Broussonetia × hanjiana (Moraceae) microsatellite markers for species and ploidy determination developed using next-generation sequencing.

BACKGROUND: Broussonetia × hanjiana has been considered a hybrid owing to its morphology, which is intermediate between that of B. papyrifera (L.) L'Her. ex Vent. and B. kazinoki Siebold. A recent study demonstrated the hybrid origin of B. × hanjiana in Korea using molecular markers. In this study, we developed microsatellite markers for B. × hanjiana using next-generation sequencing and cross-species transferability analysis. METHODS AND RESULTS: A total of 432 primers were designed from 205,819 contigs. Among them, 24 microsatellite markers showing polymorphisms were used to evaluate the population genetic characteristics. The observed heterozygosity (HO) and expected heterozygosity (HE) were 0.835 and 0.628, respectively. The cross-species transferability of these markers was evaluated in two closely related species of Broussonetia; all 24 markers showed cross-species amplification. Using flow cytometry, diploid and triploid individuals were identified in B. × hanjiana. In particular, the BR137 marker showed evidence of two parent species (B. papyripera and B. kazinoki), with a hybrid pattern observed in B. × hanjiana, demonstrating its utility for species identification and ploidy assessment. CONCLUSIONS: The new B. × hanjiana microsatellite markers can be useful in genetic studies of closely related B. papyripera, B. kazinoki, and B. × hanjiana.

Identification of the PP2C gene family in paper mulberry (Broussonetia papyrifera) and its roles in the regulation mechanism of the response to cold stress.

OBJECTIVES: To study the possible roles of type-2C protein phosphatases (PP2Cs) which have been confirmed to play roles in the response to diverse abiotic stresses in paper mulberry, we launched a series of genomic and functional studies of BpPP2Cs. RESULTS: Sixty-three PP2C proteins in paper mulberry (Broussonetia papyrifera) were classified into 13 clades. Four BpPP2Cs with kinase domains were verified to be highly conserved in organisms ranging from algae to dicots. Seven pairs of BpPP2C genes were found to be expanding, and 18 BpPP2C genes had orthologues in Arabidopsis. BpPP2Cs showed broad expression in different tissues; the expression levels of 18 BpPP2Cs were changed and the phosphorylation levels of seven BpPP2C proteins increased at low temperature. Cold-response elements were found in the promoter region of 31 BpPP2Cs. Finally, Bp01g0320 was found to act as a hub protein and Bp01g0512 and Bp09g1278 played key roles in the ABA-signaling pathway and MAPK cascades, respectively. CONCLUSION: These results suggest that the PP2C gene family of paper mulberry is evolutionarily conserved and participates the regulation of the response to cold stress, which will play a vital role in further research on phosphatases in paper mulberry.

Genetic diversity of Broussonetia papyrifera populations in southwest China.

Broussonetia papyrifera is an important native tree species with high economic value in southwest China. Its resources are drastically reduced because of over-harvesting and habitat fragmentation. In this study, 17 natural populations of B. papyrifera were analyzed using inter-simple sequence repeat (ISSR) markers to assess the genetic diversity and population structure. In total, 100 bands were obtained from 16 ISSR primers. The B. papyrifera populations showed relatively high genetic diversity at the species level [percentage of polymorphic bands (PPB): 96%; Nei's genetic diversity (HE): 0.3074; Shannon's information index (I): 0.4617], while the genetic diversity at the population level was relatively low (PPB: 53.2%; HE: 0.1826; I: 0.2735). Relatively high level of genetic differentiation among populations (41%) was disclosed by analysis of molecular variance, which agrees with the Nei's genetic diversity statistics (40.59%) and Shannon's information measure (40.76%). Gene flow among populations (NM) was only 0.7318. A significant correlation was observed between genetic and geographic distance among the studied populations (r=0.2948). We conjectured that the genetic diversity of B. papyrifera resulted from human disturbance, habitat fragmentation, small effective population size, and geographic barrier. Given the high genetic differentiation among populations, some utilization and conservation strategies were proposed. This study provides a reference for the sustainable use of the species in southwest China.

Functional analysis of BpDREB2 gene involved in salt and drought response from a woody plant Broussonetia papyrifera.

The dehydration-responsive element binding proteins (DREBs) are important transcription factors in the regulation of plant responses to abiotic stresses. In this study, BpDREB2, an AP2/DREB-type transcription factor gene, was cloned from a woody plant, Broussonetia papyrifera by RACE-PCR. Sequence analyses revealed that BpDREB2 protein has three characteristic domains, including an AP2/EREBP, a nuclear localization signal and an acidic activation domain. Yeast one-hybrid assays showed that BpDREB2 protein specifically binds to the DRE sequence and activates the expression of reporter genes in yeast. These results suggested that BpDREB2 protein could function as a transcription factor of DREB family. The expression of BpDREB2 gene was remarkably induced by dehydration and high-salt treatments, but no significant change was observed under ABA or low-temperature conditions. Importantly, transgenic expression of BpDREB2 gene in Arabidopsis significantly enhanced its tolerance to salt and freezing without causing growth retardation. Taken together, these results suggested that BpDREB2 is a novel member of the AP2/EREBP trans-acting factor family which could enhance salt stress tolerance of plants and has the potential application in the improvement of crops and economical tree species.

[Identification of fructus broussonetiae from different habitats and its confused species with clustering analysis by near-infrared diffuse reflectance spectrometry].

OBJECTIVE: To provide a new method for identifying Fructus Broussonetiae and its confused species. METHODS: Cluster analysis was adopted for the identification. RESULTS: The method can identify Fructus Broussonetiae and the results are coincident with traditional phytotaxnomy. CONCLUSION: This method can be used for the rapid and accurate differentiation of Fructus Broussonetiae.

[Pharmacognostic identification of the root bark of Broussonetia papyrifera, an adulterant of cortex mori].

The article reports the morphological characters, the histological structure, TLC and UV spectrum identification for the adulterant of Cortex Mori, the root bark of Broussonetia papyrifera. The results show that they can be distinguished each other.