QTL analysis and candidate gene mapping for skin and flesh color in sweet cherry fruit (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

Sweet cherry (Prunus avium L.) skin and fruit colors vary widely due to differences in red and yellow pigment profiles. The two major market classes of sweet cherry represent the two color extremes, i.e., yellow skin with red blush and yellow flesh and dark mahogany skin with mahogany flesh. Yet, within these extremes, there is a continuum of skin and flesh color types. The genetic control of skin and flesh color in sweet cherry was investigated using a quantitative trait locus (QTL) approach with progeny derived from a cross between cherry parents representing the two color extremes. Skin and flesh colors were measured using a qualitative color-card rating over three consecutive years and also evaluated quantitatively for darkness/lightness (L*), red/green (a*), and yellow/blue (b*). Segregations for the color measurements (card, L*, a*, and b*) did not fit normal distributions; instead, the distributions were skewed towards the color of the dark-fruited parent. A major QTL for skin and flesh color was identified on linkage group (LG) 3. Two QTLs for skin and flesh color were also identified on LG 6 and LG 8, respectively, indicating segregation for minor genes. The significance and magnitude of the QTL identified on LG 3 suggests the presence of a major regulatory gene within this QTL interval. A candidate gene PavMYB10, homologous to apple MdMYB10 and Arabidopsis AtPAP1, is within the interval of the major QTL on LG 3, suggesting that PavMYB10 could be the major determinant of fruit skin and flesh coloration in sweet cherry.     

Wild and Rare Self-Incompatibility Allele S17 Found in 24 Sweet Cherry (Prunus avium L.) Cultivars

The pollination of self-incompatible diploid sweet cherry is determined by the S-locus alleles. We resolved the S-alleles of 50 sweet cherry cultivars grown in Estonia and determined their incompatibility groups, which were previously unknown for most of the tested cultivars. We used consensus primers SI-19/20, SI-31/32, PaConsI, and PaConsII followed by allele-specific primers and sequencing to identify sweet cherry S-genotypes. Surprisingly, 48% (24/50) of the tested cultivars, including 17 Estonian cultivars, carry the rare S-allele S₁₇, which had initially been described in wild sweet cherries in Belgium and Germany. The S₁₇-allele in Estonian cultivars could originate from ‘Leningradskaya tchernaya’ (S₆|S₁₇), which has been extensively used in Estonian sweet cherry breeding. Four studied cultivars carrying S₁₇ are partly self-compatible, whereas the other 20 cultivars with S₁₇ have not been reported to be self-compatible. The recommended pollinator of seven self-incompatible sweet cherries is of the same S-genotype, including four with S₁₇-allele, suggesting heritable reduced effectiveness of self-infertility. We classified the newly genotyped sweet cherry cultivars into 15 known incompatibility groups, and we proposed four new incompatibility groups, 64–67, for S-locus genotypes S₃|S₁₇, S₄|S₁₇, S₅|S₁₇, and S₆|S₁₇, respectively, which makes them excellent pollinators all across Europe. Alternatively, the frequency of S₁₇ might be underestimated in Eastern European populations and some currently unidentified sweet cherry S-alleles might potentially be S₁₇.

     

Controlled Expression of Recombinant Proteins in <Emphasis Type="Italic">Physcomitrella patens</Emphasis> by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using β-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 °C. In contrast, a short non-damaging heat-treatment at 38 °C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25 °C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.     

Detection and Identification of ‘Candidatus Phytoplasma prunorum’, ‘Candidatus Phytoplasma mali’ and ‘Candidatus Phytoplasma pyri’ in Stone Fruit Trees in Poland

A survey was made to determine the incidence of phytoplasmas in 39 sweet and sour cherry, peach, nectarine, apricot and plum commercial and experimental orchards in seven growing regions of Poland. Nested polymerase chain reaction (PCR) using the phytoplasma‐universal primer pairs P1/P7 followed by R16F2n/R16R2 showed the presence of phytoplasmas in 29 of 435 tested stone fruit trees. The random fragment length polymorphism (RFLP) patterns obtained after digestion of the nested PCR products separately with RsaI, AluI and SspI endonucleases indicated that selected Prunus spp. trees were infected by phytoplasmas belonging to three different subgroups of the apple proliferation group (16SrX‐A, ‐B, ‐C). Nucleotide sequence analysis of 16S rDNA fragment amplified with primers R16F2n/R16R2 confirmed the PCR/Restriction Fragment Length Polymorphism (RFLP) results and revealed that phytoplasma infecting sweet cherry cv. Regina (Reg), sour cherry cv. Sokowka (Sok), apricots cv. Early Orange (EO) and AI/5, Japanese plum cv. Ozark Premier (OzPr) and peach cv. Redhaven (RedH) was closely related to isolate European stone fruit yellows‐G1 of the ‘Candidatus Phytoplasma prunorum’ (16SrX‐B). Sequence and phylogenetic analyses resulted in the highest similarity of the 16S rDNA fragment of phytoplasma from nectarine cv. Super Queen (SQ) with the parallel sequence of the strain AP15 of the ‘Candidatus Phytoplasma mali’ (16SrX‐A). The phytoplasma infecting sweet cherry cv. Kordia (Kord) was most similar to the PD1 strain of the ‘Candidatus Phytoplasma pyri’ (16SrX‐C). This is the first report of the occurrence of ‘Ca. P. prunorum’, ‘Ca. P. mali’ and ‘Ca. P. pyri’ in naturally infected stone fruit trees in Poland.     

Upstream regulatory regions from the maize Sh1 promoter confer tissue-specific expression of the ,-glucuronidase gene in tomato

A promoter fusion (Sh35) combining upstream regulatory regions from the maize Sh1 promoter with a truncated 35S promoter, Δ9035 (–90 to +8) has been compared with the original Sh1 promoter for its capacity to promote expression of the β-glucuronidase (GUS) gene in stably transformed tomato plants. For both promoters, very faint GUS expression was detected in the vegetative tissues, and no expression was detected in the fruit pericarp tissues. However, in the seed, Sh1 promoted low GUS expression but Sh35 directed 25-fold higher GUS expression. For both constructs, the profile of GUS expression was similar to that of endogenous sucrose synthase activity, but maximal GUS activity was reached 15 days after the peak of sucrose synthase activity. Received: 20 October 1998 / Revision received: 1 December 1998 / Accepted: 14 December 1998     

Cotton α-Globulin Promoter: Isolation and Functional Characterization in Transgenic Cotton,Arabidopsis, and Tobacco

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton -globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.     

Characterization of self-compatibility in sweet cherry varieties by crossing experiments and molecular genetic analysis

Self-compatibility is a major breeding objective in sweet cherry. The identification and characterization of new sources of self-compatibility will be useful for breeding and research purposes. In this work, self-compatibility of four local Spanish sweet cherry varieties was investigated by crossing experiments and molecular genetic analysis of two self-incompatibility loci. Crossing experiments included self- and cross-pollinations in the laboratory followed by microscopic observation of pollen tube growth and fruit set assay in the field. After crossing experiments, two accessions, ‘Son Miró’ and ‘Talegal Ahín’, were self-compatible while the other two were self-incompatible. Inheritance of S-locus and microsatellite EMPaS02 (linked to self-compatibility, Sc) were investigated in self-pollination progeny of both self-compatible genotypes. Results indicate that self-compatibility in ‘Talegal Ahín’ is similar to self-compatibility described in sweet cherry ‘Cristobalina’ and may be caused by the same mutation. That is a pollen part mutation not linked to the S-locus but linked to microsatellite EMPaS02 in cherry LG3. In ‘Son Miró’ self-compatibility seems more complex, affecting pollen and style function, and probably involving more than one mutation not described previously in sweet cherry. Together with ‘Cristobalina’, the newly described self-compatible varieties ‘Son Miró’ and ‘Talegal Ahín’ confirm the existence of unique self-compatible plant material in local germplasm from Spain that should be conserved and characterized for its use in breeding and research.     

Tissue-specific and ABA-regulated Maize GIN gene expression in transgenic tobacco

Summary To study the regulatory functions of the ON promoter region, a ppG1b1GUS construct, consisting of 1402 bp 5 flanking sequence ofGlbl, 1919 by GUS coding sequence, and 283 by 3 NOS terminator, was cloned into a binary vector and introduced into tobacco plants byAgrobacterium-mediated transformation. Histochemical GUS assays of T_o tobacco mature seeds indicate that theGlbl promoter drives GUS expression in ABA treated seeds. Further GUS assays of the T, seeds at different developmental stages revealed that without ABA treatment, theGibl promoter drives GUS expression in immature seeds. The results from both T_o and T₁ tobacco plants indicated thatGlbl-driven GUS expression in tobacco is embryo specific.     

Transforming petals into sepaloid organs in<Emphasis Type="Italic"> Arabidopsis</Emphasis> and oilseed rape: implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner

Oilseed rape (Brassica napus L.) genotypes with no or small petals are thought to have advantages in photosynthetic activity. The flowers of field-grown oilseed rape form a bright-yellow canopy that reflects and absorbs nearly 60% of the photosynthetically active radiation (PAR), causing a severe yield penalty. Reducing the size of the petals and/or removing the reflecting colour will improve the transmission of PAR to the leaves and is expected to increase the crop productivity. In this study the hairpin RNA-mediated (hpRNA) gene silencing technology was implemented in Arabidopsis thaliana (L.) Heynh. and B. napus to silence B-type MADS-box floral organ identity genes in a second-whorl-specific manner. In Arabidopsis, silencing of B-type MADS-box genes was obtained by expressing B. napus APETALA3 (BAP3) or PISTILLATA (BPI) homologous self-complementary hpRNA constructs under control of the Arabidopsis A-type MADS-box gene APETALA1 (AP1) promoter. In B. napus, silencing of the BPI gene family was achieved by expressing a similar hpRNA construct as used in Arabidopsis under the control of a chimeric promoter consisting of a modified petal-specific Arabidopsis AP3 promoter fragment fused to the AP1 promoter. In this way, transgenic plants were generated producing male fertile flowers in which the petals were converted into sepals (Arabidopsis) or into sepaloid petals (B. napus). These novel flower phenotypes were stable and heritable in both species.Abbreviations PAR photosynthetically active radiation - ST-LS1 potato light-inducible tissue-specific ST-LS1 gene - GUS -glucuronidase     

大麦与油菜种皮特异启动子表达模式的比较

启动子位于转录起始位点上游并能特异性地结合RNA聚合酶,其作为调控序列驱动外源基因在异源植物中表达,从而实现转基因的高效性,具有时空表达特异性的启动子对获得有效转基因植物及产物具有重要意义。为了解种皮特异启动子的表达模式,该研究基于前期报道的序列,通过同源克隆的方法分别从大麦和油菜中克隆获得Gerb和Bntt两个种皮特异性启动子,并对其进行生物信息学分析,构建了Gerb::GUS和Bntt::GUS植物表达载体并转化拟南芥,通过组织化学染色观察了GUS的表达情况。结果表明:两种启动子序列中都含有多拷贝种皮特异表达启动子元件以及多种胁迫诱导响应元件;转基因拟南芥幼苗期,大麦Gerb种皮特异启动子驱动GUS全株表达且子叶和下胚轴较真叶和根中表达量高;油菜Bntt种皮特异启动子表达较弱;成株期,Gerb在不同组织(叶片、茎、花序和角果)中均有表达,未显示组织特异性;Bntt仅在叶片及角果维管束中有微弱表达。在各种非生物胁迫下,Gerb表达模式未发生显著变化,而Bntt仅在盐胁迫下显示很强的角果和种子特异性表达,其他胁迫未见明显表达。以上结果显示,大麦种皮特异性启动子Gerb和油菜种皮特异性启动子Bntt在时间和空间表达模式上存在差异,这对今后选择种皮特异启动子具有参考作用,但其具体机制仍需进一步研究验证。     

A rapid<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transient assay system

Stable transformation ofArabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality ofcis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-oldArabidopsis seedlings were vacuum-infiltrated withAgrobacterium tumefaciens cultures carrying various upstream regulatory regions controllinguidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3–5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV)35S promoter, the upstream regulatory region of ribosomal protein geneL23A-1, and a temperature-inducible regulatory region (HSP101B) also fromArabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with theHSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d followingAgrobacterium infiltration than those collected 3–4 d postinfiltration.     

Assessment of cultivated cherry germplasm in Iran by multivariate analysis

Key message

This work is an important step in the conservation of genetic cherry resources, which showed distinctive and interesting agronomical characters. Also it introduces suitable genotypes for cultivation and breeding studies.

Abstract

The purpose of this study was to characterize cherry germplasm that is cultivated in Iran. Thirty-three morphopomological parameters were studied in this germplasm, consisting of 70 cherry genotypes (41 sweet cherry, 24 sour cherry and 5 duke cherry genotypes). A wide variation was found in blooming time, ripening time, fruit weight, fruit color, anthocyanin, total soluble solids (TSS), titratable acidity (TA), fruit dimensions and flesh firmness and stone size. There were close positive correlations between fruit weight and fruit dimensions, and between fruit weight and fruit stalk weight, fruit flesh firmness and cracking and also a negative correlation between pH and TA. Dendrogram gave a clear separation between the sour, duke and sweet cherry species and also showed existing intraspecific morphological variation. Based on fruit size and organoleptic properties, the sweet cherry genotypes ‘Siah-Mashhad’, ‘Takdaneh-Mashhad’, ‘Shabestar’, ‘Siah-Daneshkade’, ‘Ghazvin’ and ‘Droongezna’ are recommended for fresh consumption. Good fruit chemical composition and late-ripening time stands out genotypes ‘Dirres-Italia’, ‘Dirres-Pardis’, ‘Maremoot’, ‘Abardeh’ and ‘Rorshon’ and make them suitable for processing. Also, ‘Gilas46’ and ‘Gilas49’ were substantially late-ripening, a characteristic that makes these genotypes highly suitable for breeding studies in case of ripening time. Furthermore, sour cherries ‘Hashtgerd2’ and ‘Hashtgerd3’ and duke cherries ‘Pardis1’ and ‘Pardis3’ were the best genotypes. This work is an important step in the conservation of genetic cherry resources in Iran, which showed distinctive and interesting agronomical characters such as low susceptibility to fruit cracking, high levels of total soluble solids, early fruit maturity and high fruit quality.     

Isolation and characterization of <Emphasis Type="Italic">Brittle2</Emphasis> promoter from <Emphasis Type="Italic">Zea Mays</Emphasis> and its comparison with <Emphasis Type="Italic">Ze19</Emphasis> promoter in transgenic tobacco plants

ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression in embryos, which is different from similar promoters tested in maize.     

Use of a nuclear-targeted β-glucuronidase fusion protein to demonstrate vegetative cell-specific gene expression in developing pollen

To examine the site of expression of the tomato anther-specific gene, LAT52, in the developing male gametophyte, the LAT52 gene promoter was fused to a nuclear-targeted version of the β-glucuronidase (GUS) gene and introduced into tobacco. Transformed plants expressing GUS activity showed nuclear localization of the GUS reaction product to the vegetative cell of the pollen grain. No staining or localization was detected in the generative cell, at pollen maturation or during pollen tube growth in vitro. These results clearly demonstrate differential gene expression within the male gametophyte, and highlight regulatory events which determine the differing fates of the vegetative and generative cells following microspore mitosis.     

Studies on water transport through the sweet cherry fruit surface: IX. Comparing permeability in water uptake and transpiration

Water uptake and transpiration were studied through the surface of intact sweet cherry (Prunus avium L.) fruit, exocarp segments (ES) and cuticular membranes (CM) excised from the cheek of sweet cherry fruit and astomatous CM isolated from Schefflera arboricola (Hayata) Hayata, Citrus aurantium L., and Stephanotis floribunda Brongn. leaves or from Lycopersicon esculentum Mill. and Capsicum annuum L. var. annuum Fasciculatum Group fruit. ES and CM were mounted in diffusion cells. Water (deionized) uptake into intact sweet cherry fruit, through ES or CM interfacing water as a donor and a polyethyleneglycol (PEG 6000, osmotic pressure 2.83 MPa)-containing receiver was determined gravimetrically. Transpiration was quantified by monitoring weight loss of a PEG 6000-containing donor (2.83 MPa) against dry silica as a receiver. The permeability coefficients for osmotic water uptake and transpiration were calculated from the amount of water taken up or transpired per unit surface area and time, and the driving force for transport. Permeability during osmotic water uptake was markedly higher than during transpiration in intact sweet cherry fruit (40.2-fold), excised ES of sweet cherry fruit (12.5- to 53.7-fold) and isolated astomatous fruit and leaf CM of a range of species (on average 23.0-fold). Partitioning water transport into stomatal and cuticular components revealed that permeability of the sweet cherry fruit cuticle for water uptake was 11.9-fold higher and that of stomata 56.8-fold higher than the respective permeability during transpiration. Increasing water vapor activity in the receiver from 0 to 1 increased permeability during transpiration across isolated sweet cherry fruit CM about 2.1-fold. Permeability for vapor uptake from saturated water vapor into a PEG 6000 receiver solution was markedly lower than from liquid water, but of similar magnitude to the permeability during self-diffusion of ³H₂O in the absence of osmotica. The energy of activation for self-diffusion of water across ES or CM was higher than for osmotic water uptake and decreased with increasing stomatal density. The data indicate that viscous flow along an aqueous continuum across the sweet cherry fruit exocarp and across the astomatous CM of selected species accounted for the higher permeability during water uptake as compared to self-diffusion or transpiration.