Exploitation And Identification Of Two Mutants In ’Dongkui’ Chinese Bayberry (MYRICA RUBRA)

The single fruit weight of Myrica rubra cv. Dongkui is 1 to 5 times larger than that of other varieties. However, the alternate bearing, cracking, rotting and anti-litter problems are its weakness. Breeding a super variety with superior comprehensive traits to the maternal germplasm is very important. The systematic study on the formation of variation mechanism is also useful. In this study, the methods of morphology, cytology, molecular biology, fruit functional ingredients and transcriptomics were used comprehensively to analyze the difference between two Myrica rubra cv. Dongkui mutants (DB1 and DB2) and the wild-type one DK, DongKui). The main results are listed as follows:1. Morphological detection method was used for detecting the flowers, leaves, shoots, fruits and other tissues of Chinese bay berry. The flowering time of DB1 was 3d earlier than that of DB2, but 3d later than of CK. Based on large samples of leaves and fruits, it was discovered that CK was significantly different from the other two mutants in stamens, calyx, inflorescence, flowers, leaves, fruits and porosity. The average single fruit weight at full-ripen stage of DB1 and DB2 was 25.3g and 28.3g, respectively, significantly larger than CK, which is only 20.5g. Furthermore, flow cytometry and chromosome counting method were used to detect the chromosome number of CK and mutants. It was found that both DB1 and DB2 were tetraploid (2n= 4x= 32), while the DK was diploid (2n= 2x= 16), which could explain the phenomenon that the mutants have larger leaves and heavier fruit than CK.2. iPBS and ScoT molecular markers were used to detect the genetic relationship between the two mutants.452 repeatable bands and 401 polymorphic bands with a polymorphism of 88.72% were amplified from 38 primers, which were screened from 78 ones. The genetic similarity (GS) of 31 Myrica rubra materials was ranged between 0.55 and 0.94, mostly between 0.63 and 0.80.31 varieties could be divided into three groups with a GS threshold of 0.70. The GS of Dongkui variation materials DB1 and DB2 were 0.77 and 0.80, respectively, so they had closer genetic relationship. The GS of Xiyegaozhuang and Jiazhaizao were both 0.94, so they might be synonyms. The GS of Linhai Yangping was 0.55, which had the farthest genetic relationship with DK.3. TR-FRET were used to identify the content of Cy-3-glu, total phenolics and flavonoids in two mutants and CK. The results showed the Cy-3-glu content was highest in fruit at full-ripen stages, and it is significantly higher in DB1 and DB2, compared with DK. In addition, RNA-Seq of 6 libraries from fruit of two different development stages of two mutants and CK was carried out to reveal the whole transcriptical changes. 3708809 (DB1-1),3606358 (DB1-2),3575119 (DB2-1),3787895 (DB2-2),3610954 (DKl) and 3594742 (DK2) clean reads were obtained. And furthermore,28407 (DB1-1), 28040 (DB1-2),28043 (DB2-1),22256 (DB2-2),28683 (DK1) and 27351 (DK 2) genes were detected. There were 281 differentially expressed genes between DB1 and DK, with 123 up-regulated ones and 158 down-regulated ones, respectively. While 82 and 390 genes were up-regulated and down-regulated between DB2 and DK respectively, totally 472 genes were differentially expressed. Moreover, Real-time PCR were performed to confirm the gene expression levels of eight genes related to pigment regulation (MrUFGT, MrF3’H, MrDFR1, MrCHS, MrCHI, MrF3H, MrDFR2, MrANS).