Molecular Identification And Analysis Of Genetic Diversity On Skin Colour Variation In Pyrus

Peel color is the core indicators of appearance quality and the very important indicator of commodity value. In China fruit consumption market, the brightly-colored fruits are always favored more by consumers. In recent years, it’s a hot spot to cultivate high-quality red pear varieties and pear germplasm in breeding new red pear varieties becomes an important research. Compared with apples, oranges, grapes and other fruits, studies in pear peel color formation is less, research progress is lagging behind. Therefore, the study tested several groups of red and green variants pear cultivars, carried out the identification of molecular marker and expression of genetic analysis, expected to reveal the difference between the variation of color varieties (lines) and the original varieties in molecular level, so as to lay the foundation of the molecular mechanism of red skin formation. Specific results were as follows:1. In order to reveal the genetic difference of skin color mutants with original variety, molecular identification and analysis techniques were conducted on three groups of skin colour variation in pear by AFLP and SRAP techniques. A total of1089gene loci were screened by54AFLP primer-pairs, of which688were polymorphic with the polymorphism percentage of63.2%. And210gene loci were screened by20SRAP primer-pairs, of which76were polymorphic with the polymorphism percentage of36.2%. The results showed that seven AFLP and three SRAP primer-pairs could distinguish between’Nanguo’and Red variant strain; three AFLP and one SRAP primer-pairs could identify the difference among ’Doyenne du Cornice’,’Red Doyenne du Cornice’,’Early Red Doyenne du Comice’and its Green mutant; Red D’Anjou and Green mutant could not be distinguished by all AFLP and SRAP primer-pairs. Combining the data revealed by AFLP and SRAP, specific patterns of4cultivars or mutants were constructed. All cultivars or mutants, except Red D’Anjou and mutant, could be identified by SRAP primer emll/me5, this primer could be used as useful marker in molecular identification2. Using ’Early red Doyenne du Cornice’and’Green mutant strain of Early red Doyenne du Cornice’as materials, the anthocyanin were analyzed and compared. The anthocyanin contents trend showed that:’Early red Doyenne du Cornice’lowered then rised up after first rised, the anthocyanin content of’Early red Doyenne du Cornice’reached maximum in40days after flowering, respectively298mg/g; the anthocyanin content of ’Green mutant strain of Early red Doyenne du Cornice’ reached maximum in55days after flowering, respectively35.4mg/g. during the entire developmental phase, the anthocyanin content of’Early red Doyenne du Cornice’always higher than its green mutant strain. Anthocyanin content:the biggest difference of’Early red Doyenne du Cornice’s anthocyanin content appeared in70days after flowering, original varieties is9.90times over mutants. Therefore, we suggested that the original varieties are higher than radiation variant strain, and Anthocyanin content is an important factor of forming the final color differences.3. To reveal the key genes of regulating anthocyanin synthesis, the cDNA-AFLP method was employed to analyze the differential genetic expressions between the’Early red Doyenne du Cornice’and’Green variant strain of Early red Doyenne du Cornice’. In this study,64AFLP primer combinations was used for PCR amplification of expression analyses between the tested varieties. Of64combinations,60AFLP primer combinations could amplify stable and clear bands, and obtained47molecular loci for stability differences. The47differential bands were recovered、cloned and sequenced, compared to BLAST with the EST-sequence which obtained. The results showed that20bands were the known-function genes, the other20bands were the unknown-function genes, and remained7bands have no result. Furthermoer, the functional classification of20EST-sequences with known-function genes were analysed, and the results showed that one band was the anthocyanin synthesis related gene, one band was the primary metabolism regulatory protein, one band was the signal transduction gene, one band was the Transcription factor, four bands were the marker genes and the other eleven bands were the disease-resistant and defensive gene. The one band which involved in the anthocyanin synthesis, namedly PyMADS18, is highly homologous with MADS-box transcription factor.4. Using’Early red Doyenne du Cornice’ and ’Green mutant strain of Early red Doyenne du Comice’as materials, Expression of PyMADSl8gene in fruit developmental period was analyzed by qPCR. In the developmental of the’Early red Doyenne du Cornice’ fruit, Expression of PyMADS18gene lowered then rised up. Expression of PyMADS18gene reached maximum in40days after flowering. In the developmental of the’Green mutant of Early red Doyenne du Cornice’fruit, Expression of PyMADS18gene lowered then rised up after first rised. Expression of PyMADS18gene reached maximum in55days after flowering. Analyzing expression of PyMADS18gene and anthocyanin content showed that expression of PyMADS18gene run contrary to anthocyanin content, expression of PyMADS18gene increases, the anthocyanin content increased. The results suggest that the expression of PyMADS18gene had positive correlation with anthocyanin content, which was involved in the synthesis of anthocyanin and influenced the accumulation of anthocyanin. During the same period the comparison of the red peel and green variants shows that40days after flowering PyMADS18gene in the red peel was highly expressed, while the green variety was low, significantly difference, which is related with an obvious color difference in fruit set early stage. But the subsequent developmental stages, PyMADS18gene expression in green fruit were higher than the red variety, speculating PyMADS18involved the regulation of anthocyanin synthesis in early fruit development.