The object of study:
Malus sieversii, Erwinia amylovora
The purpose of the work:
To identify genotypes of Malus sieversii resistant to Erwinia amylovora.
Methods for DNA extraction, PCR, fragment analysis, in vitro culture of apple trees.
Results obtained in 2021:
- Malus sieversii samples were collected in the population of Almaty region. Collected samples from wild apple populations were obtained. Leaf samples of Malus sieversii for DNA analysis were collected in one of the populations of the Zaili Alatau, in particular, in the population of Tau Turgen. Previously, our studies showed that this population of this region was the least susceptible to genetic erosion by domestic apple trees. The collected leaf samples were stored in an ultra-low (-80°C) refrigerator for long-term storage and subsequent isolation of genomic DNA.
- Extracted and assessed the quality of DNA from the collected sample. DNA prepared for further studies was obtained from the collected samples. DNA was isolated according to our previously optimized protocol, which was as follows: 100 mg. leaves were homogenized in 1 ml. buffer consisting of: 10% SDS, 50 mM Tris/HCl, 100 mM NaCl, 10 mM EDTA, pH 8.0, just before use, 20 µl/ml β-mercaptoethanol and 10 mg per ml PVP were added to the buffer. The homogenate was incubated in a water bath at 65°C for 45 minutes. After 45 minutes, sodium acetate was added to the homogenate to a final concentration of 1 M, and the extract was incubated on ice for 20 minutes. The homogenate was centrifuged to remove debris and the DNA was precipitated with isopropanol. The DNA precipitate was dissolved in water and treated with RNase. DNA was reprecipitated with ethanol in the presence of 0.3 M sodium acetate. The DNA pellet was dissolved in 50 µl of water. Using spectrophotometric analysis, the quantity and quality of the isolated DNA were determined by the absorption ratio at wavelengths of 260/280 nm. The optimal indicator was taken: 1.8-1.9. Its decrease indicated protein and phenol contamination, since these compounds have an absorption maximum at 280 nm. Samples isolated from wild apple leaves had a 260/280 ratio from 1.57 to 1.9, with concentrations ranging from 520 ng/µl to 150 ng/µl. Agarose gel electrophoresis was performed to confirm the quality of the isolated DNA. The quality and quantity of the isolated DNA corresponds to the result of spectrophotometry. In this case, the concentration is from 52 μg to 30 μg of DNA of one sample. Samples with a low ratio of 260/280 (below 1.8) were tested for the absence of inhibitors of the amplification reaction by PCR using primers for the 18S ribosomal RNA genes. Analysis showed no inhibition in most of these samples. For samples with a low yield of PCR product, DNA was re-isolated from leaves stored at -80°C.
- Samples were analyzed using SSR markers associated with fire blight resistance. Sievers apple genotypes were obtained, selected by molecular markers associated with resistance to fire blight. DNA isolated from the samples collected in the Zaili Alatau populations was analyzed for the presence of loci associated with resistance to fire blight. The analysis was done for the following Hi07f01 loci GACTATGGGCGTGAGTGCATGGAGGGCTTTAGTTGGGAAC-3′, 5′-GTTTGAGCTCCACTTCCAACTCC-3′); Hi23d1 (5′-GACTATGGGCGTGAGTGCATGACAGCCAGAAGA ACCCAAC-3′, 5′-GTTTATTGGTCCATTTCCCAGGAG-3′); CH03e03 (5′-GCACATTCTGCCTTATCTTGG-3′, 5′-GGCTAGGAAAGGTTA GTGGCAAAACCCACAAATAGCGCC-3′); CH03g07 (5′-GAC TATGGGCGTGAGTGCATAATAAGCATTCAAAGCAATCCG-3′, 5′-TTTTTCCAAATCGAGTTTCGTT-3′); CH-F7-Fb1 (5′-AGCCA GATCACATGTTTTCATC-3′, 5′-ACCAACCTAGGAAACACAG ACAACGGCCACCAGTTTATC-3′); GE-8019 (5′-GGCTAGGA AAGGTTAGTGGCTTGAGACCGATTTTCGTGTG-3′, 5′-TCTCTC CCAGAGCTTCATTGT-3′); AE10-375 (5′-ACCAACCTAGGAA ACACAGCTGAAGCGCACGTTCTCC-3′, 5′-CTGAAGCGCATCA TTTCTGATAG-PCR was performed in a volume of 20 µl containing a thermostable DNA polymerase buffer, 0.2 µl (10 µM) of forward and reverse oligonucleotides, 0.4 µl (10 mM) of nucleotide solutions, 40 ng of DNA, and 0.4 units of oligonucleotides. DNA polymerase. Amplification was done according to the following program: one cycle 15 min. at 95°C; 30 cycles consisting of the following steps – 30 sec. at 94°C, 1.5 min. at 60°C and 1 min. at 72°C; and finally 1 cycle of 30 min. at 72°C. At the end of the PCR, the products were diluted twice and 2 µl were taken, mixed with the size standard labeled with LIZ dye (Size standard 500 LIZ Applied Biology), the total volume of the mixture was 14.5 µl. Capillary electrophoresis of PCR products was performed on a SeqStudio DNA analyzer (Applied Biosystems). Loci allele sizes were determined using the GeneMapper v.6 program. The identified samples containing loci associated with fire blight resistance will be used in further studies in accordance with the schedule.
- Samples of Malus sieversii were introduced into in vitro culture, which showed positive results of molecular genetic analysis for resistance to fire blight. Clonal micropropagation includes several steps. Seeds, prior to introduction into the culture, were stratified for 1.5 months under conditions of low temperature and relatively high humidity to accelerate seed germination and increase their germination before planting. To prevent mold, all seeds were treated with sterilizers, and the substrates, filter paper, and water were autoclaved. Apple seeds were cleaned from the seed coat before introduction into the culture. Removing the seed coat also significantly increased seed germination. The first leaves appeared 2 weeks after planting on MS Wednesday. As a result of seed stratification, a high percentage of germination was noted in stratified samples in vermiculite. Seed germination in Petri dishes with filter paper averaged 54%. The percentage of germination in seeds stratified in vermiculite was significantly higher and averaged 84%. The lowest seed germination rate was in the stratification variant in silica gel, and it amounted to 36%. More aseptic plants are obtained from seeds than from buds and shoots. A high yield of pure plants requires less labor and reagents for daily transplanting of plants into fresh growth media. At the moment, all studied samples of Malus sieversii have been cultured in vitro.
- Nurbol Galiakparov, Ph.D., project supervisor, leading researcher. Hirsch index: 9. ORCID: https://orcid.org/0000-0003-1404-535X, Researcher ID in Publons – 34234.
- Karlygash Aubakirova, Ph.D., leading Researcher. H-index: 1, Researcher ID: M-6609-2015, Researcher ID in Publons — 34234) and/or Scopus (H-index 1, Scopus Author ID -56097194200), ORCID: https://orcid.org/0000- 0003-0162-3697.
- Ruslan Kryldakov, Ph.D., senior researcher. Hirsch index: 2. ORCID: https://orcid.org/0000-0002-2299-310X.
- Laura Yerbolova, Ph.D., leading researcher. Hirsch index: 1. ORCID: https://orcid.org/0000-0002-4364-8080.
- Saule Bayzhumanova, junior researcher. ORCID: https://orcid.org/0000-0001-8715-2055.
- Akbota Rakhatkyzy, MSc, technician. ORCID: https://orcid.org/0000-0003-1890-1096.