Genetic Analysis Of The Wild Cherry Germplasm And Identification Of Cultivated Cherry Varieties Using DNA Fingerprints

There are rich wild cherry germplasm resourses distributed in China, especially in the provinces of Yunnan, Guizhou, Sichuan and Shaanxi. few studies has been conducted to research on the genetic structure and diversity of the wild cherry species, which has hindered the progress of the protection and utilization of all the rare cherry germplasm. Because many cherry clones and mutants looks quite similar by appearances, the phenomena of "homonym" and "synonym" often occurred in cherry production, cherry cultivar nomenclature in the present state showes many inconsistencies which has caused great loss. On the aspect of cherry breeding, it always takes quite long time for a breeder to preselect and to identify good characteristics of hybrids. A fast and effective method has not been found on the molecular level for genetically marking and identifying a cherry variety. Cherry fruit, with good economic potentiality, also need to be analyzed so as to establish a set of cherry quality evaluation standard for cherry breeding and fruit quality evaluation. According to the main research items mentioned above, a series of research work has been completed, some key research results are as follows:1. Use of Amplified DNA Sequences for the Genetic Analysis of the Cherry GermplasmA PCR method using arbitrary oligonucleotide primers and totalDNA was employed to analyze cherry germplasm polymorphisms. Out of 130, 48 10-mer primers were selected and 8 cherry species and 2 interspecific progenies were analyzed. The phylogenetic analysis was carried out using two distance-matrix methods and a dendrogram was generated to show the relationships among species and cultivars. The results showed that there were 840 amplified loci in total;23 sweet cherry and 4 sour cherry cultivars were clustered together with 569 and 247 polymorphic loci respectively which accounted for 67.74% and 29.40% of the total variation. P. tomentosa T, P. fruticosa var. aucta P. and P. humilis B. formed a monophyletic group. A close relationship between P. pseudocerasus L. and Colt, which formed another monophyletic group, was observed while P. avium L., P. cerasus L. and other cherry species were more divergent. The range of genetic distances was from 0.0623 to 0.2719 among Prunus species, which were genetically distinct. The topology of the tree was generally in agreement with toxonomical classification. The results indicated that there were one or more specific RAPD markers in 8 cherry species and 2 interspecific progenies. Using these specific markers, cherry species and interspecific progenies could be identified and good characteristics of hybrids could be selected early.2. Analysis of the genetic diversity and genetic differentiation of wild Prunus pseudocerasus Lindl. Distributed in Yunnan, Guizhou, Sichuan and Shaanxi provincesA study upon the genetic diversity and genetic differentiation of wild Prunus pseudocerasus Lindl.distributed in the provinces of Yunnan, Guizhou, Sichuan and Shaanxi has been carried out by means of RAPD markers. Out of 130, 21 10-mer primers were selected. 248 loci, with 246 of them polymorphic, have been detected in 46 individuals corresponding to 8 P. pseudocerasus Lindl. populations. The proportion of polymorphicloci (PPB) is 99.19%, A range of PPB among populations were from 24.6% - 61.7%, indicating that P. pseudocerasus Lindl. has a relatively high level of genetic diversity. Shannon diversity index (0.3808), NeVs gene diversity index (0.2437) and gene differentiation coefficient (GS7=0.4181) together reveal a relatively high level of among-population genetic differentiation despite the existence of most genetic variation within populations. The results of AMOVA analysis showed that within-population and among-population genetic variations of P. pseudocerasus Lindl. accounted for 71.51% and 28.49% of the total genetic variation respectively, which basically complied with the results of gene differentiation coefficient analysis (Gsr=0.4181). A low gene flow among P. pseudocerasus Lindl. populations was also revealed (iVm=0.6958, Nm<\). The results of correlation analysis showed that there existed no significant correlation between among-population genetic distances and geographical isolation (r= 0.1621, P>0.05). Results of clustering analysis showed a complied trend between each cluster group and geographical difference.3. Breeding and Identification of Sweet Cherry Cultivar and Rootstock Using DNA FingerprintsRAPD (Random amplified polymorphic DNA) technique was used to reveal the genetic variation among 15 cherry cultivars and accessions, which corresponded to 4 cherry species (Prunus avium L., P. cerasus L., P. mahaleb L., P. pseudocerasus L.) and 1 interspecific progeny. Out of 130, 46 arbitrary oligonucleotide primers were screened for the analysis. The results showed that all the 46 arbitrary primers produced reliable and reproducible RAPD profile. The sizes of the amplified fragments ranged from 100 to 2625 bp. 517 polymorphic loci were detected, which accounted for 98.85% of the total loci. The number of polymorphic DNAfingerprints per primer ranged from 4 to 23. inter-varietal Nei's genetic distances ranged from 0.166 to 0.479, with the average value 0.329, while those between the new cultivar "Qinying I" and the 10 rootstocks, including the new cherry rootstock "CDR-1", ranged from 0.248 to 0.376, which were genetically distinguished. All the 15 cherry genomes analyzed exhibited 148 unique DNA fingerprint markers, and the total number of the specific markers per cultivar ranged from 2 to 17. Using these specific markers, cherry cultivars and rootstocks were identified. It was concluded that RAPD technique could be applied to the rapid identification of sweet cherry varieties and their rootstocks. Using a DNA marker-assisted cherry breeding technique, two specific DNA markers were amplified and it has been proved that these specific DNA markers can be used to identify the new sweet cherry selection "Qinying 1". Fourteen different specific DNA markers were also amplified related to eleven arbitrary oligonucleotide primers for the identification of a new cherry rootstock selection "CDR 1".4. Determination and analysis on the main fruit inclusion of main varieties of Prunus avium L.The differences of soluble protein, Vc, total soluble sugar and acidity contents in post-harvest fruit among seven main production cultivars of Prunus avium L., including Lapins, Stella, Sunburst, Van, Germersdorfer, Hedelfinger and Burlat, were investigated. Bivariate correlations between the main fruit inclusion and fruit developmental phase and fruit size or between the factors of the main fruit inclusion were analysed. The result showed that the correlation between fruit developmental phase and fruit soluble protein content and total soluble sugar content was positively significant. It means that soluble protein and total sugar contents in fruit of early ripen sweet cherry variety with a shorter fruit developmental phasewere significantly (P<0.05) lower than that of middle and late ripen sweet cherry varieties with a longer fruit developmental phases. The fruit soluble protein concentrations in fruit of sweet cherry varieties with a moderately high fruit quality classification standard were at a range from 1052.0 to 1119.3 mg.lOOg^FW. The Vc concentrations in fruit of sweet cherry varieties with a moderately high fruit quality classification standard were at a range from 9.89 to 18.01 mg.lOOg^FW. The difference of total acidity contents in fruit between any two sweet cherry varieties was significant (P<0.05). The correlation between fruit sugar-acid ratio and fruit developmental phase and total soluble sugar content was positively significant. The sugar-acid ratio could be used as an index to evaluate fruit flavor and Quality accurately. The sugar-acid ratios in fruit of sweet cherry varieties with a moderately high fruit quality classification standard were at a range from 31.37 to 40.54. Comparatively speaking, the fruit size of early ripen sweet cherry variety was significantly smaller than that of middle and late ripen sweet cherry varieties (P<0.05).