Screen Of Molecular Markers Of The Salt-tolerance And Analysis Of Related Gene Fragments In Apple Rootstock Seedling Progenies

In this study, the difference material that is the seedling progenies of two hybrid combinations in apple rootstock were identification and analysis of differentially expressed gene related salt-resistant, used SRAP and SSR analysis, used in a bulked segregation analysis (BSA) The main results were as follows:1. The genomic DNA of apple was extracted by a modified CTAB method. The modified isolation buffer(100 mmol/L Tris-HCl, 20mmol/L EDTA, 1.4mol/L NaCl, 2%CTAB)was added to anti-phenolic antioxidants such as 1%PVP-40, 10mmol/L Na2S2O5 and 1%β-Mercaptoethanal to; polysaccharides can be removed for the adding an appropriate amount high-salt. The RNA was eliminated from the solution with RNase, and the DNA was purified by chloroform/isoamyl, and absolute ethanol.2. The optimal SRAP system in apple rootstock was established. In a total volume of 15μL SRAP system, it contains 60ng template DNA 10×PCR Buffer (with Mg2+) 1.5μL, dNTP 0.3mmol·L-1,50ng Primer, and 0.75U TaqDNApolymerase.3. The optimal SSR system in apple rootstock was established. In a total volume of 15μL SRAP system, it contains 60ng template DNA 10×PCR Buffer (with Mg2+) 1.5μL, dNTP 0.25mmol·L-1, 0.33μmol·L-1primer, and 1.0U TaqDNApolymerase.4. There were 4 pairs of primers ((Me1Em2, Me1Em8, Me1Em12 and Me1Em14), which expressed polymorphism in parents and DNA pool and totally produced 4 polymorphic bands, in 128 ones of SRAP. In low-salt-tolerance and high-salt-tolerance groups, the four markers (Me1Em2, Me1Em8, Me1Em12 and Me1Em14) also expressed polymorphism. The results of identification of 144 hybrids with Me1Em2, Me1Em8, Me1Em12 and Me1Em14 suggested that exchange took place in 15, 18, 16 and11 plants, respectively, of 96 low-salt-tolerance ones, and in 4, 8, 5 and 0 plants of 48 high-salt- tolerance ones, respectively. The results above indicated the markers selected could be used in identification of salt tolerance for apple rootstock.5. There were 3 pairs of primers (P61, P63, and P64), which expressed polymorphism in parents and DNA pool and totally produced 10 polymorphic bands, in 79 ones of SSR. In low-salt-tolerance and high-salt-tolerance groups, the three markers (P61, P63 and P64) also expressed polymorphism. The results of identification of 144 hybrids with P61, P63 and P64 suggested that exchange took place in 13, 15 and 8 plants, respectively, of 96 low-salt-tolerance ones, and in 2, 5 and 8 plants of 48 high-salt-tolerance ones, respectively. The results above indicated the markers selected could be used in identification of salt tolerance for apple rootstock.6. The gene fragments were recovered and sequenced. BLAST search showed that No. 1 gene sequence was highly homologic with a gene encoding aβ- subunit of ATP synthetase and an EST originating from a cDNA in relation to water deficit stress, which both were closely associated with the protein functioning in stress resistance and ion transportation. The results suggested that No. 1 gene sequence was involved in stress resistance of apple rootstock. No. 2 gene sequence had high homology with a gene encoding a serine/ threonine kinase protein, indicating perhaps it was a new gene encoding the serine/ threonine kinase protein in apple rootstock. The gene sequences of A1 and A2 were highly homologic with a gene encoding aβsubunit of ATP synthetase.