Assessment of genetic diversity among headed cabbage (Brassica oleracea L.) accessions by using SSR markers

Out of all cole crops in Russian Federation traditional cultivars and hybrids of headed cabbage B. oleracea L. сonvar. capitata (L.) are the most widespread. The classification of breeding material with the use of DNA markers enables to find out valuable genotypes and to detect genetic relationships among them in order to develop genetically distinguishing breeding forms. Microsatellite (SSR) markers are known to have been broadly used for genetic identification and genotyping in crops. These markers have shown their efficiency to reveal the polymorphism among varieties, cultivars and within cultivars in B. oleracea L. In the present work we have estimated for the first time the genetic relationship among local accessions of headed cabbage on the basis of SSR-loci polymorphism. The goal of the work was to reveal genetic relationship between breeding accessions of B. oleracea L. сonvar. capitata (L.) Alef. var. capitata L. f. alba , var. capitata L. f. rubra , and var. sabauda L. based on DNA typing and genetic classification using SSR markers, and to compare DNA data of studied genotypes with defined cabbage varietal and maturity groups. Twenty-four breeding accessions of headed cabbage including red and Savoy varieties from collection of Federal Research Center for Vegetable Growing (FRCVG) and also developed at FRCVG were involved. Genomic DNA was extracted from young plant leaves at 2-3 leaf stage with DNA extraction kit Sorb-GMO (Syntol, Russia). Final DNA purity and concentration were identified with the SmartSpec Plus spectrophotometer (Bio-Rad, USA). Twenty-one microsatellite loci with known primer sequences were chosen to perform SSR analysis. The amplification was run in C1000 Touch thermocycler (Bio-Rad, USA). PCR products were separated in a 6 % polyacrylamide sequencing gel with the use of Sequi-Gen GT electrophoresis system (Bio-Rad, USA). The fragments sizes were detected in comparison with molecular weight markers GeneRuler100 bp plus DNA ladder (Thermo Fisher Scientific, USA). The digital images of electrophoregrams were analyzed with Image Lab 3.0 software (Bio-Rad, USA). STRUCTURE 2.3.4 (https://web.stanford.edu/group/pritchardlab/home.html) software was used to study population structure. The genetic distances were calculated using GenAlEx 6.5 software for Microsoft Excel by Nei’s method. To construct the UPGMA dendrogram the algorithm of MEGA 5.2 program was used. As a result of analysis 103 alleles were obtained with an average 4.9 alleles per locus. PCR product sizes were between 130 and 410 bp. The PIC value varied from 0.3 to 0.9. Population analysis revealed six clusters to distribute all breeding accessions. Calculated Nei’s genetic distances varied from 0.060 to 0.186. The UPGMA deprogram constructed on distances matrix reflected the origin of cabbage accessions taken. Thus, cultivars Belorusskaya 455, Podarok 2500, Amager 611 and Zimovka 1474 originated from Northwestern Europe were joined into one cluster, there was also hybrid Severiynka F1 developed with the use of these cultivars. Early-maturing varietal group Ditmarskaya Raniya represented by cultivars Ijunskaya 3200, Stakhanovka 1513, Nomer Perviy Gribovskiy 147 formed a separate cluster which also included an early-maturing hybrid Avrora F1 being of a partial origin from Ijunskaya 3200. Two breeding lines obtained from Avrora F1 were genetically distant and disposed in another subcluster. Cultivars Slava 1305 and Slava 231 belonging to the separate varietal group Slava formed a branch of the dendrogram. Cultivar Parus and hybrids Zarnitsa F1, Mechta F1 developed relatively recently were disposed distantly from other accessions. Moscovskaya pozdnyaya 15 a local cultivar formed its own branch of the dendrogram. Three cultivars of Savoy cabbage were grouped together with sufficient genetic distance between each other, where a new early-maturing cultivar Moskovskaya kruzhevnitsa was more distant from others. The group of red cabbage accessions situated distantly from other clusters with great difference inside the group. The obtained results based on SSR marker variation were in accordance with data on the origin of headed cabbage accessions confirming that they belong to defined varietal and maturity groups. This provides information for nearest breeding program for new cabbage breeding forms.