Fast and reliable strawberry cultivar identification using inter simple sequence repeat (ISSR) amplification

ISSR amplification was evaluated for its applicability to strawberry varietalidentification. Eighteen primers based on various di- tri- or tetra- SSR motifswith 3 or 0 5'-selective nucleotides for anchoring were screened against thestrawberry genome by agarose gel electrophoresis. PCR conditions wereoptimised to obtain high quality patterns. Five primers that gave informativepatterns were selected and used to characterise, by polyacrylamide gelelectrophoresis, 30 strawberry varieties of various geographic and geneticorigins. A total of 390 bands, 113 of which were polymorphic (30%),were generated using these five primers. Genetic similarity between varietieswas estimated using Jaccard's coefficient of similarity. The associationsbetween varieties revealed by UPGMA analysis were consistent withpedigree data. With only one primer, all the varieties were distinguishedincluding those with a common pedigree. Banding patterns were highlyreproducible for DNA samples extracted from different tissues (leaves,sepals, and rhizomes) of the same plant, or from different plants (clones)of the same variety. ISSR technique is therefore a potentially useful tool forthe identification of strawberry varieties because it is simple, fast,cost-effective, highly discriminant and highly reliable.     

Simplified AFLP procedure as a tool for identification of strawberry cultivars and advanced breeding lines

DNA polymorphisms among 6 cultivars of Fragaria × ananassa (Duch.) and 13salinity tolerant clones were evaluated using simplified – PstI based Amplified Fragment Length Polymorphism procedure(^PstIAFLP). Out of 129 amplification products obtained with 10 selective primers, 116 markers were polymorphic and could be used to distinguish all analyzed materials. Coordinate and cluster analyses revealed 2 main groups of clones and divided strawberry cultivars (CUL) and tested F₁ hybrids of ‘Sweet Heart’(HYB). Mean genetic similarities in groups of cultivars and selected breeding lines (SEL) were significantly higher (0.722 and0.706, respectively, p < 0.05) than in group of SH hybrids (0.485). Results suggest that ^PstIAFLP method is sufficient for effective identification and useful for assessing the level of genetic diversity in strawberry cultivars and breeding lines. The presented method can bean alternative multilocus marker system to widespread RAPD method. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bermudagrass cultivar identification by use of isoenzyme electrophoretic patterns

Summary Isoenzyme analysis has been demonstrated as an effective tool for definitive identification of plant cultivars, but it has not been applied to pasture bermudagrass (Cynodon spp.) cultivars in the USA. Polyacrylamide gel electrophoresis (PAGE) was used to study five isoenzyme systems in mitochondrial, microsomal, and soluble cell fractions of actively growing leaves, stems, and roots of seven vegetatively-propagated pasture bermudagrass (Cynodon spp.) cultivars used in the southern half of the USA. Peroxidase, esterase, and, with one exception, acid phosphatase successfully differentiated between the cultivars in all leaf and stem cell fractions. Fewer cultivar differences were found for amino- and endo-peptidases. Only peroxidase and acid phosphatase were resolved from root cell fractions; and only the microsomal fraction differentiated between all cultivars. Within plant parts, cultivars were distinguishable on the basis of peptidase banding in some cellular fractions, but not in others. Plant part and subcellular fraction-specific isoenzyme variations suggest the existence of multiple molecular forms of various enzymes within the same plant.Journal Article 5746 of the Okla. Agric. Exp. Stn.     

Resistance to a single UK isolate ofColletotrichum acutatum in strawberry germplasm from Northern Europe

Summary Colletotrichum acutatum on strawberry was first introduced to the UK on runners of cultivar Brighton, which were imported from California in 1983. Recently further outbreaks have been reported following the importation of infected runner plants from mainland Europe. Phytosanitary precautions have so far been successful in controlling the disease but the current trend in the UK is for increasing late season production. This increases the likelihood of there being fruiting plants when the environmental conditions are favourable toC. acutatum. Fifteen cultivars and 19 HRI breeding lines were tested for susceptibility toC. acutatum using the most pathogenic isolate found on plants imported into the UK. It is likely this isolate was originally of Californian origin. Potted plants were spray inoculated and subsequently rated in the glasshouse using a disease severity rating (DSR) with the range from 0–4, where a rating of 4 indicated severe disease symptoms. In contrast to results from California and Italy standard deviations were variable and often large and consequently it was considered unsafe to classify the susceptibility of the genotypes on the basis of mean DSR alone. Classification was thus based on a combination of the mean value and the distribution of the scores but in some cases the results were inconclusive. It was possible to classify five lines as having a high level of resistance (Elvira, Honeoye, EM99, EM224, EM255); six lines had a useful level of resistance but did show symptoms on some plants (Cambridge Favourite, Gorella, Pandora, Pantagruella, EM17 and EM290); five lines were highly susceptible (Elsanta, Redgauntlet, Tamella, EM237 and EM319). The remaining 18 lines could not be classified with the same degree of certainty but 10 were probably resistant.     

The use of synthetic octoploids in strawberry breeding

Summary A number of methods of producing synthetic octoploids from Fragaria species of lower levels of ploidy are described. Six synthetic octoploids were produced from various combinations involving one hexaploid species, two tetraploid species and four diploid species. Three of the synthetic octoploids are male fertile and were used successfully as male parents in crosses with octoploid strawberry cultivars.     

The use of reversed-phase high performance liquid chromatography as an aid to the identification of European barley cultivars

Summary A reversed-phase high performance liquid chromatography (RP-HPLC) system was used to separate the storage proteins (hordeins) extracted from European barley cultivars. From a total of 38 barleys tested, 26 types of hordein patterns could be distinguished after RP-HPLC. This appears to be a marked improvement in resolution over that achieved in a similar survey of European barley cultivars using SDS polyacrylamide electrophoresis (32 hordein patterns resolved by SDS PAGE from a total of 160 spring and winter barleys tested).Different hordein patterns were resolved by RP-HPLC within each of two groups of barley previously classified by SDS PAGE as indistinguishable within groups (three distinct patterns identified in a total of five cultivars tested from group 1A and five patterns observed among eight cultivars from group 3B). Thus RP-HPLC achieves a higher resolution than undirectional electrophoresis and promises to be a valuable aid in the identification of European barley cultivars.     

In vitro screening of strawberry plants for cold resistance

Formation of embryo autonomy of strawberry, plant regeneration fro membryo components, plant freezing conditions in vitro and the possibility to differentiate objectively genotypes by freezing them in vitro and in vivo were studied to create strawberry screening technology in vitro for cold resistance. It was established that autonomy of strawberry embryos manifests itself not earlier than on 14–16^th day after pollination and full autonomy is reached on 20–22^nd day. Plants regenerated from 26 days old embryos grew most intensively. At the highest rate strawberry plants regenerated from an isolated embryo axis on MS medium without phytohormones, and from rescued cotyledons x on the medium with 1.0 BA and 0.5 NAA. The temperature interval, at which genotypes differentiated according to cold resistance in vitro, was -8 to 12 ^°C. Differentiation of strawberry genotypes according to this character conformed to their differentiation in vivo, provided hardening proceeded not less than 21 days. The correlation between cold resistance in vitro and in vivo reached 0.93. Domination of cold resistance manifested itself in strawberry seedlings from various crossing combinations. This revised version was published online in August 2006 with corrections to the Cover Date.     

The use of RAPD technique for the identification and classification of Pisum sativum L. genotypes

Summary The genomic DNAs of 42 Pisum sativum genotypes, representing four wild and cultivated subspecies were used as templates in RAPD reactions. Amplification with eight decamer primers generated 149 polymorphic products. Genetic similarities of RAPD profiles were estimated via a coefficient of Jaccard and then the data were processed by cluster analysis (UPGMA). Each genotype was clearly identified and separated from the others. Our results show that RAPD technology is a rapid, precise and sensitive technique for identification of pea genotypes. However, the phylogenetic relationships within the Pisum sativum, which we tested by bootstrap analysis (Wagner parsimony), must be interpreted with caution.     

Proposal for the identification of barley varieties based on the genotypes for 2 hordein and 39 isoenzyme loci of 47 reference varieties

Summary Fifty-nine spring and 7 winter barley varieties in The Danish List of Varieties of Agricultural Crops, 1983/84 were examined for variation at 39 isoenzyme and two hordein loci. Twenty-three isoenzyme loci had one allele only, and 16 loci had from two to five alleles. One hordein locus had 12 and the other 15 alleles. The variation in the 16 enzyme loci permitted the division of the 66 varieties into 63 groups, while the two hordein loci produced 34 groups. A study of 20 individuals from each variety showed that 22 varieties were polymorphic in at least one locus. Eight starch gel electrophoresis with various buffer systems, one agar gel electrophoresis (for amylases), and one polyacrylamide gel electrophoresis (for hordein) were performed to develop the patterns associated with the 41 loci. The polyacrylamide gel electrophoresis developing hordein patterns was clearly the most powerful single system for identifying barley varieties because of the large number of alleles.     

Strawberry cultivar identification based on hypervariable SSR markers

We genotyped strawberry cultivars by two newly selected and two previously reported SSR markers. All four markers produced interpretable electropherograms from 75 accessions consisting of 72 Fragaria × ananassa cultivars or lines and three octoploid Fragaria species accessions. These SSR markers were highly polymorphic; in particular, one of the newly developed markers, FxaHGA02P13, was capable of distinguishing all of the accessions except for a mutant strain that was derived from another accession in the set. When two markers were combined, all 48 full-sib individuals could be distinguished. Fingerprinting patterns were reproducible between multiple samples, including the leaves, sepals, and fruit flesh of the same accession. Principal-coordinate analysis of the 75 accessions detected several groups, which reflect taxon and breeding site. Together with other available markers, these SSR markers will contribute to the management of strawberry genetic resources and the protection of breeders’ rights.     

Effect of in vitro propagation methods on field performance of two strawberry cultivars

Summary Strawberry cultivars, Redcoat and Veestar, propagated by meristem culture (MC), callus culture (CC) and direct shoot regeneration (DR) from leaf disks were compared for their vegetative and reproductive characters with standard runner (SR) propagated plants under field conditions. In the planting year, in vitro propagated plants of both cultivars had the same number of leaves as SR plants, but in vitro propagated Redcoat produced fewer stolons per plant than SR plants. However, in the following year, in vitro propagated mother plants of both cultivars had more leaves and higher runner production than SR mother plants. Flowering and fruiting behaviour of Veestar was not appreciably influenced by in vitro propagation methods. However, in vitro propagated plants of Redcoat flowered earlier and produced more flowers and fruits than SR plants, but still maintained normal berry weight. Among in vitro propagated plants, DR plants of Redcoat were the earliest to flower, whereas MC plants produced more flowers and fruits. The field performance of the first daughter plants derived from the in vitro propagated plants was consistent with their respective mother plants. Leaf shape of both cultivars was not altered by in vitro propagation. Phenotypic abnormalities were mainly confined to occurrence of yellow leaf variants in MC and CC plants and occasional appearance of plants with irregular flowering and growth habit among CC plants.NRCC No. 38004     

In vitro selection of strawberry (Fragaria × ananassa Duch.) clones tolerant to salt stress

The method of in vitro selection for increased salt tolerance at the seed germination and early growth phase of strawberry seedlings is proposed. Clone Pau/27 was selected on medium containing 200 mM of NaCl from population obtained by free pollination of cv. ‘Paula2019;. Subsequently, on the same medium 18 salt tolerant clones were attained from the population of seeds collected from self pollinated Pau/27 plants. In the next step we examined the influence of a mild salt stress (75 mM of NaCl) on vegetative growth parameters of selected clones and two cvs.; ‘Paula2019; and ‘Senga Sengana’. All materials in the study on the basis of calculated indexes were divided into four groups differing in reaction to salt. First group (clustered together cv. ‘Paula’, Pau/27 and three second generation clones: Pau/27/11, Pau/27/24, Pau/27/30) represents sensitive genotypes. Second group, including ‘Senga Sengana’, Pau/27/06, Pau/27/10, Pau/27/12, Pau/27/13, Pau/27/15, Pau/27/18, Pau/27/20, Pau/27/21, Pau/27/26, Pau/27/27, Pau/27/31 and Pau/27/32 was designated tolerant. Third group contains only one highly tolerant clone Pau/27/08. The last group comprises two highly sensitive clones (Pau/27/01 and Pau/27/03). This revised version was published online in August 2006 with corrections to the Cover Date.     

Utilizing wild Fragaria virginiana in strawberry cultivar development: Inheritance of photoperiod sensitivity, fruit size, gender, female fertility and disease resistance

The genetics of photoperiod sensitivity, flowering date, fruit size, gender, female fertility, and disease resistance were investigated in progeny between sets of elite F. virginiana selections and F. × ananassa cultivars and selections planted at sites in Michigan, Minnesota and Ontario. Progeny means varied considerably for all the production traits. Most notable were the large fruit and high fertility observed in crosses with High Falls 22 at all three sites, and Montreal River 10 in Ontario and Michigan. Fragaria virginiana ssp. virginiana parents yielded progeny with much larger fruit than F. virginianassp. glauca parents. General combining ability was significant for all traits at all locations, while specific combining ability was significant for only fruit diameter, ovule set and fruit set in Michigan. Overall, the highest number of day-neutral genotypes were detected in Ontario (mean =44%) compared to Minnesota (31%) and Michigan (26%). In progeny populations of day-neutral F. × ananassa × short-day F. virginiana almost all fit the 1:1 ratio expected if day-neutrality is regulated by a single dominant gene; however, only a few families of short-day F. × ananassa ×day-neutral F. virginianacrosses fit a 1:1 ratio. Likewise, in progeny of day-neutral F. virginiana ×day-neutral F × ananassa crosses, only a few of them fit the 3:1 ratio expected if day-neutrality is regulated by a single dominant gene. These data suggest that it should be relatively easy to useF. virginiana germplasm in strawberry cultivar improvement, and that several different sources of day-neutrality may exist in natural populations. This revised version was published online in August 2006 with corrections to the Cover Date.     

A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLPs, RAPDs, and pedigree data

Nineteen of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the UnitedStates and Canada were examined for AFLP markerpolymorphisms. For the AFLP reactions, the EcoRI-ACC primer was used in combination with fourMseI primers (MseI-CAC, MseI-CAG,MseI-CAT, or MseI-CTT). Each set ofprimers produced 46–66 scorable fragments ranging insize between 50 and 500 bp. The polymorphic fragmentsproduced from each set of primers were more thansufficient to distinguish among all the cultivars,demonstrating the usefulness of AFLP markers forcultivar identification. Similarity coefficients werecalculated based on data from 228 AFLP markers anddata from 15 previously characterized RAPD markers. The RAPD markers had been specifically selected forfingerprinting purposes because they succesfullydistinguish 41 strawberry cultivars, including the 19cultivars analyzed in this study. Separatedendrograms were constructed based on analysis of theAFLP and RAPD marker data using a neighbor-joiningalgorithm. The dendrograms were compared and found tobe very different. Correlations between similaritycoefficients calculated from AFLP marker data,similarity coefficients calculated from RAPD markerdata, and coefficients of coancestry calculated frompedigree information were evaluated. Interestingly,a better correlation with the coefficients ofcoancestry was observed with the RAPD marker data thanwith the AFLP marker data.     

Evolution of cauliflower types grown in Great Britain as indicated by the isoenzyme composition of the cauliflower curds

Summary The curds of cauliflowers (Brassica oleracea var. botrytis L.) which are representative of the European biennials, European annuals and Australian types were used to extract 12 of the enzymes involved in carbohydrate and amino acid metabolism. Each enzyme was separated into their isoenzymes using polyacrylamide gel electrophoresis. Two enzymes, acid phosphatase EC 3.1.3.2. and aspartate aminotransferase EC 2.6.1.1. were shown to have different numbers of isoenzymes depending upon which of the three main groups of cauliflower cultivars were used. The enzymes examined showed evolutionary divergence of the cauliflower types during the selection for different times of development.     

Utilization of isozyme polymorphism for cultivar identification of 45 commercial peas (Pisum sativum L.)

Forty five Pisum sativum cultivars were analysed by isozyme electrophoresis with the objective to find protein markers for exact and reproducible discrimination of individual genotypes. The combination of six enzyme systems (acid phosphatase, amylase, esterase, leucine aminopeptidase, shikimate dehydrogenase and phosphoglucomutase) with two electrophoretic techniques (NATIVE-PAGE, isoelectric focusing) and use of seed and leaf tissue enabled to identify all 45 studied cultivars. Critical factors which may affect utilization of isozyme electrophoresis for commercial applications in pea breeding and seed production and testing are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The production of multispecific octoploids from Fragaria species and the cultivated strawberry

Summary A method of incorporating genetic material from five species of Fragaria and the cultivated strawberry into fertile octoploids is described. A synthetic octoploid derived from a hexaploid x diploid hybrid was crossed to octoploid cultivar breeding lines until a BC₂ hybrid was produced. A second synthetic octoploid in which two diploid species and a tetraploid species were combined was crossed to a cultivated strawberry to produce a hybrid breeding clone. The two breeding clones were crossed and 222 seedlings were produced. The seedlings were generally vigorous and fruitful, some having commercial potential.     

玉米杂交种及其亲本自交系的生化指纹鉴定

本试验以浙单9号等五个玉米杂交组合及其亲本自交系为材料,进行种子盐溶蛋白聚丙烯酰胺凝胶电泳等多种电泳鉴定方法的研究,以揭示玉米杂交种及其亲本自交系的“生化指纹”(biochenucal fingerprint),以及筛选出适合于玉米杂交种及其亲本自交系真实性和纯度鉴定的方法。结果表明,各供试玉米杂交种及其亲本自交系都具有相应的、唯一的种子盐溶蛋白聚丙烯酰胺凝胶电泳所显现的生化指纹。对于有些组合。玉米芽鞘和叶片绿色组织过氧化物酶同工酶电泳图谱存在阴极第4、第5酶带差异,因这两条酶带的差异稳定,并且重现性好,故能用过氧化物酶同工酶技术对其进行有效地鉴定。上述两种方法,尤其是前者,因技术要求不高,费用低,快速及重现性好等特点,能满足我国目前种子检验室日常玉米品种纯度快速测定工作的要求,具育良好的应用前景。     

Development of cleavage amplified polymorphic sequence (CAPS) markers for identification of strawberry cultivars

We developed 6 cleavage amplified polymorphic sequence (CAPS) markers to identify strawberry (Fragaria ×ananassa Duch.) cultivars, with an aim of protecting breeders' rights. We successfully used them to distinguish 14commercial cultivars. The results were highly reproducible among different extraction methods, different organs and different researchers. The stability and simplicity of CAPS analysis makes it a good tool for the identification of strawberries. This revised version was published online in July 2006 with corrections to the Cover Date.     

Accelerating and synchronizing germination of strawberry seeds by osmotic pre-treatments

Summary Pre-soaking strawberry seed (Fragaria ananassa Duch.) in osmotic solutions accelerated and partially synchronized their germination. Pre-soaking advanced 50% germination from about 4 weeks to 4 or 5 days. Two to three weeks pre-soaking in a mineral solution of -10⁶Pa nominal osmotic potential at about 20 C was satisfactory. The resulting synchronization of germination could allow seedlings to be selected for rate of growth by directly comparing seedling in bulk sowings.