones, are discussed.     

Characterization of glutamine synthetase in the apple

Glutamine synthetase (l -glutamate: ammonia ligase, ADP-forming, EC 6.3.1.2) in bark tissue of the apple (Malus domestica Borkh. cv. Golden Delicious) was partially purified and characterized. The Mn²⁺- and Mg²⁺-dependent activities were maximal at pH 7.2 and 7.5, respectively. The enzyme was almost completely inactivated within two weeks at 0°C. Both Mg²⁺ and β-mercaptoethanol were effective in stabilizing the enzyme during storage. The enzyme was protected from thermal inactivation at 60°C by the addition of Mg²⁺ and ATP. One-tenth mM phenylmercuric acetate inhibited the Mg²⁺-dependent activity by 50%. Equimolar dithiothreitol protected the enzyme from this inactivation. The K_m values of the enzyme were 0.27, 7.35, and 0.69 mM for ATP, glutamate, and NH₂OH, respectively. The constant for NH⁺₄ was an order of magnitude higher in the presence of Mn²⁺ than Mg²⁺. When the amino acids were externally added to the reaction mixtures, the measurement of P_i exhibited a higher degree of enzyme inhibition than the measurement of γ-glutamyl monohydroxamate (GHA). Ten mM histidine inhibited the Mg²⁺- and Mn²⁺-dependent activities by 26 and 45% respectively. Twenty mM aspartate (d,l -form) inhibited the enzyme 30% in the presence of either Mg²⁺ or Mn²⁺. Aspartate (Mg²⁺-dependent) and histidine (Mn²⁺-dependent) inhibited the enzyme competitively with respect to glutamate, the estimated inhibition constants being 17.6 and 1.6 mM, respectively. At 10 mM, amino acids such as tryptophan, arginine, alanine and citrulline inhibited enzyme activity from 1 to 18%. Glutamine stimulated the Mg²⁺-dependent activity 25% at 25 mM when GHA was measured. Glutamine above 32 mM inhibited the enzyme.     

Characterization of an apple anthocyanidin synthase gene in transgenic tobacco plants

Anthocyanin is the major color pigment in plants. The apple anthocyanidin synthase gene(ANS) manifests fruit skin-preferential expression (Kim et al., 2003). To understand the regulatory mechanism for such expression, we isolated and analyzed an appleANS genomic clone. Sequence analysis of ca. 1.4-kb of theANS promoter region predicted several cis-elements for MYB, light responsive GT-1, and the ABA Responsive Element (ABRE). Transgenic tobacco plants carrying a chimeric fusion between theANS promoter and the β-glucuronidase gene(GUS) showed that GUS was expressed in the receptacles and immature seeds as well as in the floral buds, but not in the vegetative organs.     

Validation and development of an immunofluorescence method for isolating and identifying mycobacteria L forms

The complement fixation test and the immunofluorescence test have demonstrated that the L-forms of mycobacteria retain their species-specific and genus-specific determinants and possess serological activity. The L-variants obtained by different methods differ in size, depending on the degree of the destruction of their cell wall. Specific antisera to the L-forms of mycobacteria, suitable for use in the indirect immunofluorescence test, have been obtained. These antisera are highly specific and permit not only the rapid detection, but also identification of the L-forms.     

Genomic characterization of self-incompatibility ribonucleases (S-RNases) in European pear cultivars and development of PCR detection for 20 alleles

Sexual self-incompatibility in European pear (Pyrus communis L.) is controlled by a single locus (S-locus) encoding a polymorphic stylar ribonuclease (S-RNase) that is responsible for the female function in pollen–pistil recognition. In this study, genomic DNA sequences corresponding to five new S-RNase alleles (named S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , and S ₂₄ ) and to S _m were characterized in European pear cultivars. Re-sequencing S _q from ‘General Le Clerc’ showed this S-RNase to encode the same protein as S ₁₂ . Based on these findings, a polymerase chain reaction (PCR)-based method was developed for the molecular typing of cultivars bearing 20 S-RNases (S ₁ –S ₁₄ , S _m , and S ₂₀ –S ₂₄ ) using consensus and allele-specific primers. Genomic PCR with consensus primers amplified product sizes characteristic of the S-RNases S ₁ , S ₂ , S ₄ , S ₁₀ , S ₁₃ , and S ₂₀ . However, the allele groups S ₃ /S ₁₂ , S ₆ /S ₈ /S ₁₁ /S ₂₂ and S ₅ /S ₇ /S ₉ /S ₁₄ /S _m /S ₂₁ /S ₂₃ /S ₂₄ amplified PCR products of similar size. To discriminate between alleles within these groups, primers to specifically amplify each S-RNase were developed. Application of this approach in 19 cultivars with published S-alleles allowed re-evaluation of one of the alleles of ‘Passe Crassane,’ ‘Conference,’ and ‘Condo.’ Finally, this method was used to assign S-genotypes to 37 cultivars. Test crosses confirmed molecular results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phylogeny of Leavenworthia S-alleles suggests unidirectional mating system evolution and enhanced positive selection following an ancient population bottleneck

The adoption of self-fertilization from an ancestral outcrossing state is one of the most common evolutionary transitions in the flowering plants. In the mustard family, outcrossing is typically enforced by sporophytic self-incompatibility (SI), but there are also many self-compatible species. The genus Leavenworthia contains taxa that either possess or lack SI. Here, we present data showing that SI is associated with strict outcrossing and that there is widespread trans-specific sequence polymorphism at the locus involved in the recognition of self-pollen (the S-locus). This ancestral polymorphism is consistent with the presence of an outcrossing mating system in the common ancestor of Leavenworthia species, and suggests that there have been several independent losses of SI in the group. When compared with other mustard species, the bulk of Leavenworthia S-allele sequences are highly diverged from those found in other Brassicaceae and show relatively low levels of nucleotide diversity, a pattern that suggests the common ancestor of the genus likely underwent a strong population bottleneck. The hypothesis of postbottleneck S-locus rediversification is supported by tests showing stronger positive selection acting on S-alleles from Leavenworthia than those found in other Brassicaceae.     

Mapping of quantitative trait loci for fire blight resistance in the apple cultivars 'Florina' and 'Nova Easygro'

Fire blight is a devastating bacterial disease of rosaceous plants. Its damage to apple production is a major concern, since no existing control option has proven to be completely effective. Some commercial apple varieties, such as 'Florina' and 'Nova Easygro', exhibit a consistent level of resistance to fire blight. In this study, we used an F1 progeny of 'Florina'?× 'Nova Easygro' to build parental genetic maps and identify quantitative trait loci (QTLs) related to fire blight resistance. Linkage maps were constructed using a set of microsatellites and enriched with amplified fragment length polymorphism (AFLP) markers. In parallel, progeny plants were artificially inoculated with Erwinia amylovora strain CFBP 1430 in a quarantine glasshouse. Shoot length measured 7?days after inoculation (DAI) and lesion length measured 7 and 14 DAI were used to calculate the lesion length as a percentage of the shoot length (PLL1 and PLL2, respectively). Percent lesion length data were log10-transformed (log10(PLL)) and used to perform the Kruskal-Wallis test, interval mapping (IM), and multiple QTL mapping (MQM). Two significant fire blight resistance QTLs were detected in 'Florina'. One QTL was mapped on linkage group 10 by IM and MQM; it explained 17.9% and 15.3% of the phenotypic variation by MQM with log10(PLL1) and log10(PLL2) data, respectively. A second QTL was identified on linkage group 5 by MQM with log10(PLL2) data; it explained 10.1% of the phenotypic variation. Genotyping the plants of 'Florina' pedigree with the microsatellites flanking the QTLs showed that the QTLs on linkage groups 5 and 10 were inherited from 'Jonathan' and 'Starking' (a 'Red Delicious' sport mutation), respectively. Other putative QTLs (defined as QTLs with LOD scores above the chromosomal threshold and below the genome-wide threshold) were detected by IM on linkage groups 5 and 9 of 'Nova Easygro'.     

Use of an arbitrarily primed PCR product in the development of a Campylobacter jejuni-specific PCR.

Development of a PCR assay for Campylobacter jejuni is based on the isolation of species-specific DNA. An arbitrarily primed PCR incorporating 10-mer primers was used to generate fingerprints of C. jejuni M129 genomic DNA. Fingerprint products were then screened individually for their species specificity in dot blot hybridizations with 6 C. jejuni isolates, 4 Campylobacter species other than C. jejuni, and 27 enteric bacterial species other than Campylobacter spp. A 486-bp fingerprint product hybridized specifically to C. jejuni DNA under stringent conditions; no binding to Campylobacter DNA other than that of C. jejuni or to DNA from enteric bacteria was detected. The 486-bp fingerprint product was sequenced, and primers corresponding to three overlapping regions of the DNA probe were synthesized. Evaluation of the three primer pairs for specificity to C. jejuni DNA identified an oligonucleotide primer pair which amplified a 265-bp product from six C. jejuni isolates only. In sensitivity studies using a crude M129 lysate as the template, the C. jejuni-specific PCR amplified the 265-bp product in a lysate with as few as 100 bacteria.     

Discovery of a new fragrance allele and development of functional markers for identifying diverse fragrant genotypes in rice

Discovery of new fragrance alleles provides important genetic resources for breeding fragrant rice. In this study, a hybrid complementation test demonstrated the association of a new fragrance allele without mutation in the coding region with flavor formation in a fragrant rice variety Nankai 138. The new allele (badh2-p-5′UTR) has a 3-bp deletion in the 5′ untranslated region and an 8-bp insertion in the promoter (?1,314 site upstream from the initiation codon). Surprisingly, we found that there is also an 8-bp insertion in the promoter of the badh2-E7 allele. We developed a new sequence tagged site functional marker to identify the badh2-p-5′UTR and badh2-E7 alleles according to the 8-bp insertion in their promoters. A cleaved amplified polymorphic sequence (AluI) functional marker targeting a common base substitution in the intron 2 of three badh2 alleles, viz. badh2-p-5′UTR, badh2-E7 and badh2-E2, was developed to identify diverse genotypes for fragrance in rice. Based on the results of sequence alignments among the three badh2 alleles, we suggest that the badh2-E7 and badh2-p-5′UTR alleles may have the same genetic origin. In addition, the genetic distance between the badh2-E7 and badh2-p-5′UTR alleles may be closer than that between the badh2-E2 and the badh2-p-5′UTR alleles, or between the badh2-E2 and the badh2-E7 alleles.