Development of specific primers for cpSSR analysis in caper,olive and grapevine using consensus chloroplast primer pairs

Chloroplast SSR (cpSSR) markers have demonstrated utility in studying genetic relationships. DNA sequence information of the chloroplast genome is necessary for the development of cpSSR primer pairs. To overcome this limitation, “consensus” primers have been developed to amplify the homologous regions in plants where chloroplast sequences are not available. However, 80% Pinus thunbergi and Nicotiana tabacum developed “consensus” primers tested with grapevine, olive and caper showed multi-locus patterns. The presence of multi-locus patterns requires the use of agarose gel electrophoresis followed from isolation and sequencing of the bands. Herein, a PCR-strategy is proposed to construct specific cpSSR primer pairs without genomic sequence information, giving single-band amplifications that can be directly sequenced. Twelve new specific cpSSR primer pairs were developed for Capparis spinosa L., Olea europea L. and Vitis vinifera L. PCR products were sequenced to confirm the presence of microsatellite sequences, and their transportability was tested on six V. vinifera cultivars. Both single-nucleotide polymorphisms and polymorphic cpSSR were observed in the six grapevine cultivars using the specific cpSSR primers.     

Short days and temperature effects on growth and flowering in strawberry (Fragaria × ananassa Duch.)

Summary

‘Korona’, ‘Elsanta’, ‘Bounty’ and ‘Senga Sengana’ strawberry (Fragaria × ananassa Duch.) plants, were placed at constant temperatures of 9, 15 or 21°C and daylengths of 8 h (short day) or 24 h (long day). The plants were given different numbers of short-day (SD) cycles, and flowering and growth were studied. ‘Korona’ and ‘Elsanta’ were responsive to both short-day treatment and temperature, with optimum flowering at 15°C and 24 SD. ‘Bounty’ was more responsive to temperature, inducing flowers independently of the number of SD cycles at 9°C and 15°C. In ‘Senga Sengana’ flowering was induced independently of temperature and the number of SD cycles, indicating that it had a stronger dependence on other environmental effects. The effect of the number of short-day cycles and the temperature on vegetative growth variâtes such as the number of stolons and daughter plants, the length of flower trusses and petiole length were also studied.     

Phloem unloading follows an extensive apoplastic pathway in developing strawberry fruit

Summary

Strawberry (Fragaria × ananassa) is widely used as a model for fleshy fruit biology research; nevertheless, the pathway of phloem unloading in strawberry fruit remains unknown. Using transmission electron microscopy (TEM) and the symplastic tracer, carboxyfluorescein, the phloem unloading pathway was studied in ‘Akihime’ strawberry fruit from post-anthesis to the ripening stage. Ultrastructural analysis showed that many plasmodesmata existed between the sieve element cells (SEC) and the companion cells (CC), but plasmodesmata were absent between the sieve element cell – CC complexes and their surrounding phloem parenchyma cells (PPCs). Interestingly, an abundance of plasmodesmata was observed between parenchyma cells. Imaging of carboxyfluorescein unloading revealed that the dye was confined only to the phloem vascular bundles in developing receptacles. In conclusion, the results obtained from the present study indicate that sugar unloading from the phloem occurs through an apoplastic pathway. Post-phloem transport of sugar then follows a symplastic pathway in strawberry fruit.     

Molecular marker analyses of pistachio rootstocks by Simple Sequence Repeats and Sequence-Related Amplified Polymorphisms

Summary

Simple Sequence Repeat (SSR) and Sequence-Related Amplified Polymorphism (SRAP) molecular marker systems were used to analyse four commercially important pistachio rootstocks: two species of Pistacia atlantica (cv. ‘Standard Atlantica’), P. integerrima (cv. ‘Pioneer Gold’) and two interspecific hybrids of the same, ‘Pioneer Gold II’ (‘PGII’) and ‘University of California at Berkeley 1’ (‘UCB-1’). A total of 35 putative alleles were detected by 12 SSR primer pairs with an average of 2.9 alleles per locus. The number of putative alleles ranged from 2 to 5 in the pistachio rootstocks tested. The number of bands produced by the SRAP protocol was highly variable, ranging from 11 to 38, with an average of 25.2 per primer combination. Eight primer combinations resulted in 104 (51%) polymorphic markers in these samples. SSR and SRAP markers successfully identified all pistachio rootstocks tested from their unique fingerprints. Both SSR and SRAP molecular markers confirmed that the observed variation in ‘UCB-1’ rootstock is genetic.Thus, there will always be variation among ‘UCB-1’ hybrid seedling progeny due to the segregation of alleles when propagated by seed.We also found evidence of contaminating pollen other than from P. integerrima in some hybrid ‘UCB-1’ rootstock progeny produced by closed pollination. Only alleles from the cultivar ‘Standard Atlantica’ were observed in abnormal ‘UCB-1’ rootstock in the nursery. We found that the poor performance of the scion cv. ‘Kerman’ on ‘UCB-1’ rootstock was not due to ‘UCB-1' rootstocks displaying abnormal behaviour in the nursery. We have successfully developed two efficient marker systems for genome analyses in pistachio, which can be used for identification and management in pistachio rootstock production.     

Comparison of cell wall components between Fragaria × ananassa and Fragaria chiloensis grown in Chile

Summary

Fruit firmness and cell wall composition were compared among five cultivars of F. × ananassa (‘Chandler’, ‘Seascape’, ‘Tudla’, ‘Hokowase’ and ‘Reiko’) and two land races of F. chiloensis (‘94BAU-1A’ and ‘97PUR-1A’) grown in central Chile. Fruits of F. chiloensis were smaller than those of F. × ananassa. Firmness of epidermal tissue did not differ significantly between F. × ananassa and F. chiloensis. Firmness of cortical tissue, however, was significantly lower in F. chiloensis than in F.× ananassa and as a result, difference in the firmness between epidermal and cortical tissues was significantly greater in F. chiloensis than in F. × ananassa. Calcium concentration was low in ‘Reiko’ and ‘Hokowase’ while high in the other varieties of F. × ananassa. F. chiloensis showed intermediate values between these two groups. Higher levels of total uronic acid and neutral sugars in cell wall fractions of F. chiloensis as compared with F. × ananassa were mainly due to its high concentration in the water soluble fraction. Total uronic acid in cell wall fraction was significantly higher in F. chiloensis than in F. × ananassa, mainly due to significantly higher concentrations in the water-soluble fraction. Non-cellulosic neutral sugars, especially arabinose in the water, CDTA, and Na₂CO₃-soluble fractions were significantly higher in F. chiloensis than in F. × ananassa. On the other hand, galactose in the CDTA-, Na₂CO₃-, and KOH-soluble fractions was significantly lower in F. chiloensis than in F. × ananassa. Moreover, cellulose concentration of F. chiloensis was also significantly lower than that of F. × ananassa. Results obtained from cell wall analysis suggest that softer cortex in F. chiloensis might be due to either lower cellulose content, higher solubilization rate of pectic substances, and/or compositional differences in the branching of neutral sugars.     

Evaluation of Strawberry Genotypes for Chaetosiphon fragaefolii (Strawberry Aphid) Preference

ABSTRACT

Thirty-one strawberry genotypes were evaluated for supporting the reproductive success of the strawberry aphid (Chaetosiphon fragaefolii), a vector of several strawberry viruses. A pure colony of C. fragaefolii was initiated from eggs collected from field strawberry leaves in Fall 2013. In Spring 2014 greenhouse-grown strawberry plants with four to five leaves were placed in screened cages (16 genotypes/cage) and five aphids were placed on each plant. After 30–32 days, the number of aphids in each of four developmental stages was counted on each plant. Total aphid numbers/plant ranged from a mean of 33 on Fragaria chiloensis CFRA 48 (PI 551459) to 279 on F. × ananassa ‘AAC Lila’. Cultivars with relatively low numbers of aphids included ‘Bounty’ (106 aphids), ‘Mira’ (114 aphids), and ‘Annapolis’ (115 aphids). This experiment, part of a larger project on aphids and virus diseases associated with the cultivated strawberry, will inform decisions in the strawberry breeding program.     

Application of five DNA marker techniques to distinguish between five apple (Malus × domestica Borkh.) cultivars and their sports

Summary

Genetic variation between five apple cultivars (‘Golden Delicious’, ‘Gala’, ‘Jonagold’, ‘?ampion’, and ‘Idared’) and ten of their sports (‘Golden Delicious Reinders’, ‘Goldrosio’, ‘Gala Must’, ‘Gala Schniga Schnitzer’, ‘Jonagored’, ‘Jonagold Excel’, ‘Szampion Arno’, ‘Szampion Reno Malinowy’, ‘Idaredest’, and ‘Red Idared’) was investigated using five types of DNA markers: Inter-Simple Sequence Repeats (ISSR), Simple Sequence Repeats (SSR), Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), and Inter-Primer Binding Site (iPBS) amplification. In total, 941 polymorphic amplified fragments were obtained using 12 ISSR, 12 SSR, ten AFLP, 19 iPBS, and 15 S-SAP primers or primer pairs. Four of the above-described techniques (except for SSRs with the primer pairs used in this study) were able to distinguish between the sports and their parental cultivar. The most effective technique to distinguish between the genotypes analysed was S-SAP, which detects variations in DNA regions flanking retrotransposon insertion sites.The combined use of ISSR,AFLP, iPBS, and S-SAP markers identified and distinguished all of the sports tested.     

Conditioning is not needed for late summer and early fall production of June-planted ‘Albion’ strawberry (Fragaria X ananassa Duch.) in the plasticulture system

Two plant types [direct planted, cold-stored dormant crowns (crowns) or dormant crowns grown in a greenhouse for 3 weeks prior to field planting (plugs)] of ‘Albion’ strawberry (Fragaria x ananassa Duch.) were evaluated in off-season field production under LD (long day: natural daylength supplemented with 24 h incandescent radiation) or ND (natural daylength) in New Jersey, USA. After 1 week under LD or ND plants received either 100 or 800 ppm N for 4 weeks. Inflorescence, runner, and branch crown production were monitored and fruit harvested from July through September. Field conditioning was ineffective for enhancing total yield of June-planted ‘Albion’ in off-season production. If early summer planting is anticipated, conditioning is not needed. Growing plugs in the greenhouse before transplanting to the production field is not beneficial and reduces productivity with the early summer plantings. Extending the daylength with continuous incandescent lighting to mimic LD during production is not recommended for early summer planting as productivity and fruit size are reduced under continuous low level lighting.     

An inbred line of the diploid strawberry Fragaria vesca f. semperflorens for genomic and molecular genetic studies in the Rosaceae

Background  

The diploid woodland strawberry (Fragaria vesca) is an attractive system for functional genomics studies. Its small stature, fast regeneration time, efficient transformability and small genome size, together with substantial EST and genomic sequence resources make it an ideal reference plant for Fragaria and other herbaceous perennials. Most importantly, this species shares gene sequence similarity and genomic microcolinearity with other members of the Rosaceae family, including large-statured tree crops (such as apple, peach and cherry), and brambles and roses as well as with the cultivated octoploid strawberry, F. ×ananassa. F. vesca may be used to quickly address questions of gene function relevant to these valuable crop species. Although some F. vesca lines have been shown to be substantially homozygous, in our hands plants in purportedly homozygous populations exhibited a range of morphological and physiological variation, confounding phenotypic analyses. We also found the genotype of a named variety, thought to be well-characterized and even sold commercially, to be in question. An easy to grow, standardized, inbred diploid Fragaria line with documented genotype that is available to all members of the research community will facilitate comparison of results among laboratories and provide the research community with a necessary tool for functionally testing the large amount of sequence data that will soon be available for peach, apple, and strawberry.     

Long-day and nitrogen conditioning of ‘Albion’ strawberry (Fragaria X ananassa Duch.) enhances off-season field production

Experiments conducted over 2 years evaluated photoperiod and nitrogen conditioning of ‘Albion’ strawberry (Fragaria x ananassa Duch.) plants for off-season field production in New Jersey, USA. Plants were conditioned the first year with natural days (ND, natural daylength) or long days (LD, natural daylength supplemented with 24 h incandescent radiation) for one week followed by 100 or 800 ppm N for 4 weeks under ND or LD. The second year, plants were conditioned with ND or LD for 1 week followed by 1 to 6 weeks of 100 or 800 ppm nitrogen (N) under ND or LD. After conditioning, plants were established in plasticulture. Ripe fruit were harvested from August through October both years. LD conditioning enhanced precocity by approximately 9 days in both studies. Productivity was also enhanced by conditioning with LD or elevated N. Plants grown on silver or white mulch were more productive than those grown on black mulch in the first study, but not the second.     

Detection of variation among clonal selections of grapevine (Vitis vinifera L.) ‘Kishmish Chernyi’ by AFLP analysis

Summary

Clonal selection is an important method for varietal improvement in grapevine. Ampelometric and morphological markers fail to differentiate clones from their parent genotype. Molecular markers offer the opportunity to identify the clonal material. In this study, five clones of the grapevine variety ‘Kishmish Chernyi’ were analysed using microsatellite (SSR) and AFLP markers. These clones differed significantly in their bunch characteristics including berry size, shape, and colour. Microsatellite (SSR) analysis using 24 primers could not distinguish between these clones. The allele profiles of the clones and the parent variety were identical. AFLP analysis using 13 primer pair combinations yielded 592 markers ranging in size from 50 – 500 bp. Of these, 79 markers (13%) were polymorphic. The majority of the polymorphic markers (75/79) were detected in the clone ‘Sharad Seedless’. Three AFLP primer combinations detected unique markers in three clones which could be useful for future identification.     

Exploiting Illumina sequencing for the development of InDel markers in watermelon (Citrullus lanatus)

Watermelon [Citrullus lanatus (Thunb.) Matsum. &; Nakai var. lanatus] is a cross-pollinating diploid species and is one of the most important dicotyledonous plants belonging to the family Cucurbitaceae due to its high nutritional value, rapid growth, sweet and juicy taste, and refreshing nature as a summer fruit. However, the insertion–deletion (InDel) markers available for watermelon cannot satisfy the demand to identify and map resistance genes in this major crop. In this study, we have characterised and developed numerous polymorphic InDel markers using Illumina re-sequencing of a male parental line that is resistant to Fusarium wilt. In total, 25,701 potential InDel markers were identified, with a mean size of 18 bp. From these, 7,387 potential InDel markers >?10 bp were selected and tested as candidate InDel markers to locate resistance genes for map-based cloning and polymorphism analysis. We designed and synthesised 69 primer pairs using Primer 3 software. Overall, 54 primer pairs detected polymorphism between the Fusarium-susceptible female parent ‘JSw01-1’ and the Fusarium-resistant male parent ‘JRw08-3’. Among the 69 InDel markers, 54 (78.3%) exhibited polymorphism at 179 PCR loci. Polymorphic information content (PIC) values ranged from 0.04 to 0.91, and the number of alleles per primer ranged from 1 to 11. The 54 InDel markers that were mined and exploited via Illumina re-sequencing of watermelon could accelerate the development of marker-assisted breeding and the progression of genetic research to understand resistance to Fusarium wilt in watermelon.     

The Genus <Emphasis Type="Italic">Fragaria </Emphasis>in Croatia

Only three native species of genus Fragaria (F. moschata, F. vesca and F. viridis) are recorded in three regions of Croatia. These species as well as many of their hybrids, are, or once were, cultivated for their edible fruits. The majority of cultivated strawberries in Europe belong to garden strawberries F. x ananassa (hybrids of F. chiloensis and F. virginiana). The most expanded wild strawberry species in Croatia is a woodland strawberry (F. vesca) whose berries are gathered seasonally as wild edible fruits. They often contain higher amount of nutrients and bioactive compounds in comparison to cultivated varieties. The research on the genus Fragaria species distribution in Croatia has not been carried out, and so is the case with many others wild growing fruit species in Croatia. By summing up a number of individual citations and observations, it is possible to get a perspective regarding the current state of their distribution.     

Multiple Avenues to Gender in Strawberries

ABSTRACT

Common strawberry cultivars are hermaphrodites, producing both anthers and pistils in their flowers. However, octoploid Fragaria species are trioecious and different genotypes can be female with pistillate flowers, hermaphrodites, or males with staminate flowers. One female selection, F. virginiana High Falls 22 and three hermaphrodite selections, N8688, RH23, and Montreal River 10 were hybridized with five June-bearing cultivars. Seedlings of each family were planted in Ontario and Michigan. Two F. chiloensis selections Pigeon Point (female) and FRA1267 (hermaphrodite), were hybridized with 14 F. virginiana selections. Seedlings from each family were planted in Ontario and Minnesota. In all progeny, the presence or absence of anthers was recorded and a subjective scale used to estimate the percentage of fruit set (1–10 representing 10% intervals). Also, FRA1267 was crossed with one F. × ananassa selection and the progeny intercrossed and grown in Ontario. In the sib-crosses gender was recorded. In two female parents, the female progeny had on average a higher fruit set than the hermaphrodite progeny. F. virginiana genotypes varied in their fruit set, which suggested that there are a number of alleles involved in the fertility of hermaphrodites. In crosses, FRA1267 produced 80% females when used as a female parent and 100% hermaphrodites when used as a male parent. It is thought that the trait is inherited cytoplasmically or that the F. chiloensis alleles involved are epistatic when F. virginiana or F. × ananassa is used as a male parent.     

苹果柱型基因的ISSR分子标记研究

 以普通型苹果品种‘富士’和柱型苹果‘舞姿’以及其杂交后代的柱型与非柱型实生苗为试材, 建立了苹果的ISSR ( Inter-Simple Squence Repeat polymorphic DNA) 分子标记体系, 并将ISSR 标记用于苹果柱型基因Co 的遗传分析。结果表明, 在20μL 反应体系中各组分的用量为Taq DNA 聚合酶1U、Mg²⁺ 的浓度为2.5 mmol·L^-1 、模板DNA 用量20 ng、引物浓度0.2μmol·L - 1及退火温度52 ℃, 80 %引物具有良好的扩增能力。调整模板DNA 的用量、引物浓度及退火温度能够优化苹果ISSR-PCR 扩增体系。从所筛选的65 个引物中获得了35 个ISSR 标记, 其中33 个标记呈现1∶1 分离, 可用于苹果柱型基因的遗传分析。     

Comparative analysis of genetic diversity in Prunus L. as revealed by RAPD and SSR markers

RAPD and SSR markers were used for genetic diversity evaluations among 15 genotypes selected from the genus Prunus L. Altogether 40 RAPD primers and 21 primer pairs designated for microsatellite loci were applied on the whole group of genotypes.     

Studies on the variability,inheritance, and inter-relationships of mineral macro-nutrients and micro-nutrients in strawberry (Fragariaananassa Duch.)

Summary

Strawberry (Fragaria ananassa Duch.) is a rich source of minerals, vitamins, and anti-oxidants, and has a unique flavour and aroma. However, there is little information on the genetic variability and inheritance of mineral elements in strawberry. This is important as it can influence the selection of better genotypes and suitable breeding approaches for improvements in the nutritional quality of fruit. The objectives of the present study were, therefore, to estimate the variability, inheritance, and inter-relationships of mineral elements, to identify mineral-rich and productive genotypes, and to suggest suitable breeding approaches. A significant genotypic mean square for all the minerals studied indicated sufficient variation among genotypes. Our results also indicated that the effect of genotype on the mineral content of fruit was stronger than that of environment. Six genotypes, namely ‘Ofra’, ‘Sweet Charlie’, ‘Elista’, ‘Festival’, ‘Camarosa’, and ‘Douglas’ were found to have higher mineral contents as well as better fruit yields, and therefore could be useful in further breeding programmes. N, K, Ca, Mg, Fe, Mn, Zn, and Cu contents, together with fruit yield, could be improved by selection and hybridisation as these traits showed high heritability (> 80%) and high genetic advance as a percentage of the mean (> 40%), while recurrent selection was suitable for increasing P contents, which showed moderate heritability (> 79.81%) and low genetic advance as a percentage of the mean (> 33.86%). Moreover, Mg and N contents could be used as reliable tools with which to identify the most productive and mineral-rich genotypes, as indicated by correlation and path analysis. Studies on such genetic estimates will increase our understanding of the inheritance of mineral contents and affect the design of crop improvement strategies.     

Romina and Cristina: Two New Strawberry Cultivars with High Sensorial and Nutritional Values

ABSTRACT

The two cultivars (Romina and Cristina) recently released from the Marche Polytechnic University (Ancona, Italy) strawberry (Fragaria × ananassa) breeding program, show high adaptability to non-fumigated soil. Furthermore, ‘Romina’ showed very early ripening, conic or bi-conic fruit shape, good taste with high sweetness, high firmness and shelf life, and high vitamin C and folate contents. ‘Cristina’ showed very late ripening, high productivity, large fruit of conical shape, good taste, and high vitamin C and folate contents. The European Union patents of ‘Romina’ and ‘Cristina’ have been transferred to the United States, and nursery production has begun following United States rules to determine future market interest.