Analysis And Establishment AFLP Fingerprinting Of SH Line Apple Rootstocks

SH line apple Rootstocks were developed from the species hybrids between Rail (Malus domestica var. Rail) and Honan crabapples (Malus Honanensis Rehder). They have outstanding characters in stress-resistantant (drought-resistant, bolting-resistantant), dwarfing, high-yielding, fruit quality, flavor and fruit coloring, which are adaptable to China natural environment. The previous studies about SH line roostocks focused on morphological character, anatomy, physiological characteristic to research dwarfing trait and productivity. However, the report about characters of different SH line rootstocks on DNA molecular level hasn't been seen. AFLP (amplified fragment length polymorphism) is a new DNA molecular marker technique with high efficiency, promptitude, stability and accuracy. It has been widely used in reaearch of fruit germplasm. In order to provide scientific basis for the identification, conservation and utilization of SH line rootstocks, AFLP silver-staining technique system suitable for apple was established. The fingerprinting of 7 SH line apple rootstocks and their parents were constructed, and the dendrogram based on AFLP bands was builted using UPGMA cluster analysis.The main research results obtained are as followings:1. The genetic DNA was extracted from apple leaves by modified CTAB method. Adding 1%β-Mercaptoethanal, 2% PVP₄₀ and 2% Na₂S₂O₅ to the modified isolation buffer. The DNA was deposited with 3M NaAc and 100% cooled ethanol and was purified by chloroform/isoamyl alcohol (24:1, v/v).The purified genomic DNA was suitable for AFLP analysis.2. AFLP silver-staining analysis system suitable for apple genomic DNA was established: The purified apple genomic DNA 450 ng, NEB Buffer2 2.0μL, BSA (10 mg/mL) 0.2μL, EcoRI 3.0 U, MseI 3.0 U and ddH₂O were in a reaction volume of 20μL, and incubated at 37℃for 5 h. Double-stranded adaptor were added to restriction fragment at 37℃for 10h (or overnight). The linked produce was diluted 10 times with ddH_O and used as templates for pre-amplification. The pre-amplification produce was also diluted 10 times with ddH₂O 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 10min, the cooled on ice immediatedly. The PCR reaction were analysis on 6% denatural acrylamide/bisacrylimade gel and the stained by silver.3. Twenty EcoRI/MseI primer pairs showing high level of polymorphism and scorable strong band were selected out from 130 primer combinations and used for further practical AFLP analysis. 548 bands were scored, 47% of which (261) were polymorphic.The fingerprinting of materials were established based on AFLP bands, most of them have unique band patten, which can be used to their identification.4. Clustering of UPGMA analysis indicates that the SM's coefficient of the SH line apple rootstocks ranged from 0.47～0.76. The results show that the materials can be divided into 2 categories and 4 groups according to the SM coefficient 0.58.5. At level 0.55, the 7 SH line apple rootstocks and their parents are divided in 3 categories, and the 7 SH line apple rootstocks themselves cluster together. The SH line apple roostocks are close to male-parent on the dendrogram. The result observed in the field that SH line apple rootstocks and their male-parent had the same deep incised and etiolated leaves was accordant to cluster analysis.