Cloning and characterization of the UBC gene from lotus (Nelumbo nucifera).

Protein ubiquitination is extensively involved in the regulation of a considerable number of physiological processes in plant cells. E2 (ubiquitin-conjugating enzyme, UBC), one of the essential enzymes of eukaryotic ubiquitination, catalyzes protein ubiquitination together with E1 and E3. In this study, we cloned four full-length cDNA NnUBCs of Nelumbo nucifera. With the same coding sequence length of 459 bp and coding 153 amino acids, these four genes are highly homologous with the AtUBC1 and AtUBC2 of Arabidopsis thaliana. Quantitative fluorescence polymerase chain reaction showed that these four genes exhibited different expression patterns in different tissues of N. nucifera. Overall, the expression of NnUBC3 was the highest in all plant tissues. Tests of different stress treatments showed that NnUBC3 plays an important role in response to heat, salt, and drought stresses in N. nucifera. Moreover, transgenic Arabidopsis plants (Atubc1-1Atubc2-1 mutant) expressing NnUBC3 presented a wild-type phenotype, indicating that NnUBC3 performs the same function as AtUBC1 and AtUBC2.

Development and characterization of genic-SSR markers from different Asia lotus (Nelumbo nucifera) types by RNA-seq.

Nelumbo nucifera is an important economic vegetable and traditional medicine, but available genetic resources remain limited. Next generation sequencing has proven to be a rapid and effective means of identifying genic simple sequence repeat (genic-SSR) markers. This study developed genic-SSRs for N. nucifera using Illumina sequencing technology to assess diversity across cultivated and wild lotus. A total of 105,834 uni-contigs were produced with an average read length of 722 bp. Exactly 11,178 genic-SSR loci were identified in 9523 uni-contigs. Di-nucleotide (64.5%) was the most abundant SSR, followed by tri-nucleotide (23%), tetra-nucleotide (8.9%), penta-nucleotide (2.5%), and hexa-nucleotide (1%) repeat types. The most common di- and tri-nucleotide repeat motifs were AG/CT (51%) and AAG/CTT (8%), respectively. Based on these SSRs sequences, 6568 primer pairs were designed, of which 72 primers were randomly selected for synthesis and validation, and 38 in-silico polymorphic primers were obtained using in-house perl scripts. A total of 110 primers were screened in the lotus samples and the results showed that 101 primers yielded amplification products, of which 80 were polymorphs. The number of alleles ranged from 2 to 17 and the PIC (polymorphism information content) ranged from 0.19 to 0.87 with a mean value of 0.55. An Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrogram based on Jaccard's similarity coefficients showed that the correlation between geographical source and genotype was low. This study describes the distribution of genic-SSRs in the expressed portion of the lotus genome. These genic-SSRs have an important role to play in molecular mapping, diversity analysis, and marker-assisted selection strategies in Nelumbo.