doi: 10.15389/agrobiology.2015.1.30eng

UDC 633.367.2:632.4:575.174.015.3:577.21

(Lupinus angustifolius L.) USING DNA-MARKERS AnSeq3 AND AnSeq4

S.Yu. Grishin1, V.V. Zayakin1, I.Ya. Nam1, P.A. Ageeva2, M.I. Lukashevich2, N.S. Kuptsov3

1Bryansk State University, 14, ul. Bezhitskaya, Bryansk, 241036 Russia,
e -mail;  
2All-Russian Research Institute of Lupine, 2, ul. Berezovaya, pos. Michurinskii, Bryansk, 241524 Russia, e-mail;
3Scientific and Practical Center on Agriculture of NAS of Belarus, 1, ul. Timiryazeva, Zhodino, 222160 Republic of Belarus, e-mail
Supported by the Ministry of Education and Science of the Russian Federation

Received November 14, 2013

Anthracnose is one of the fungal diseases of the narrow-leafed lupine (Lupinus angustifolius L.) caused by Colletotrichum lupini. Resistance to anthracnose is not absolute in character, as the plants with high resistance can be affected by the pathogen but in less extent than those non-resistant. Recent suggestions of the total number of genes involved in control of anthracnose tolerance are discrepant. Current approach in breeding anthracnose-tolerant lupine is based on combination of non-allele genes of resistance in a single genotype. Specific DNA markers are being developed which are linked to the genes of resistance and can be used for rapid and effective selection of resistant plants, but prior to their application the efficacy of DNA marker should be specifically tested with the breeding material of interest to avoid false positive responces. The AnSeq3 and AnSeq4 DAN markers flanking Lanr1 gene at 0.9 cM distance are considered the closest to it (H. Yang et al., 2012). In this article we report the possibility of using DNA markers AnSeq3 and AnSeq4, the single nucleotide polymorphisms (SNPs), in selecting forms resistant to anthracnose among varieties of narrow-leafed lupine. A total of 50 Russian, Belarusian, Polish and Australian varieties and samples were tested to detect the allele DNA markers of susceptibility or resistance to anthracnose. DNA was individually isolated from seeds in three replicates. Polymerase chain reaction (PCR) was used to detect alleles of DNA markers linked to the Lanr1 gene. The list of tested plants, PCR mix composition and protocol are specified. PCR enzymes and reagents of the SibEnzyme company (Russia) were used. The praimers for AnSeq3 and AnSeq4 markers were site-specific and synthesized by the Syntol company (Russia). Polyacrylamide gel electrophoresis was used for visualization of the allele markers. The Australian varieties resistant and susceptible to anthracnose were used as a control for AnSeq3 and AnSeq4 alleles. DNA fragments of 92 and 87 base pairs corresponding to the markers AnSeq3 and AnSeq4, respectively, were obtained for all 50 breeding samples included in the study. For 13 Russian and 10 Polish varieties the marker alleles of susceptibility to anthracnose were detected. For BGB-6 Belarusian sample the resistance alleles were identified by AnSeq3 and AnSeq4 markers, and for Myrtan variety only the AnSeq3-specific pattern was shown. The rest of 21 Belarusian samples possessed the alleles of susceptibility to anthracnose. Earlier by means of other DNA markers, AntjM1 and AntjM2, we showed the absence of alleles linked to Lanr1 in currently registered varieties originated from All-Russian Research Institute of Lupine. Thus, the DNA markers AnSeq3 and AnSeq4 linked to gene Lanr1 may be useful in breeding Russian and Belarusian anthracnose-resistant lupine varieties.

Keywords: Lupinus angustifolius L., anthracnose, polymerase chain reaction (PCR), DNA markers AnSeq3, AnSeq4.


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