Identification Of Apple Rootstocks And Paternity Of SH40 Natural Seedlings Using AFLP And SSR Molecular Markers

Taking advantage of high salt-tolerant apple rootstock is a key to improve the apple tolerance to salt. Cultivating salt-tolerant rootstock is the base of heightening salt tolerance. Cultivating and identifying salt-tolerant rootstock have met with a series of problems, such as choice and identification of parents. This test did the research on 30 important rootstocks in apple production by AFLP and SSR molecular markers, which provided the scientific basis for further selecting and using of excellent rootstock resources. Meanwhile, it made identification on SH40 seedlings of salt-tolerant, which not only offered the basis on the parent choice during the cross-breeding, but also established the foundation for salt-tolerant screening and character identification later on. The main research results as follows:1. The genomic DNA of apple was extracted by a modified CTAB method. The modified isolation buffer was: 100 mmol/L Tris-HCl, 20mmol/L EDTA, 1.4mol/L NaCl, 2%CTAB, 1%PVP40, 10mmol/L Na₂S₂O₅ and 2%β-Mercaptoethanal. The RNA was eliminated from the solution with RNase, and the DNA was purified by chloroform/isoamyl alcohol (two). The purified apple genomic DNA was suitable for AFLP analysis.2. After systematically study on the main factors involved, an AFLP silver-staining analysis system suitable for apple genomic DNA was established. The purified apple genomic DNA 450ng, EcoRI 3U, MseI 3U and ddH₂0 were in a reaction volume of 20μL, and incubated at 37℃for 4h. Double-stranded adaptors were added to the restriction fragments at 37℃for more than 10h (or overnight). The linked produce was diluted 10 times with ddH₂0 and used as templates for pre-amplification. The pre-amplification produce was also diluted 10 times with ddH₂0 and used for selective amplification. At the end of the selective amplification, the apple samples were denatured by adding 10μL loading buffer and heated up at 95℃for 10 min, then cooled on ice immediately. The PCR reaction products were analysed on 6% denatural acrylamide/bisacrylimade gels and then stained by silver.3. A SSR analysis system of apple was established and optimized. 75ng genomic DNA was used as the template for polymerase chain reaction (PCR). The optimized concentration of dNTP, primer and Taq Polymerase was 0.25mmol/L, 0.33μmol/L and 1U/15μL respectively.4. Seven EcoRI/MseI primer pairs showing high level of polymorphism and clear strong band were selected out from 64 primer combinations and used for further practical AFLP analysis. 382 bands were scored, 86.1% of which (328) were polymorphic. As analyzed by NTSYSpc2.1, parents of 81 SH40 natural seedlings were identified by similarity coefficient and characteristic bands. Three primer pairs of the seven were used for identification of apple rootstocks, 199 bands were scored, 88.4% of which were polymorphic, any primer combination can identify all the rootstocks and the efficiency is 100%. There were 12 rootstocks have characteristic bands, which was 40% among all of the rootstocks.5. Two pairs of high polymorphism and high quality primers for SSR analysis were selected from 12 pairs of SSR primers: An SSR fingerprinting of 30 apple rootstocks was constructed, all the rootstocks were identified, and some of them have unique band patterns, which can be used to their identification; An SSR fingerprinting of 118 SH40 natural seedlings was established, two primers can identify parent of 11 SH40 natural seedlings and the identification rate is 9%.6. Make comparison between AFLP and SSR molecular markers for the efficient identification of apple rootstocks and the paternity of SH40 natural seedlings.