The Mechanisms Of Fruit Appearance Characters Formation In Sand Pear(Pyrus Pyrifolia Nakai)

Appearance quality of pear fruits is mainly composed of skin color, size and density of fruit dot, the fruit surface waxiness, epidermal rust and other factors. The sand pear ’Cuiguan’ is a cultivar with early maturity. It usually generates surface rust, which largely damages the fruit appearance quality. In order to reveal the mechanism of the appearance quality formation, we used ’Cuiguan’ and other cultivars with different fruit peel color as materials and studied fruit peel pigment content dynamic changes and microscopic and ultrastructural features of epidermal tissue during different developmental stages of fruits; screened the AFLP markers linkage with fruit color genes using BSA classification; analyzed the application of apple fruit peel color related genes in pear homologues cloning, and based on which the preliminary study of molecular regulation of sand pear lignin biosynthesis was carried out. The major results are as follows:1. Using ’Xizilv’ and ’Qingxiang’ as materials, we compared the efficiency of ethanol and acetone in chlorophyll and carotenoid extracting in peel and pulp. The results showed that the extracting efficiency of80%acetone is nearly similar with that of95%ethanol. Compared with80%acetone,95%ethanol resulted slightly lower pigment extraction, but there was no significant difference in the statistic analysis (except for chlorophyll a). The comparison of chlorophyll and carotenoid content in peel and pulp between the four green and four russet varieties at different development stages showed that the content of the pigments has no relation with the fruit skin color phenotype.’Cuiguan’ and ’Yuguan’ were used as materials to measure the dynamics changes of fruit vertical and horizontal diameter, pigments content in peel and pulp during the fruit development, and to measure titratable acid, soluble solids, and sugar contents during fruit maturing and harvest stage. The results showed that the development of fruit size of two varieties presented "S" curve; there was no significant differences of total chlorophyll and carotenoid content in peel and pulp between russet and green pear. During maturation (14days before harvest of’Cuiguan’,28days before harvest of’Yuguan’) and havest stage, fruit growth rate decreased, and the contents of titratable acid and sugars dramatically increased. The properties of these were difference between the two cultivars. Different harvest date should be determined according to the character of fruit inner quality formation for certain cultivar.2. The epidermis structure observation was carried out at microscopic and ultra-microscopic levels using green-skin fruit sand pear cultivars’Cuiguan’ and ’Cuiyu’ and russet-skin fruit sand pear cultivars ’Qingxiang’ and ’Yuguan’. The results showed that rain shelter cultivation could significantly reduce’Cuiguan’fruit dot size, while the’Cuiyu ’the fruit dot size does not significantly affect.the epidermal cell shape and arrangement of ’Cuiguan’ is different with that of’Cuiyu’. The distribution of cuticle thickness in ’Cuiguan’ is uneven, and its cuticle thickness is half of that of’Cuiyu’; the cuticle of both two cultivars ruptured and formed netted texture. But ’Cuiyu’ generated uniform and shallow cracks,’Cuiguan’ generated disorder cracks and deep into the inner epidermis.’Yu Guan’ and ’Qingxiang’ fruit formation period than ’Cuiguan’ and ’Cuiyu’ early about50d.’Cuiguan’ fruit rust is composed of single-layer corky epidermal cells that constitute the ’Yu Guan’ brown leather by multilayer corky consists of cells, the cell patchy distribution. The ultra-structure of epidermal cells showed that the1-2layer cells under the cuticle had dense cytoplasm, and rich of liposomes. The liposomes contain small lipid droplets. The structural feature indicates that the1-2layer cells under the cuticle had strong ability to secrete waxes, but the ability differed with cultivars. The fatty granule was intensive in ’Cuiyu’ liposomes, but’Cuiguan’lacked of liposomes. The research also showed that the fruit rust of ’Cuiguan’ mainly formed in the latter period of fruit development; moisture is a main external factor resulted in the rust formation.3. Two markers linked with fruit skin color were obtained through screening64pairs of AFLP primers using ’Akibae’×’Chuxialu’ hybrid offspring. One of the markers (E-AAG/M-CAG) amplified missing band in80%green-skin-fruit offspring, and amplified the target band in74%of non-green materials; the other maker (E-ACT/M-CTT) amplified missing band in77%russet-skin-fruit offspring, and amplified the target band in89%non-pure-russett-skin-fruit materials.4. Eight of three types genes including vacuolar proton pump PPase, subunit A of ATPase, auxin receptor gene, ABA receptor gene, ethylene receptor gene and the genes involved in fruit weight and disease resistance were selected for PCR analysis of apple (Malus domestica Borkh) and pear (Pyrus pyrifolia). For each gene, the primers were designed in exon and intron regions as well as in the regions upstream of start codon and downstream of stop codon, respectively. These primers were used to amplify apple and sand pear genomic DNA. It was found that all the genes have at least one pair of primers amplifying specific bands in pear genome DNA except the disease-resistant gene. Compared to the products amplified at gene other regions, the PCR products at upstream of start codon were more polymorphics, and the PCR products at exon and intron regions showed higher conservation, among different sand pear genotypes. Results of exon PCR product sequencing showed that the fragments of sand pear and apple corresponding genes in the area shared89.6%to97.5%homology. Four putative promoter sequences of lignin biosynthetic regulatory factor gene MYB were cloned from the sand pear (P. pyrifolia) with homology method. The four clones have the length of821bp,874bp,972bp and914bp, and their GenBank accession number was JF900710, JF900709, JF900707and JF900708, respectively. These sequences have the typical characteristics of gene promoters. The allelic analysis of the promoter sequences between green-and russet-skin fruit sand pear genotypes revealed that a SNP (A/G) site in PpMYBx2promoter region may have correlation with the fruit skin character.