Using AFLP Markers To Analyse Genetic Diversity In Armeniaca Sibirica (L.) Lam.

Armeniaca sibirica (L.) Lam. is Rosaceae, Prunoideae, Armeniaca Mill, usually deciduous arbor or shrub. It has has these characteristics of tolerance to cold, drought, infertile, and so on. Almond is high of nutrition, has highly economic and medical value. Leaves, flowers, bark and roots can be used as medicine. It is mainly distributed in Hebei, Liaoning and Inner Mongolia, as one of the dominant species of breakwind. In the past the studies on A. sibirica (L.) Lam. were being mostly concentrated on the cultivation, graft and so on, less concentrated on seed selection, breeding, and resource classifiction. In the county of Chicheng, Zhangjiakou, it was primarily selected that high-yield, late-flower and other specific types. Classification and remembership of genealogy were analyzed using RAPD markers with phenotypic character, which provided a theoretical basis of endospecies classification, characteristics of germplasm, breeding and genetic improvement. The main results are as follows:1. The genomic DNA of Armeniaca sibirica (L.) Lam. was extracted by a modified CTAB method.The modified isolation buffer was:100 mmol/L Tris-HCl, 20mmol/LEDTA, 1.4mol/L NaCI, 2%CTAB, 1%PVP-40, l%Vc, 10mmol/LNa₂S₂O₅ and 2%P-Mercaptoethanal.The RNA was eliminated from the solution with RNase,and the DNA was purified by phenol/chloroform(one), chloroform/isoamyl alcohol (one).The purified Armeniaca sibirica (L.) Lam genomic DNA was suitable for AFLP analysis.2. After systematically study on the main factors involved, an AFLP silver-staining analysis system suitable for Armeniaca sibirica (L.) Lam. genomic DNA was established. The purified Armeniaca sibirica (L.) Lam genomic DNA450ng, EcoRI 3U, MseI 3U and ddH₂O were in a reaction volume of 20uL, 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 10times with TE buffer and used as templates for pre-amplification.The pre-amplified produce was also diluted 10 time with TE buffer and used for selective amplification.At the end of the selective amplification, the Armeniaca sibirica (L.) Lam 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. Ten EcoRI/MseI primer pairs showing high level of polymorphism and scorable strong band were selected out from 64 primer combinations and used for further practical AFLP analysis.274 bands were scored, 58.3% of which were polymorphic.4. As analyzed by NTSYSpc21, the similarity coefficient of 22 Armeniaca sibirica (L.) Lam. cultivars ranged from 0.68 to 0.83.UPGMA cluster analysis showed that the tested materials could be divided into six groups when the simple matching coefficient is 0.72.The genetic relationship of 22 Armeniaca sibirica (L.) Lam. cultivars was analyzed by this method. The aim of this paper was to lay foundation for the research of germ plasm identification, varieties classification and genetic relationship.