Molecular Identification And Enhancement Of Germplasms In Hawthorn

Hawthorn (Crataegus spp.) is one of important germplasm resources of fruit trees. This research took hawthorn germplasm resources which are originally from China as the materials and RAPD system and ISSR system of hawthorn were established and optimized. Base on that, genetic diversity of the genus Crataegus was analyzed by RAPD markers and ISSR markers with the 96 accessions of the genus Crataegus growing at the National Hawthorn Germplasm Repository in Shenyang. Besides these, the in vitro regeneration system of hawthorn was established and the tetraploid plants were inducted. The main results were as follows:1. The modified CTAB method (3% CTAB in the extraction buffer) and the QIAGEN Plant DNeasy Kit were the suitable protocols for the extraction of total DNA of hawthorn. The production of total DNA was lower with the modified CTAB method, but the purity was relatively high and OD₂₆₀/OD₂₈₀ value was between 1.638 and 1.889. Both the production and the purity of total DNA was high with the QIAGEN Plant DNeasy Kit, since total DNA of the 8 samples tested in this research were higher than 100μg/g and OD₂₆₀/OD₂₈₀ value were between 1.758 and 1.850. RAPD and ISSR amplifications were carried on using total DNA of hawthorn with the modified CTAB method and the QIAGEN Plant DNeasy Kit, and several clear bands with better polymorphism, stability and repeatability could be obtained. Meanwhile, RAPD and ISSR amplifications were carried on using total DNA of hawthorn with the SDS method, but no band was obtained.2. A number of factors affected the amplification results of RAPD were investigated such as cycle numbers, Mg²⁺ concentration, quality of DNA template and kinds and concentration of Taq DNA polymerase etc. The optimized RAPD analysis system of the genus Crataegus was established for the first time, i.e., reaction mixture contained I×PCR buffer (Promega), 2.0 mM MgCl₂, 0.2 mM dNTPs (Promega), 0.3μM primer, 0.5 U Taq enzyme (Tiangen) and 50 ng total DNA of hawthorn extracted with the modified CTAB method or the QIAGEN Plant DNeasy Kit in a total volume of 20μl. The cycling conditions consisted of an initial denaturation step at 94℃for 30 s, followed by 45 cycles at 94℃for 30 s, 40℃for 2 min and 72℃for 3 min, and then a final elongation step at 72℃for 7 min. Thirty primers screened out from 146 RAPD primers were suitable for the genetic analysis of the genus Crataegus based on the number, polymorphism and clarity of the bands.3. A number of factors affected the results of ISSR were investigated such as annealing temperature, cycle numbers, Mg²⁺ concentration, quality of DNA template and kinds and concentration of Taq DNA polymerase etc. The optimized ISSR analysis system of the genus Crataegus was established for the first time, i.e., reaction mixture contained 1×PCR buffer (Promega), 3.0 mM MgCl₂, 0.2 mM dNTPs, 0.3μM primer, 0.5 U Taq enzyme (Tiangen) and 50 ng total DNA of hawthorn extracted with the modified CTAB method or the QIAGEN Plant DNeasy Kit in a total volume of 20μl. The cycling conditions consisted of an initial denaturation step at 94℃for 3 min, followed by 38 cycles at 94℃for 30s, suitable annealing temperature (screened out by temperature gradient PCR) for 1 min and 72℃for 2 min, and then a final elongation step at 72℃for 7 min. Thirteen primers, in which 8 primers were added 1～2 anchor bases in 3' end based on the repeated sequence of (GA/CT)_n or (AG/TC)_n, screened out from 36 ISSR primers were suitable for the genetic analysis of the genus Crataegus based on the number, polymorphism and clarity of the bands.4. After 1 polymorphism band of RAPD and 16 polymorphism bands of ISSR were recovered, cloned and sequenced, 9 different genome sequences of hawthorn were obtained. Based on the analysis of genome sequences of hawthorn, specific primers were designed and SCAR conversions of one RAPD marker and five ISSR markers were done successful.5. It is the first time to analyze the genetic diversity of Crataegus germplasms with molecular marker techniques and the results indicated that the genus Crataegus had abundance genetic diversity. One hundred and thirty-six polymorphism loci were detected from the 96 accessions of the genus Crataegus with 12 RAPD primers. The dissimilarity coefficients of different germplasms of hawthorn were between 0 and 0.73, and dendrogram with UPGMA method displayed that distance coefficients of different germplasm resources of hawthorn were between 0 and 0.58; 130 polymorphism loci were detected from the 96 accessions of the genus Crataegus with 13 RAPD primers. The dissimilarity coefficients of different germplasms of hawthorn were between 0 and 0.80, and dendrogram with UPGMA method displayed that distance coefficients of different germplasm resources of hawthorn were between 0 and 0.65.6. Dendrograms of the genus Crataegus were drawn separately based on the RAPD marker and the ISSR marker. Dendrogram based on the RAPD marker was nearly the same as that based on the ISSR marker. But compared with the analysis result of ISSR marker, the classification result based on the RAPD marker was more closed to traditional botany classification result which was mainly based on the morphological characteristics, therefore, RAPD marker was more suitable to study on the genetic diversity of intraspecies and interspecies of the genus Crataegus.7. Both the results of RAPD marker and ISSR marker indicated that C. brettschneideri Schneid. was a variety or a subspecies of C. pinnatifida Bge., but not a new independent species. Lvroushazha, a hawthorn resource introduced from Xiongyue Town, Gaizhou City, Liaoning Province, with black seed case and green flesh belonged to C. chlorosarca Maxim; Moreover, the hawthorn resource Zhangwushanlihong was a new species.8. In vitro regeneration from embryo, hypocotyl and cotyledon explants of hawthorn was investigated comprehensively and the results were as follows:â‘ The regeneration abilities of immature cotyledon, mature cotyledon and cotyledon leaf were obviously different, especially the regeneration ability of cotyledon leaves was higher and regeneration percentage of its adventitious bud was six times higher than that of mature cotyledon. On MS medium supplemented with 1.0 mg/L BA and 1.0 mg/L TDZ, the percentage of bud regeneration from cotyledon leaves of cultivar 'Qiujinxing' was 33.3%.â‘¡Adventitious bud regenerated from hypocotyl of hawthorn and on radicel no adventitious bud but only callus formed.â‘¢The percentage of seedling forming in vitro from immature embryo of hawthorn harvested after 40 d of flowering was very low and it was lower than 20% for 15 accessions tested in this research. But the percentage of seedling forming in vitro from mature embryo was relatively high—more than 50%.9. The mature embryo of Longhuafenrou was disposed by the mixed solution of 0.5% colchicine and 1% dimethyl sulfoxide for 48 h and transferred to SC medium supplemented with 0.1 mg/L IBA and 1.0 mg/L TDZ to induce bud differentiation. The result of chromosome number indicated that 3 out of 67 tested plants were tetraploid and the variation rate reached 4.5%.