ISSR marker-assisted determination of the genetic polymorphism of an original sterile form of winter rape

Abstract

The aim and tasks of the study. To identify the genetic differences in descendants of an original sterile form of winter rape bred at the Plant Production Institute nd a VYa Yuryev and based on the Ogura- and Polima-type male sterility using microsatellite analysis.

Materials and methods. The study was conducted under laboratory conditions in the Plant Production Institute nd a VYa Yuryev of NAAS in 2014-2016. PCR with ISSR markers was carried out in a horizontal Hoefer HE 100 SuperSub unit (USA). Seven accessions based on Ogura- and Polima-type male sterility and the original sterile form were investigated. Molecular weight markers 1 Kb ("SibEnzim", Russia) were used. Gels were documented using a Nikon D50 camera. Amplicon numbers and sizes were determined using a demo version of Totallab100. Frequencies of locus alleles were calculated in Excel. Amplification profiles were evaluated by fingerprinting using informative parameters, such as the index of primer informativeness (IPI). The Botstein’s classification and Shannon’s indices were used. Polymorphism of ISSR markers was assessed using the Nei index of genetic diversity (He). To evaluate the divergence between accessions by ISSR markers, the PHYLIP program was used. Dendrograms were constructed by the nearest neighbor algorythm.

Results and discussion. Amplification with five ISSR primers identified 49 DNA fragments, 31 of which were polymorphic, in the accessions under investigation. The amplicon number varied from 5 to 14 fragments, on average 9.8 per primer. The maximum number of loci was identified using primers UBC810 and UBC 834.

Amplification with five ISSR primers found 12 allelic variants (UBC810 [587 bp], UBC834 [988, 774, 709 bp], UBC842 [1127, 475 bp], UBC847 [1349, 969, 496 bp] and UBC857 [791, 520, 446 bp]). The Polima accession had no marker loci. The results proved that there were no identical genotypes in the test sample.

The absolute and normalized values of theShannonindex showed that UBC 810 was the most polymorphic ISSR marker was (H '= 0.495, H'norm = 0.715). The ISSR marker UBC 857 had the lowest level of polymorphism (H '= 0.364, H'norm = 0.525). The total level of variability in ISSR markers was H'= 0.445 and H'norm = 0.642.

The dendrogram distinguished three opposite clusters of rape accessions. Cluster 1 included Bekr (1) D 21, Bekr (1) D 2, Bekr (2) D 16/2 (descendants of the original sterile form); cluster 2 - Polima and MON accessions (descendants with Polima sterility); cluster 3 - Inra and D8 (in vivo) (descendants with Ogura sterility).

Conclusions. A high range of intraspecies variability among the rape accessions under investigation was detected by ISSR markers. We established the general patterns of grouping rape accessions, using classification, which was formulated by cluster analysis using the nearest neighbor (NJ) algorithm. The study proved that descendants of the original sterile form had one root and showed that there were significant genetic differences from accessions with the Ogura- and Polima-type male sterility