Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.     

Influence of temperature on the rhythmic emission of volatiles from Ribes nigrum flowers in situ

The floral volatiles of blackcurrant (Ribes nigrum) were collected in situ using the headspace technique. Eleven compounds were identified, including monoterpenes hydrocarbons and monoterpene ethers. The fragrance was emitted in a rhythmic manner, the maximum being in the middle of the photoperiod. Emission at 20°C was significantly higher than at 10°C. The rhythmic nature of emission seemed to cease under conditions of constant light. However, a slight rise in emission during the second period in continuous light may indicate a circadian control of emission. The significance of flower volatiles in relation to pollination is discussed.     

Anticandidal effect of berry juices and extracts from Ribes species

The biological activities of fruit juices and pomace (skin, seeds) extracts from blackcurrant (Ribes nigrum), gooseberry (Ribes uva-crispa) and their hybrid plant (jostaberry, Ribes × nidigrolaria) were evaluated against the most frequently isolated twelve human pathogenic Candida species by broth dilution tests. The phenolic content of juice, water and methanol extracts were measured and the relationship with antifungal activity was assessed. Growth of the most Candida species was inhibited, with the exception of C. albicans, C. krusei, C. lusitaniae and C. pulcherrima. R. nigrum, with the highest phenol content, was observed to have the highest anticandidal activity, indicating a positive correlation between phenol content and antifungal activity.     

Anti‐viral and anti‐bacterial activities of an extract of blackcurrants (Ribes nigrum L.)

The inhibitory effects of an extract of the blackcurrant (Ribes nigrum L.) against pathogens associated with oral, nasopharyngeal and upper respiratory infectious diseases; namely respiratory syncytial virus (RSV), influenza virus A and B (IFV‐A and IFV‐B), adenovirus (AdV), herpes simplex virus type 1, Haemophilus influenzae type B, Streptococcus pneumoniae and Streptococcus mutans, were investigated. Less than 1% concentration of extract of blackcurrant inhibited replication of RSV, IFV‐A and ‐B and HSV‐1 by over 50% and a 10% extract inhibited adsorption of these viruses onto the cell surface by over 95%. The effects on AdV were much less pronounced; the half minimal inhibitory concentration of AdV replication was 2.54 ± 0.26, and a 10% concentration of the extract inhibited AdV adsorption on the cell surface by 72.9 ± 3.4%. The antibacterial activities of the blackcurrant were evaluated based on its efficacy as a disinfectant. A 10% extract disinfected 99.8% of H. Influenzae type B and 78.9% of S. pneumoniae in 10 min, but had no demonstrable effect against S. mutans. The blackcurrant extract still showed antiviral and antibacterial activities after the pH had been made neutral with sodium hydroxide, suggesting that these activities are not the result of acidic reactions or of components precipitated at a neutral pH. These findings demonstrate the potential of blackcurrant extract as a functional food for oral care.     

Estimation of heterozygosity in Ribes nigrum L. using RAPD markers

Heterozygosity in three cultivars of the blackcurrant (Ribes nigrum L.) was estimated. A selfed population of each cultivar was screened for Random Amplified Polymorphic DNA markers (RAPDs) and heterozygous loci were identified by band segregation in contrast with the non-segregation of homozygous loci. On average, 21% of the loci scored in each cultivar were heterozygous. The implications for mapping studies in Ribes nigrum are discussed.     

Linking ascorbic acid production in Ribes nigrum with fruit development and changes in sources and sinks

Background and Aims

Understanding the synthesis of ascorbic acid (l-AsA) in green tissues in model species has advanced considerably; here we focus on its production and accumulation in fruit. In particular, our aim is to understand the links between organs which may be sources of l-AsA (leaves) and those which accumulate it (fruits). The work presented here tests the idea that changes in leaf and fruit number influence the accumulation of l-AsA. The aim was to understand the importance of leaf tissue in the production of l-AsA and to determine how this might provide routes for the manipulation of fruit tissue l-AsA.

Methods

The experiments used Ribes nigrum (blackcurrant), predominantly in field experiments, where the source–sink relationship was manipulated to alter potential leaf l-AsA production and fruit growth and accumulation of l-AsA. These manipulations included reductions in reproductive capacity, by raceme removal, and the availability of assimilates by leaf removal and branch phloem girdling. Natural variation in fruit growth and fruit abscission is also described as this influences subsequent experimental design and the interpretation of l-AsA data.

Key Results

Results show that fruit l-AsA concentration is conserved but total yield of l-AsA per plant is dependent on a number of innate factors many of which relate to raceme attributes. Leaf removal and phloem girdling reduced fruit weight, and a combination of both reduced fruit yields further. It appears that around 50 % of assimilates utilized for fruit growth came from apical leaves, while between 20 and 30 % came from raceme leaves, with the remainder from ‘storage’.

Conclusions

Despite being able to manipulate leaf area and therefore assimilate availability and stored carbohydrates, along with fruit yields, rarely were effects on fruit l-AsA concentration seen, indicating fruit l-AsA production in Ribes was not directly coupled to assimilate supply. There was no supporting evidence that l-AsA production occurred predominantly in green leaf tissue followed by its transfer to developing fruits. It is concluded that l-AsA production occurs predominantly in the fruit of Ribes nigrum.     

Development and characterization of microsatellite markers for the tropical tree species Tabebuia aurea (Bignoniaceae)

In this study, we report the development and characterization of 21 polymorphic microsatellite loci for Tabebuia aurea, using genomic library enrichment. Number of alleles per locus and expected heterozygosity ranged from nine to 26 and from 0.808 to 0.955. The high combined probability of genetic identity (1.03 × 10^?37) and probability of paternity exclusion (0.9889) showed that multilocus genotypes are likely to be unique and will allow detailed parentage studies in natural populations of T. aurea. Additionally, a high percentage of transferability was achieved for the four species of the same genus studied.     

Development and characterization of EST‐SSR markers for the genus Rhododendron section Brachycalyx (Ericaceae)

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) for Rhododendron section Brachycalyx in order to elucidate its evolutionary processes and reproductive ecology. Nineteen polymorphic EST‐SSR markers were developed from EST libraries of R. amagianum and R. hyugaense. Polymorphisms for these markers were assessed using four species of section Brachycalyx. The number of alleles ranged from 1 to 14, and the observed and expected heterozygosity ranged from 0.000 to 0.931 and 0.000 to 0.904, respectively. The EST‐SSR markers developed in this study will be useful for elucidating population genetic structure and breeding systems in section Brachycalyx.     

Development and characterization of microsatellite markers for Prosopis chilensis and Prosopis flexuosa and cross‐species amplification

Prosopis chilensis and Prosopis flexuosa (Fabaceae) are closely related hardwood arboreal species that are widely distributed in the arid regions of Argentina. The development of highly polymorphic markers, such as microsatellites, is desirable for genetic studies of these species. Here, we present the development and characterization of six polymorphic microsatellite markers in P. chilensis and P. flexuosa. These markers showed a polymorphism information content between 0.14 and 0.85 and the number of alleles varied from two to 13 considering both species. All markers revealed a broad cross‐species affinity when tested in seven other Prosopis species. All primers amplified in at least five species.     

Development and characterization of 10 microsatellite markers for the semi-evergreen tree species, Ligustrum ovalifolium (Oleaceae)

Ligustrum ovalifolium is a semi-evergreen tree distributed in the western part of Japan and southern Korea. This species contains an insular endemic variety, L. ovalifolium var. pacificum; this variety occurs only on the Izu Islands located south of the Japanese mainland Honshu. We isolated 10 polymorphic microsatellite loci from the species, and characterized them for 21 individuals from a population of L. ovalifolium. The primers developed in this study yielded an average 12.2 alleles per locus and an average expected heterozygosity of 0.78. These markers will be powerful tools for studying the genetic differentiation within the species.     

Development and characterization of genomic SSR markers in Cynodon transvaalensis Burtt-Davy

Simple sequence repeat (SSR) markers are a major molecular tool for genetic and genomic research that have been extensively developed and used in major crops. However, few are available in African bermudagrass (Cynodon transvaalensis Burtt-Davy), an economically important warm-season turfgrass species. African bermudagrass is mainly used for hybridizations with common bermudagrass [C. dactylon var. dactylon (L.) Pers.] in the development of superior interspecific hybrid turfgrass cultivars. Accordingly, the major objective of this study was to develop and characterize a large set of SSR markers. Genomic DNA of C. transvaalensis ‘4200TN 24-2’ from an Oklahoma State University (OSU) turf nursery was extracted for construction of four SSR genomic libraries enriched with [CA] _n , [GA] _n , [AAG] _n , and [AAT] _n as core repeat motifs. A total of 3,064 clones were sequenced at the OSU core facility. The sequences were categorized into singletons and contiguous sequences to exclude redundancy. From the two sequence categories, 1,795 SSR loci were identified. After excluding duplicate SSRs by comparison with previously developed SSR markers using a nucleotide basic local alignment tool, 1,426 unique primer pairs (PPs) were designed. Out of the 1,426 designed PPs, 981 (68.8 %) amplified alleles of the expected size in the donor DNA. Polymorphisms of the SSR PPs tested in eight C. transvaalensis plants were 93 % polymorphic with 544 markers effective in all genotypes. Inheritance of the SSRs was examined in six F₁ progeny of African parents ‘T577’ × ‘Uganda’, indicating 917 markers amplified heritable alleles. The SSR markers developed in the study are the first large set of co-dominant markers in African bermudagrass and should be highly valuable for molecular and traditional breeding research.     

Pollen Morphology of Ribes L. and Its Taxonomic Significance

The pollen morphology of 19 species and 2 varieties in the genus Ribes Linn. was examined under light microscope (LM) and scanning electron microscope (SEM). Pollen grains of the genus are subspheroidal, 15.0-41.67 μm in diameter, more or less circular to obtusely quadrangular in outline with all sides slightly concave, or with alternating concave sides, or elliptic under SEM, 5-, 6-zonocolporate or 5-, 6-pantoporate. Exine usually thin, 1.0-1.5μm, without visible differentiation between nexine and sexine. Exine is psilate or sometimes coarsely or finely granular or verrucate. The most important characteristic of pollen in this genus is the presence of a “rugoid Area” (ectoaperture) around the endoaperture under SEM. It is different from the other taxa of Saxifragaceae (s. 1.). Based on the pollen morphology and the external morphological characteristics, the treatment of the genus Ribes as an independent family (i.e. Grossulariaceae) by many taxonomists is reasonable. The pollen grains of the genus can be divided into 4 types from the materials examined: (1) Grossularia-type. ectoaperture colpate, with two endoapertures, exine psilate. (2) Ribes-type: ectoaperture porous or colpate, with one or two endoapertures, exine mostly psilate. (3) Grossularioides-type: ectoaperture porous, circular, small, with one endoaperture, exine verrucate. (4) Berisia-type: ectoaperture porous, subcircular to circular, large, with one endoaperture exine mostly psilate. The observation made by the present authors supports the subdivision of Ribes into the subgenera Grossularia, Ribes, Grossularioides and Berisia by many taxonomists based on the other morphological characteristics, and the observation by the present authors is in accordance with this view. Pollen information also shows the close relationships among thefour subgenera and therefore they belong to the same genus — Ribes.     

Development and characterization of nine new microsatellite markers in Taraxacum (Asteraceae)

This study aims at developing and characterizing new microsatellite primer pairs in Taraxacum officinale auct. to produce polymorphic markers for genetical and evolutionary studies on apomixis in this sexual‐apomictic complex. A total of 24 diploid plants were tested for allelic polymorphism and heterozygosity. Out of nine loci three deviated significantly from the Hardy–Weinberg equilibrium expectations, probably due to the presence of null‐alleles. Successful cross‐species amplification was obtained for all markers in 29 Taraxacum microspecies from five sections.     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

Development and characterization of microsatellite markers in the Colorado potato beetle,Leptinotarsa decemlineata

The characterization of 11 Leptinotarsa decemlineata microsatellite loci isolated using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol is reported. Three to 15 alleles per locus were detected in 54 beetles collected from four populations of L. decemlineata. The mean number of alleles was 7.4 ± 3.2 and the level of expected heterozygosity ranged from 0.451 to 0.798. The total exclusionary probabilities using these loci for the first and the second parent were 0.982 and 0.999, respectively. These are the first microsatellite loci characterized from the Colorado potato beetles that can be used for estimating genetic diversity, population structure and parentage analysis.     

Some New Taxa of the Genus Ribes (Saxifragaceae) of China

Fifteen new taxa of the genus Ribes L. (Saxifragaceae) are described from China. These new taxa are R. alpestre var. eglandulosum L. T. Lu, R. burejense var. villosum L. T. Lu, R. moupinense var. pubicarpum L. T. Lu, R. himalense var. pubicalycinum L. T. Lu et J. T. Pan, R. meyeri var. pubescens L. T. Lu, R. davidi var. ciliatum L. T. Lu, R. davidi var. lobatum L. T. Lu, R. laurifolium var. yunnanense L. T. Lu, R. xizangense L. T. Lu, R. glabricalycinum L. T. Lu, R. tenue var. incisum L. T. Lu, R. vilmorinii var. pubicarpum L. T. Lu, R. rubrisepalum L. T. Lu, R. glabrifolium L. T. Lu, R.fasciculatum var. guizhouense L. T. Lu.     

Development and characterization of SSR markers in lychee (Litchi chinensis)

Sixteen microsatellite loci were isolated from lychee (Litchi chinensis), all of which exhibited polymorphism (two or three alleles per locus), with levels of heterozygosity ranging from 0.021 to 0.900. These loci can help assess the genetic structure of lychee.     

Development and characterization of 11 microsatellite markers in the rock sparrow,Petronia petronia

Eleven polymorphic microsatellite loci were isolated in the passeriform Petronia petronia using the enrichment protocol FIASCO (fast isolation by AFLP of sequences containing repeats). We detected three to 13 alleles per locus in 25 specimens collected from an Italian population. The level of expected heterozygosity ranged from 0.439 to 0.856. One locus is sex linked to the Z chromosome. The total exclusionary probabilities using these loci for the first and the second parent were 0.978 and 0.999, respectively. These are the first microsatellite loci characterized from the rock sparrow that can be used for estimating population structure and for large‐scale parentage analysis.     

Isolation of microsatellite markers in Cannabis sativa L. (marijuana)

We have identified 15 variable microsatellite loci in Cannabis sativa. In 48 samples from five fibre crop seed accessions, we detected an average of 10 alleles per locus (range 2–28) with mean heterozygosity of 0.68 (range 0.28–0.94). Significant genetic differentiation was found between accessions (F_ST = 0.12, P < 0.001). These markers have utility for characterizing genetic diversity in cultivated and naturalized Cannabis populations.     

穗醋栗dfr的克隆及其在果实成熟过程中的表达

对穗醋栗花色苷合成的分子机理知之甚少。拟探究穗醋栗花色苷合成关键基因dfr对不同颜色醋栗花色苷的影响,以黑穗醋栗(Ribes nigrum L.)、红穗醋栗(Ribes rubrum L.)和白穗醋栗(Ribes albrum L.)果实为试材,通过RACE方法克隆二氢黄酮醇4-还原酶(dfr)基因cDNA全长序列,分别命名为Rndfr、Rrdfr和Radfr(KY786100、KY786101和KY786102)。系统发育分析表明,Rndfr、Rrdfr和Radfr在进化上具有较高的同源性。测定果实发育不同时期的花色苷含量,结果显示,黑穗醋栗和红穗醋栗花色苷含量较高且随着果实的发育成熟而逐渐增加。而白色醋栗中花色苷含量极低,几乎检测不到花色苷。定量PCR分析表明,dfr在黑穗醋栗中的表达量在果实成熟的各个时期均高于红穗和白穗醋栗。随着果实直径不断变大和果皮着色加深,在黑穗醋栗中,dfr的表达量总体呈现持续上升的趋势;在红穗醋栗中,果实着色约75%时dfr的表达量最高,之后下降;在白穗醋栗中,dfr的表达量总体呈现下降趋势,其表达量最低。推测dfr基因在醋栗果实呈色中发挥作用。