The Trouble with Genetic Mapping of Raspberry

Lack of variation among black raspberry cultivars is thought to be a limiting factor in fruit production and in breeding improved cultivars. An assessment of the available diversity in black raspberry is needed to effectively develop improved cultivars. Such an assessment was done to estimate the genetic similarities for RAPD markers in 16 black raspberry genotypes and to determine the genetic diversity among these genotypes based on these markers. In addition, the ability to distinguish between the black raspberry genotypes, two red raspberry cultivars ( Rubus idaeus L.), and a blackberry cultivar ( Rubus hybrid) was determined. A similarity matrix from 379 RAPD markers was calculated, and a phylogenetic tree was constructed using the PHYLIP suite of phylogeny software, which revealed the relationship among the genotypes. An average of 81% similarity was calculated among 16 black raspberry genotypes with a maximum similarity of 98% and a minimum of 70%. The average similarity between black raspberry and red raspberry was 41% and was 26% between black raspberry and blackberry. Combined marker profiles from six RAPD primers could be used to distinguish between the 16 black raspberry genotypes. Red raspberry and blackberry could be distinguished from black raspberry by 27 and 29 of 30 RAPD primers tested, respectively. Genetic diversity was most prominent in genotypes from the extremes of the black raspberry indigenous range. Diversifying the germplasm pool for black raspberry cultivar improvement can be achieved through utilizing genotypes from the extremes of the black raspberry range and through interspecific hybridization.

WHAT'S GOING ON IN THE WORLD OF RUBUS BREEDING?

Some raspberry and blackberry breeders are interested in using molecular markers to assist with selection. Simple Sequence Repeat markers (SSRs) have many advantages, and SSRs developed from one species can sometimes be used with related species. Six SSRs derived from the weed R. alceifolius, and 74 SSRs from R. idaeus red raspberry `Glen Moy' were tested on R. idaeus red raspberry selection NY322 from Cornell Univ., R. occidentalis `Jewel' black raspberry, Rubus spp. blackberry `Arapaho', and blackberry selection APF-12 from the Univ. of Arkansas. The two raspberry genotypes are parents of an interspecific mapping population segregating for primocane fruiting and other traits. The two blackberry genotypes are parents of a population segregating for primocane fruiting and thornlessness. Of the six R. alceifolius SSRs, two amplified a product from all genotypes. Of the 74 red raspberry SSRs, 56 (74%) amplified a product from NY322, 39 (53%) amplified a product from `Jewel', and 24 (32%) amplified a product from blackberry. Of the 56 SSRs that amplified a product from NY322, 17 failed to amplify a product from `Jewel' and, therefore, detected polymorphisms between the parents of this mapping population. Twice as many detected polymorphisms of this type between blackberry and red raspberry, since 33 SSRs amplified a product from NY322, but neither of the blackberry genotypes. Differences in PCR product sizes from these genotypes reveal additional polymorphisms. Rubus is among the most diverse genera in the plant kingdom, so it is not surprising that only 19 of the 74 raspberry-derived SSRs amplified a product from all four of the genotypes tested. These SSRs will be useful in interspecific mapping and cultivar development.

A comparative study of raspberry structural and biochemical responses to late leaf rustRosaceae is a family composed of important crops such as apple, peach, strawberry, and raspberry.Among these species, red and black raspberries belong to the genus Rubus and are high-value crops with increasing worldwide demand.However, these plants are affected by several diseases that impact their quality and productivity.Late leaf rust is a fungal disease in raspberries caused by Aculeastrum americanum (Farl.)M. Scholler & U. Braun (syn.Thekopsora americana (Farl.)Aime & McTaggart).While red raspberries (Rubus idaeus L.) are susceptible, black raspberries were previously reported as more resistant (R. occidentalis L.) and immune (R. niveus Thunb.) to this pathogen.Since genetic resistance is a promising way to manage this disease, hybridizing red and black raspberries can provide new cultivars with interesting traits.However, it is important to understand how plants respond to pathogens before developing expensive and long breeding programs.The aim of this thesis was to investigate the histopathological and biochemical responses of red and black raspberries and a hybrid between them to A. americanum colonization.It was observed that the pathogen germinated and colonized all studied raspberries, however, black raspberries and the hybrid had distinctive responses compared to R. idaeus.The compact mesophyll, the pre-and post-formed phenolic compounds, post-formed pectic compounds, and cell collapse in the lesioned area were the main defense mechanisms against A. americanum.Additionally, to structural and biochemical studies of raspberries defense responses, it was performed a comparative genomics analysis to identify the calcium-dependent protein kinases (CDPKs), focusing on the CPK28 orthologs in black raspberry (R. occidentalis) and strawberry (Fragaria vesca L.) as representative of Rosaceae.Both have 26 CDPKs together.The orthologs FvCPK28 and RoCPK28 were cloned and expressed in Nicotiana benthamiana and Arabidopsis cpk28 mutants.It was found that both Rosaceae CPK28 orthologs are plasmamembrane localized, and their overexpression dampened the oxidative burst upon elicitation.These results provided a proof-of-concept to investigate the functional similarities between these proteins and Arabidopsis CPK28.

The triacylglycerol (TAG) compositions of blackberry, red raspberry, black raspberry, blueberry and cranberry seed oils were examined using ultra-performance convergence chromatography-quadrupole time-of-flight mass spectrometry (UPC2-QTOF MS). A total of 52, 53, 52, 59 and 58 TAGs were detected and tentatively identified from the blackberry, red raspberry, black raspberry, blueberry and cranberry seed oils, respectively, according to their accurate molecular weight in MS1 and fragment ion profiles in MS2. OLL was the most abundant TAG in the blackberry, red raspberry and black raspberry seed oils. Furthermore, the fatty acid compositions of the five berry seed oils were directly determined by gas chromatography coupled with mass spectrometry (GC-MS). In addition, the seed oils had total phenolic contents ranging 13.68–177.06 µmol GAE (gallic acid equivalent)/L oil, and significant scavenging capacities against DPPH, peroxyl, and ABTS+ radicals. These results indicated that the combination of UPC2 and QTOF MS could effectively identify and semi-quantify the TAGs compositions of the berry seed oils with sn-position information for the fatty acids. Understanding the TAGs compositions of these berry seed oils could improve the utilization of these potentially high nutritional value oils for human health.

Influence of Variety and Location of Growth on Resulting Bramble Dried Leaf Volatiles

Amphidiploid production of hybrid between black raspberry and Rubus parvifolius L., a wild Asian species, using colchicine treatment

Rubus yellow net virus (RYNV) is a member of the genus Badnavirus (family: Caulimoviridae). RYNV infects Rubus species causing chlorosis of the tissue along the leaf veins, giving an unevenly distributed netted symptom in some cultivars of red and black raspberry. Recently, a strain of RYNV was sequenced from a Rubus idaeus plant in Alberta, Canada, exhibiting such symptoms. The viral genome contained seven open reading frames (ORFs) with five of them in the sense-strand, including a large polyprotein. Here we describe a graft-transmissible strain of RYNV from Europe infecting cultivar 'Baumforth's Seedling A' (named RYNV-BS), which was sequenced using rolling circle amplification, enzymatic digestion, cloning and primer walking, and it was resequenced at a 5X coverage. This sequence was then compared with the RYNV-Ca genome and significant differences were observed. Genomic analysis identified differences in the arrangement of coding regions, promoter elements, and presence of motifs. The genomic organization of RYNV-BS consisted of five ORFs (four ORFs in the sense-strand and one ORF in the antisense-strand). ORFs 1, 2, and 3 showed a high degree of homology to RYNV-Ca, while ORFs 4 and 6 of RYNV-BS were quite distinct. Also, the predicted ORFs 5 and 7 in the RYNV-Ca were absent in the RYNV-BS sequence. These differences may account for the lack of aphid transmissibility of RYNV-BS.

The article gives a comparative analysis of the data obtained by Russian and foreign scientists considering the content of vitamins and other valuable substances in raspberry fruit. That determines high antioxidant properties of raspberry. The goal of the study was to generalize information which shows specific nature of that plant in relation to the complex of vitamins and polyphenol compounds which it contains. The author shows that fruit of raspberry R. idaeus L. consists of 5–40 mg/100 g of vitamin C, folic acid - 26-44 mg/100 g. Consumption of only 100 g of raspberries satisfies daily requirement of vitamin C (60 mg) by 8.3 - 66.7%, in folates (200 mg) by 13–22%. Total anthocyanin content in red raspberry varies within wide range (20–100 mg/100 g), in black raspberry – form 200 to 600 mg/100 g. Raspberry accumulates from 38 to 270 mg/100 g of ellagic acid. In small amounts the cultivated red raspberry consists of the following substances: carotenoids (β-carotene – 9.3 mg/100 g, zeaxanthin – 11 mg/100 g, lutein – 320 mg /100 g), vitamin E – 0.15–0.44 mg/100 g tocopherol equivalents, thiamine – 0,020 mg/100 g, riboflavin – 0.034 mg/100 g, niacin – 0.036 mg/100 g, pyridoxin – 0.05 mg/100 g, biotin – 5.7 mg/100 g. Total content of antioxidants in the cultivated raspberry fruit is 1.71 mg/g (standard quercetin). As a result of the analysis of different literary sources the author determined that raspberry antioxidant properties are mainly connected with high polyphenol content. Contribution of vitamin C into total antioxidant activity is relatively low. Though raspberry fruit chemical composition has already been studied quite well it is necessary to perform further more detailed research of different raspberry cultivars considering their total antioxidant activity as well as certain biochemical components which comprise antioxidant complex of raspberry fruit. The obtained results will form the base for further research aimed at investigating phytochemical compounds of fruit crops which have become an essential part of healthy human diet and developing nutraceutical products.

SUMMARYThe apparent immunity of five cultivars of red raspberry and two of black raspberry from graft inoculation with raspberry vein chlorosis virus (RVCV) was established or confirmed. The segregations obtained from selfs and crosses with infectible cultivars of four of the red raspberry cultivars and the black raspberry cultivar Cumberland indicated that the apparent immunity was not determined by a single major gene. The range in severity of the symptoms expressed in the segregates suggested either that sensitivity to infection is under separate genetic control, or that immunity and sensitivity are opposite expressions of a character which varies continuously rather than discontinuously. Nevertheless, although the precise mechanism of immunity remains unclear, the high proportion of immune segregates obtained in crosses indicated that breeding for immunity from RVCV is feasible and offers the best prospect for control of the virus.

Glycation is associated with several neurodegenerative disorders, including Alzheimer’s disease (AD), where it potentiates the aggregation and toxicity of proteins such as β-amyloid (Aβ). Published studies support the anti-glycation and neuroprotective effects of several polyphenol-rich fruits, including berries, which are rich in anthocyanins. Herein, blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts were evaluated for: (1) total phenolic and anthocyanins contents, (2) free radical (DPPH) scavenging and reactive carbonyl species (methylglyoxal; MGO) trapping, (3) anti-glycation (using BSA-fructose and BSA-MGO models), (4) anti-Aβ aggregation (using thermal- and MGO-induced fibrillation models), and, (5) murine microglia (BV-2) neuroprotective properties. Berry crude extracts (CE) were fractionated to yield anthocyanins-free (ACF) and anthocyanins-enriched (ACE) extracts. The berry ACEs (at 100 μg/mL) showed superior free radical scavenging, reactive carbonyl species trapping, and anti-glycation effects compared to their respective ACFs. The berry ACEs (at 100 μg/mL) inhibited both thermal- and MGO-induced Aβ fibrillation. In addition, the berry ACEs (at 20 μg/mL) reduced H2O2-induced reactive oxygen species production, and lipopolysaccharide-induced nitric oxide species in BV-2 microglia as well as decreased H2O2-induced cytotoxicity and caspase-3/7 activity in BV-2 microglia. The free radical scavenging, reactive carbonyl trapping, anti-glycation, anti-Aβ fibrillation, and microglial neuroprotective effects of these berry extracts warrant further in vivo studies to evaluate their potential neuroprotective effects against AD.

Due to the lack of effective and stable reference genes, studies on functional genes in Rubus, a genus of economically important small berry crops, have been greatly limited. To select the best internal reference genes of different types, we selected four representative cultivars of blackberry and raspberry (red raspberry, yellow raspberry, and black raspberry) as the research material and used RT-qPCR technology combined with three internal stability analysis software programs (geNorm, NormFinder, and BestKeeper) to analyze 12 candidate reference genes for the stability of their expression. The number of most suitable internal reference genes for different cultivars, tissues, and fruit developmental stages of Rubus was calculated by geNorm software to be two. Based on the results obtained with the three software programs, the most stable genes in the different cultivars were RuEEF1A and Ru18S. Finally, to validate the reliability of selected reference genes, the expression pattern of the RuCYP73A gene was analyzed, and the results highlighted the importance of appropriate reference gene selection. RuEEF1A and Ru18S were screened as reference genes for their relatively stable expression, providing a reference for the further study of key functional genes in blackberry and raspberry and an effective tool for the analysis of differential gene expression.

The color difference of rubus fruits is closely related to the composition of flavonoids including anthocyanins

Simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) molecular markers were evaluated for detecting intraspecific variation in 38 commercially important peach and nectarine ( Prunus persica ) cultivars. Out of the 20 SSR primer pairs 17 were previously developed in sweet cherry and three in peach. The number of putative alleles revealed by SSR primer pairs ranged from one to five showing a low level of genetic variability among these cultivars. The average number of alleles per locus was 2.2. About 76% of cherry primers produced amplification products in peach and nectarine, showing a congeneric relationship within Prunus species. Only nine cultivars out of the 38 cultivars could be uniquely identified by the SSR markers. For SRAP, the number of fragments produced was highly variable, ranging from 10 to 33 with an average of 21.8 per primer combination. Ten primer combinations resulted in 49 polymorphic fragments in this closely related set of peaches and nectarines. Thirty out of the 38 peach and nectarine cultivars were identified by unique SRAP fingerprints. UPGMA Cluster analysis based on the SSR and SRAP polymorphic fragments was performed; the relationships inferred are discussed with reference to the pomological characteristics and pedigree of these cultivars. The results indicated that SSR and SRAP markers can be used to distinguish the genetically very close peach and nectarine cultivars as a complement to traditional pomological studies. However, for fingerprinting, SRAP markers appear to be much more effective, quicker and less expensive to develop than are SSR markers.

Several chemicals, used to induce budbreak of deciduous fruit trees in areas lacking sufficient chilling units were applied to 3 cultivars of red raspberry (Rubus idaeus L. cvs. Delmes, Mailing Exploit, and Schoenemann) grown in Bet Dagan, Israel. All treatments including oil + dinitrol-o-cresol (5%), KNO3 (5%), thiourea (1%), and cyanamide (4%) solutions improved budbreak, and increased yield. The most impressive effect was obtained with 4% cyanamide, which increased fruit size, earliness, and yield.