Use of molecular markers in detection of downy mildew resistance gene Plarg in sunflower

Plamenjače suncokreta je bolest koja može da izazove značajno smanjenje prinosa. Šteta prouzrokovana ovim patogenom može da se kontroliše uzgajanjem otpornih hibrida suncokreta. Gen otpornosti Plarg unet je u gajeni suncokret iz divljeg srodnika H. argophillus i obezbeđuje otpornost na sve do sada identifikovane rase plamenjače. Kako bi se proces unošenja gena otpornosti u različite linije ubrzao i učinio pouzdanijim, potrebno je identifikovati molekularne markere blisko vezane za gen otpornosti. Od posebnog interesa su kodominantni markeri, koji mogu da naprave razliku između homozigotno i heterozigotno otpornih biljaka. Cilj ovog rada bio je identifikacija molekularnih markera blisko vezanih za gen Plarg koji će moći da se koriste za marker asistiranu selekciju (MAS). U radu je korišćena mapirajuća populacija koja se sastojala od 103 F2 biljke dobijene iz ukrštanja RHA 419 x RHA-N-49. Molekularne analize pokazale su da se gen Plarg nalazi na LG1 genetičke mape suncokreta. Identifikovan je novi kosegregirajući SSR marker ORS675, dok je kosegregacija markera ORS716 i ORS662 sa Plarg potvrđena. Veza markera i gena potvrđena je na dodatne 22 linije sa genom Plarg. Testiranje linija HA-R4 (Pl14 i Pl16) i HA-R5 (Pl13) markerima ORS662, ORS716 i ORS675 pokazalo je da se gen Plarg nalazi odvojeno od drugih do sada identifikovanih gena sa LG1. Nakon validacije, primena markera ORS675 i ORS716 u selekciji je testirana na dve različite BC1 populacije. Markeri su se pokazali korisnim kako u molekularnom oplemenjivanju tako i za analizu genetičke čistoće linija suncokreta. Može se zaključiti da se molekularni markeri iz ove studije blisko vezani za gen Plarg mogu koristiti za MAS, i s toga se upotrebljavati u selekcionim programima kako bi se olakšao unos gena otpornosti u različite linije suncokreta.Downy mildew is a common sunflower disease that can cause significant yield reduction. The damage caused by the pathogen can be controlled by growing sunflower varieties resistant to this disease. Resistance gene Plarg was introduced into cultivated sunflower from the wild species H. argophyllus and it provides resistance against all known downy mildew races. Molecular markers can facilitate and accelerate the process of transfer of resistance gene into commercial sunflower lines. Co-dominant markers are particulary useful, since they can discriminate between homozygous and heterozygous resistant plants. The aim of this study was to identify molecular markers closely linked to gene Plarg that will be used in marker assisted selection (MAS). A mapping population of 103 F2 progeny from the cross RHA 419 x RHA-N-49 was used. Molecular analyses indicated that the gene Plarg is located on LG1 of the genetical map of sunflower. A new co-segregating simple sequence repeat (SSR) marker ORS675 was identified and the co-segregation of markers ORS716 and ORS662 with Plarg gene was confirmed. The markers were validated on twenty two resistant inbred lines (RHA 443, RHA 464 and RH 1-20). Results obtained on lines HA-R4 (Pl14 i Pl16) and HA-R5 (Pl13) with markers ORS662, ORS716 i ORS675 showed that Plarg is a resistance locus diffrent from other resistance genes mapped on LG1. Application of the markers ORS675 and ORS716 in marker assisted selection was tested on two different BC1 populations. Markers were found to be useful both for molecular breeding and genetic purity analyses. The closely linked molecular markers from this study will facilitate transfer of the resistance gene into different sunflower lines

DNA isolation from dry samples of broomrape - the effect of isolation method and sample storage on DNA yield and quality

Accurate characterization of broomrape races is of a great importance for creation of sunflower hybrids resistant to this parasite. In molecular diversity studies, it is necessary to isolate sufficient quantity of good quality DNA. As collecting and transporting fresh broomrape tissue and seed samples for molecular analyses can be problematic, we have tested the possibility of DNA isolation from dry tissue of mature broomrape plants, with the aim to find optimal method for sample storage and isolation of sufficient quantity of DNA for multiple PCR reactions needed for diversity studies. Mid parts of dry broomrape stalks were collected and stored in four different ways. Ten DNA isolation protocols were tested and the obtained results compared. The best results regarding DNA yield and quality were obtained with dry samples stored at room temperature in ventilated space. DNA suitable for RAPD analysis was isolated with three of ten tested methods. As protocol of Somma (2006) is labour intensive and produced the lowest DNA yield, only DNA isolation with DNeasy (R) Plant Mini Kit, Qiagen and protocol by Rogers and Bendich (1985) could be recommended for future studies

Stvaranje i korišćenje genotipova suncokreta sa promenjenim kvalitetom ulja

Sunflower oil is among the highest quality oils of plant origin. The oil of standard sunflowers has an average of 10% saturated fatty acids, 20-30% oleic acid and 60-70% linoleic acid. The total content of tocopherols in standard sunflower oil is 700-1000 mg/kg with the predominant being alpha-tocopherol (vitamin-E). Following the trends of the food and non-food industries sunflower breeders have been able to significantly change the fatty acid composition of the oil. The oil of high-oleic hybrids (more than 80% of oleic acid in sunflower oil) has excellent nutritional properties, as well as oxidative stability, and is a suitable raw material for many derivatives of the chemical industry and for the production of high quality biodiesel. In addition to creating high-oleic sunflower hybridis also possible to direct selection towards increasing or decreasing other fatty acids content (linoleic, palmitic and stearic). Achievements in sunflower breeding have allowed changes in the type and content of tocopherols in the oil.Suncokretovo ulje spada među najkvalitetnija ulja biljnog porekla.Ulje standardnog suncokreta u proseku ima 10% zasićenih masnih kiselina, 20% oleinske kiseline i oko 60-70% linolne kiseline. Ukupan sadržaj tokoferola u ulju standardnog suncokreta je 700-1000 mg/kg, a preovladava alfa-tokoferol (vitamina E). Prateći zahteve prehrambene i drugih industrija oplemenjivači suncokreta uspeli su da bitnije izmene sastav masnih kiselina ulja. Ulje visokooleinskih hibrida suncokreta ima odlične nutritivne karakteristike, pogodna je sirovina za proizvodnju mnogih derivata hemijske industrije i za proizvodnju kvalitetnog biodizela. Pored stvaranja visokooleinskih hibrida suncokreta moguće je selekciju usmeriti i na povećanje ili smanjenje sadržaja drugih masnih kiselina (linolne, palmitinske i stearinske). Dostignuća u oplemenjivanju suncokreta omogućila su promenu tipa i sadržaja tokoferola u ulju

Use of SSR markers in identification of sunflower isogenic lines in late generations of backcrossing

In the present study, thirteen SSRs (Simple Sequence Repeats) were employed for screening the level of similarity between sunflower parental lines and progeny obtained in final stages of selection. Another aim of the study was to compare the success of making isogenic lines through selection. Line P (donor line) was used as a donor of downy mildew gene Pl6 and backcrossed with four commercial inbred lines (A, B, C and D) susceptible to downy mildew. HAP3, a DNA-specific primer, was used to test the success of transfer of Pl6 gene from the donor line to the progeny lines in the F6 and F7 generations. It was found that all progeny lines were resistant to downy mildew. Results obtained by using thirteen primers showed that progeny lines were similar to their respective recipient parent, but further studies have to be conducted using a larger number of primers and a larger number of genotypes

Evaluation of Combining Ability in Ornamental Sunflower for Floral and Morphological Traits

Ornamental sunflowers are widely cultivated for use as cut flowers and/or as garden plants. The objective of the study was to investigate breeding values of new F-1 ornamental sunflower hybrids using incomplete diallel crossing of four ornamental inbred lines: Heliopa, Talia, Iskra and Neoplanta. Six traits important for ornamental market use were studied. Additive gene effects prevailed in most tested traits except for lateral floral diameter and duration of lateral flowering, which exhibited non-additive effects. The results indicated that the Talia x Neoplanta hybrid combination was the most promising for cut flowers due to long and strong branches, relatively big lateral flowers while Heliopa x Iskra and Heliopa x Talia hybrids were suitable for use as garden plants due to strong plant habit, long flowering time and desirable plant height. The results obtained from this study will be helpful for further ornamental sunflower breeding

Towards sustainable downy mildew resistance in sunflower

Breeding for downy mildew resistance is one of the major goals in sunflower breeding programmes. In the last few decades new sources of resistance have been discovered and the position of 11 resistance genes was established on the genetic map of sunflower. For most of these resistance genes sequence specific markers have been developed, which facilitate their detection and make the selection process faster and more reliable. By combining the partial resistance provided by minor genes with specific resistance genes, durable resistance could be achieved

Brzi test za ispitivanje otpornosti suncokreta na tribenuron metil

A rapid laboratory test for detection of tribenuron-methyl resistant sunflower genotypes was developed. Four homozygous and four heterozygous tribenuron-methyl resistant hybrids, as well as a susceptible one were grown on MS medium supplemented with different concentrations of herbicide (2.0 µM, 2.5 µM, 3.0 µM, 3.5 µM and 4.0 µM) and with pH either 7 or 8. The effect of medium pH and herbicide concentration on above-ground part and root mass of sunflower seedlings was observed. The test enabled visual discrimination between resistant and susceptible genotypes in 5 days, and discrimination between homozygous and heterozygous resistant genotypes in 12 days. All tested herbicide concentrations and morphological parameters were found to be suitable for discrimination of tribenuron-methyl susceptible genotypes. The best pH and herbicide concentration combination and morphological parameter for discrimination between homozygous and heterozygous resistant sunflower genotypes were 3.0 µM at pH 7 and root mass, respectively.Razvijen je brzi laboratorijski test za detekciju genotipova suncokreta otpornih na tribenuron metil. Po četiri hibrida suncokreta homozigotno i heterozigotno otporna na tribenuron metil, kao i jedan neotporan hibrid su gajeni na MS podlozi sa pH vrednošću ili 7 ili 8 i sa različitim koncentracijama herbicida Express 50X, čija je aktivna materija tribenuron-metil (2.0 µM, 2.5 µM, 3.0 µM, 3.5 µM i 4.0 µM). Ispitivan je uticaj pH i koncentracije herbicida u podlozi na masu nadzemnog dela i korena klijanaca testiranih hibrida. Vizuelne razlike između otpornih i neotpornog hibrida su uočene nakon 5 dana kulture, dok su se razlike između homozigotnih i heterozigotnih otpornih hibrida pojavile nakon 12 dana kulture. Sve testirane koncentracije herbicida i testirani morfološki parametri su se pokazali kao dobri za identifikaciju neotpornih genotipova. Najbolja kombinacija pH i koncentracije herbicida za razlikovanje homozigotnih od heterozigotnih otpornih genotipova je bila 3.0 µM na pH 7, a morfološki parametar masa korena

Fenotipska i molekularna evaluacija genetičke raznolikosti NS kolekcije šafranjike (Carthamus tinctorius L.)

Safflower (Carthamus tinctorius L.) belongs to the Asteraceae (Compositae) family. It is primarily grown for seeds used for bird feed or as edible oil. Stamens are used in traditional medicine and nutrition. Breeding for high resistance to dry growing conditions has initiated intensive studies of this plant species in recent years. Six safflower genotypes of different geographical origins (Ukraine, Italy, Turkey) were collected and added to the collection of less cultivated oil plant species of the Institute of Field and Vegetable Crops in Novi Sad. Phenotypic observations during two growing seasons revealed that analysed genotypes differed in flower colour (yellow, orange, red), in the presence of spines, and in seed oil and protein content. Oil and protein content differed between years and genotypes, indicating large influence of genotype and environmental conditions on variations of these quantitative traits that are negatively correlated. Genetic variability of the analysed genotypes was tested by use of molecular markers. Given that sunflower and safflower belong to the same family, the possibility of applying SSR markers developed for sunflower for molecular analysis of safflower was analysed. The obtained results proved that sunflower markers can be successfully transferred to safflower. Future studies should include larger number of markers in order to identify polymorphic and informative ones. Significant variations within a relatively small number of the analysed safflower genotypes justify further work on the evaluation of the collection, taking into account both genetic and environmental factors.Šafranjika (Carthamus tinctorius L.) pripada porodici Asteraceae (Composita). Gaji se prvenstveno za dobijanje semena koje se koristi za ishranu ptica ili proizvodnju jestivog ulja. Prašnici se koriste u tradicionalnoj medicini i ishrani. Visoka otpornost na sušne uslove gajenja inicirala je poslednjih godina intenzivnija istraživanja ove biljne vrste. Za kolekciju manje gajenih uljanih biljnih vrsta Instituta za ratarstvo i povrtarstvo, prikupljeno je šest genotipova šafranjike različitog geografskog porekla (Ukrajina, Italija, Turska). Fenotipskim opažanjem u toku dve vegetacione sezone utvrđeno je da se genotipovi među sobom razlikuju u boji cveta (žuta, narandžasta, crvena), u prisustvu bodlji, kao i sadržaju ulja i proteina u semenu. Sadržaj ulja i proteina se razlikovao između godina i genotipova, što ukazuje na veliki uticaj i genotipa i spoljašnje sredine u variranju ovih kvantitativnih svojstava koja se nalaze u negativnoj korelaciji. Genetička varijabilnost genotipova je ispitana molekularnim markerima. S obzirom na to da suncokret i šafranjika pripadaju istoj porodici, proučavana je mogućnost upotrebe SSR markera suncokreta za molekularnu analizu šafranjike. Dobijeni rezultati pokazali su da se markeri suncokreta mogu uspešno koristiti za šafranjiku i da buduća istraživanja treba da sadrže veći broj markera u cilju identifikovanja polimorfnih i informativnih markera. Značajne razlike u relativno malom broju analiziranih genotipova šafranjike opravdavaju dalji rad na evaluaciji kolekcije, uzimajući u obzir i uslove sredine i genetičku varijabilnost

Evaluation of rapd markers as a marker-assisted selection tool for variety type and erucic acid content in rapeseed

Random amplification of polymorphic DNA (RAPD) analysis was performed on twelve rapeseed genotypes from Institute of Field and Vegetable Crops, Novi Sad, Serbia, genepool in order to identify markers that could be used in marker assisted selection (MAS) for different growing type and selection of the varieties with low or zero level of erucic acid. Out of fifteen RAPD markers, three were monomorphic, whereas twelve had polymorphic profiles. Three primers amplified specific fragments in spring varieties. UBC 25 and UBC 191 amplified the fragments of 450 and 750 bp, respectively, in all tested spring varieties, except in JR-NS-36. Primer UBC 72 generated a fragment of 700 bp that was present in all spring varieties. These fragments were not present in any of winter varieties. None of the tested RAPD primers amplified fragment(s) uniquely present either in varieties with or without (0%) erucic acid or with different erucic acid content. Cluster analysis showed a concordance between the position of varieties in the cluster and their pedigree information, but also enabled separation of spring and winter varieties. Contingency analysis confirmed that fragment UBC 72_700 is specific for spring varieties, while for erucic acid content, only moderate association was found with UBC 137_750

Genetic diversity of wild sunflower (helianthus sp.) Accessions with different tolerance to mid-stalk white rot

Random amplified polymorphic DNA (RAPD) markers were used to detect polymorphism among accessions of wild sunflower species Helianthus maximiliani, Helianthus tuberosus, Helianthus mollis and Helianthus rigidus with different tolerance to mid-stalk white rot and selection of potential markers for different levels of tolerance to this disease. Estimates of genetic variation showed that genetic diversity was equally distributed between Helianthus species and within them. Cluster analysis corresponded to the phylogenetic relations within the genus Helianthus. The results obtained by principal coordinates analysis (PCoA), where the first two principal coordinates accounted for 83.7% of total variation, perfectly coincided with the results of cluster analysis. Contingency coefficient significance test showed that most of the used primers generated bands associated with some level of tolerance or susceptibility to mid-stalk white rot. Furthermore, contingency analysis showed that primer C12 generated bands associated with resistance (100%) to mid-stalk white rot both in H. mollis and in all accessions, while primer X18 generated bands significantly associated with high tolerance (75%) in H. rigidus, H. mollis as well as in all tested accessions. The C15-600 bp locus was found to be significantly associated with high tolerance (75%) in all accessions, and medium tolerance (50%) in H. mollis