Research on molecular identification and genetic relationship of Zuili plum(Prunus salicina)

Abstract:【Objective】Zuili plum is a group of cultivars of Prunus salicina Lindl., which is originated in Jiaxing city, Zhejiang province, China. The fruits of Zuili plum are famous for their superior quality with melting flesh. Due to long period of propagation and cultivation, the phenomenon of homonyms and synonyms in Zuili plum is serious. The purpose of this study was to screen molecular markers for identifing the varieties of Zuili plum and and to understand the their phylogenetic relationships.【Methods】The plant DNA barcoding standard sequences of ITS, mat K and rbc L were used in this research. The methods included PCR amplification, sequence alignment and phylogenetic tree analysis. Total DNA were extracted from22 Zuili plum cultivars, PCR amplification was performed according to the ITS, mat K and rbc L sequence primers reported in the literature. PCR products were detected by 1% agarose gel electrophoresis. The DNA bands were cutting directly and sent to the Thermo Fisher Scientific(China) for bidirectional DNA sequencing. The ITS, mat K and rbc L sequences were assembled and then were blasted in the NCBI database. Multiple sequence alignment analysis was performed with DNAMAN software supplemented by manual proofreading. The UPGMA phylogenetic tree was constructed using MEGA 6 software.【Results】The636-641 bp ITS sequences were obtained. Base composition analysis showed that the content of(G+C)were above 60%. The sequence alignment showed, that there were 84 variation sites in ITS sequences, including 19 sites in ITS1 sequence, 62 sites in ITS2 sequence and 2 sites in 5.8S r DNA sequence. By the alignment of 22 ITS sequences, we found three sites for the site-specific mutation of Zuili plum which was located in 165 bp(C«T), 182 bp(G«C) and 195 bp(C«T), respectively. By analyzing the variation sites of ITS sequences between Zuili plum at different mature dates, we found that 8 variation sites could distinguish Zuili plum lines with different mature dates, which were, located in 138, 168, 182,285, 331, 430, 557 and 620 bp, respectively. The variation sites on 331 bp and 620 bp were specific for early mature Zuili plum, the sites on 168 bp and 285 bp were specific for late mature Zuili plum, the138 bp together with 182 bp and 557 bp were specific for mid mature Zuili plum. The NJ phylogenetic tree was constructed based the ITS sequences of plum, apricot and mume. The results showed that the Jiaxing Zuili had the closest relationship with Fujian‘Furongli'and USA‘Pluot'. The USA‘Pluot'and Fujian‘Furongli'were on one clade, the USA‘Methley'and Japan‘Oishi Wase'were on one clade.The mat K sequence were about 800 bp and had 6 variation sites which were located in 221, 329, 332,402, 430 and 750 bp. The rbc L sequence was 678 bp with 43.2%(G+C) content and sequence alignment showed there was no variation site in the 22 plum plants. The results also showed that there was no sitespecific mutation in mat K and rbc L sequences for Zuili plum. The ITS sequence had been widely used in plant taxonomy, origin and evolution researches for its length conservation and high nucleotide variability. The variation sites in ITS(83) was more than those of mat K(6) and rbc L(0) in 22 plums,indicating that the ITS sequence could be used in researches on intraspecific genetic relationships and plant molecular identification. In addition, the mat K and rbc L sequences were more suitable for phylogenetic analysis of interspecies. Zuili plum is an ancient local breed which has a special taxonomic status in Chinese plum. The NJ phylogenetic tree based the ITS sequences of plum showed that the Jiaxing Zuili had the closest relationship with Fujian‘Furongli'and USA‘Pluot'. However, the‘Furongli'and‘Pluot'were originated in Fujian province and USA respectively. Additionally, the‘Pluot'is a hybrid cultivar between the species of plum and apricot. Therefor, the genetic relationship of Zuili plum with‘Furongli'and‘Pluot'should be comprehensively evaluated with other DNA barcoding sequences or DNA molecular markers.【Conclusion】The plant DNA barcoding standard sequences of ITS, mat K and rbc L were used for Zuili molecular identification and genetic relationship anlaysis. The results showed there were84 variation sites in ITS sequences. Of which, 3 sites(located in 165 bp, 182 bp and 195 bp) were sitespecific mutation for Zuili, 8 sites(located in 138, 168, 182, 285, 331, 430, 557 and 620 bp) could distinguish early, mid and late mature Zuili plum lines. The mat K sequences had 6 variation sites and rbc L sequences had no variation sitee. Zuili plum had the closest relationship with Fujian‘Furongli'and USA‘Pluot'.