Collection And Development Of SSR From Peach ESTs And Its Application

The improvement of peach cultivars by traditional cross breeding techniques has been constrained by long juvenility factors. The emergence of embryo culture techniques and molecular marker technologies provide convenience for saving the hybrids with embryo abortion, early identification of hybrids and improvement of breeding efficiency. In this study, a procedure of embryo culture and transplanting seedlings of ’Huangshuimi’ which is an early-mature yellow peach cultivar were developed by using embryos as explants. Moreover,96 hybrids derived from the cross ’Huangshuimi’×’Zhongyou 14’progeny were obtained by using the procedure of embryo culture and transplanting. In addition, peach EST-SSR markers from network databases with abundant ESTs were developed, collected and analyzed. The development of peach EST-SSRs with polymorphism were provided efficient markers for early identification of hybrids by using MAS (molecular marker assisted selection). The main results are as follows:1. Establishment of embryo culture and transplanting system for ’Huangshuimi’ seedlings. The embryo shell freshly removed from the fruits were treated in 5% NaCIO for 20 min and removed shell with a small hammer, followed by a 75% solution of alcohol treatment for 30 s and rinsed three or four times with sterile distilled water. Then the embryo were treated in 0.1%mercuric chloride for 5 min, and rinsed three times with sterile distilled water. The embryos stripped of the seed coat were inoculated in WPM medium in 4℃ on 65 d.2. Transplanting technology of test-tube plantlets of embryo culture.1) Acclimatization. Plantlets of peach which washed the medium and cut off the partial roots were transplanted into the plastic cups with turfysoil, perlite and loam (1:1:1, v:v:v) after roots of plantlets were dipped using carbendazim (1000 times). The plastic cups were covered into the plantlets which cultured in 2-3 w.2) Control of damping-off. liquid pesticides hymexazol (1000 times) were sprayed into the plant stalks every 3-5 d until eliminated the lesion site and turned green.3) Break apical bud dormancy.100 mg/L GA3 were used to smeared into the apical bud every 3 d. The dormancy of apical buds were broken by using GA32-3 times.3. Collection and development of EST-SSR markers of peach.1) Analysis of peach SSR-EST:30756 ESTs of peach from 83837 ESTs which derived from NCBI database were obtained by using Phrap software joined these ESTs together.2911 SSRs were distributed in 30756 ESTs were mined, including 676 single nucleotide repeat,1673 dinucleotide repeat,277 trinucleotide repeat,86 tetranucleotide repeat, 133 pentanucleotide repeat,102 hexanucleotide repeat. Dinucleotide and single nucleotide were dominant, accounted for 56.23% and 23.22%, respectively. AG/CT and AT/AT were major motifs in dinucleotide. The frequency was 79.05% and 17.96%, respectively. ATC/ATG (35.38%) was the largest motif among 8 kinds of trinucleotide repeat. AAAT/ATTT and ATCC/ATGG were major motifs in tetranucleotide. The frequency was 26.74% and 19.77%, respectively. AAGGC/CCTTG (30.83%) was the largest motif in pentanucleotide, while AAAAT/ATTTT (13.53%) was the lowest one in pentanucleotid. The frequency of 55 kinds of motif among hexanucleotide were below 6.00%。2) Development of EST-SSR marker:528 pairs of EST-SSR primers were obtained by using Primer Premier 5.0 software. The effectiveness of primers was verified by using 6 peach cultivars, including Songsen, Zaomei, Hongchuizhi, Qiumihong, Shuguang and Zhongyoupan. The target fragments could be amplified by using 298 pairs of EST-SSR primers among 6 peach cultivars. The amplification efficiency of primers was 56.4%.3) Collection of peach SSR primers.134 pairs of peach SSR primers were collected from the published literature and the GDR (Genome database of Rosaceae) database, respectively.4. Application of peach EST-SSR primers. The stable and clear fragments were amplified by using primers CPPCT26 and P09 among 6 peach varieties belong to different growth types. CPPCT26 primer related to peach growth types showed polymorphism between different varieties.