Studying of plants DNA with PCR method plays an important role in the agrarian activity. Preceding stage of DNA studying is its extraction from plant material. We studied quality of DNA extracted by the different methods from seeds, seedlings, and green leaves of sunflower. We used an inbred sunflower line as a research object. For analysis we used embryos, dry and presoaked during 24 h seeds, roots, 7-day seedlings, green leaves. DNA was extracted using five methods: 1 – a standard method of extraction (1% CTAB), 2 – a modified method (2% CTAB), 3–4 – extraction with sets (Diamond DNA Plant Kit D, Russia; Lumiprobe, Germany), 5 – extraction from green leaves (2% CTAB with absorbent carbon). Amplification was conducted in thermocycler S1000тм (BioRad, USA). Spectrophotometery was done at scanning spectrophotometer LEKI SS2110UV (Russia). Analyzing all used methods we concluded they allow extracting of DNA from dry seeds, seedlings and green leaves of sunflower with sufficient reliability and repeatability, it is proved by PCR results. We couldn’t extract DNA from roots and presoaked seeds with the modified method (2% CTAB). The most economically profitable is first method (1% CTAB). Due to the results of spectrophotometery, the highest level of DNA clearance can be reached with method 3 – extraction by a set Diamond DNA Plant Kit D from seedlings and method 5 – extraction by 2% СТАВ with absorbent carbon from green leaves. The method 3 is more preferable by time necessary for DNA extraction.
Studying of sunflower lines resistant to broomrape race G, bred in V.S. Pustovoit All-Russian Research Institute of Oil Crops (VNIIMK), Krasnodar showed the genetic control of resistance is monogenic with incomplete dominance. The purpose of the research was to seek and analyze molecular markers for the genes of resistance to broomrape race G using PCRmethods. In our research we used six developed in VNIIMK lines-donors resistant to broomrape race G: RGP1, RGP2, RGB, RGL1, RGL2, RGA, and susceptible lines VK 101, VK 678, VK 680. We extracted DNA from the leaves of young sunflower plants using STAB-buffer. For PCR-analysis we used 17 pairs of primers of two types: SCAR (sequence characterized amplified region) and SSR (simple sequence repeat). Conditions of amplification are as recommended by authors of markers with experimental selection of a temperature for primers hybridization. Within the research we balanced involved into work SSR- and 12 SCAR-loci using BLAST and a set of HanXRQr1.0. Due to these data we composed a physical map of loci location. After assessment of six resistant and three susceptible parental lines, seven markers demonstrated polymorphism by a length of locus DNA and two ones – by presence/absence of amplified fragment. For markers ORS 683 and ORS 1112 we observed a relation between loci polymorphism and susceptibility of parental lines to broomrape. This was certified with a presence of an allele of 364 n.p. length in ORS 683 and an absence of an allele of 375 n.p. length in ORS 1112. These markers were noted as primary ones for hybridological analysis in F1 и F2 with selected pairs for crossing. Additional markers will be selected individually using obtained data on loci polymorphism. Thus, as a result of the research we created a physical map for the further markers selection, found hybrid combinations and markers from the studied ones for hybridological analysis.
The aim of this research was to develop molecular genetic passports of sunflower lines from the collection of the Don experimental station of V.S. Pustovoit All-Russian Research Institute of Oil Crops based on polymorphic fractions of microsatellite DNA. We used 17 lines as a research material. We used 12 pairs of primers for genotyping. We found that the ORS 559 locus was monomorphic for these samples. The rest of the loci had from 2 to 4 alleles. The average number of alleles per locus was 2.75, PIC – 0.49, the effective number of alleles – 2.16. The analysis of the DNA profiles of the lines showed the individuality of the allelic composition of each of them. The analysis of the genetic relations between the lines showed that the studied lines were divided into two groups, with a genetic distance between them of 5.9.
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