Abstract: [Objective]Rattan is one of the important forest plants, cirri and flagella are important organs for its climbing habit, which are also the important basis for rattan classification. In order to provide a basis for molecular breeding and SSR-assisted classification of rattan, study on the molecular characteristics of NAC (NAM, ATAF and CUC) transcription factor genes in Daemonorops jenkinsiana and the development of SSR markers was performed in this paper.[Method]With the aid of transcriptome data, the sequences of NAC homologue genes in D. jenkinsiana were isolated by PCR method. The analyses of gene structure, protein properties and structure as well as the prediction of SSR locus in NAC genes were conducted using bioinformatics method. The tissue specific expression of NAC genes in different tissues were analyzed using real-time quantitative PCR (qPCR). To evaluate the universality and polymorphism of the developed SSR markers, PAGE electrophoresis and sequencing analyses were used on the basis of products amplified with different rattan samples.[Result]Two homologous genes, DjNAC3 (GenBank No. KU556738) and DjNAC4 (GenBank No. KX579750), were obtained from leaves of D. jenkinsiana, of which open reading frame (ORF) were 729 bp and 1 326 bp, respectively. The genomic sequence corresponding to the ORFs of DjNAC3 and DjNAC4 were 850 bp and 1 441 bp, which all contained two exons and one intron. The proteins encoded by DjNAC3 and DjNAC4 were 242 aa and 441 aa respectively. Protein structure analysis showed that DjNAC3 and DjNAC4 had the typical structural features of NAC transcription factors, which were belonged to the CUC subfamily of NAC family. However, the similarity coefficient between DjNAC3 and DjNAC4 was only 23.6%, indicating that they might have different functions in the growth process of D. jenkinsiana. The expression patterns of DjNAC3 and DjNAC4 in different tissues were obviously different. DjNAC3 was expressed in developed cirri with the highest level, and the lowest in leaves, while that of DjNAC4 was the highest abundance in developed barbs and the lowest in early developed barbs. The SSR loci were detected in the genomic sequences of DjNAC3 and DjNAC4, the SSR locus of (TA)₆ was located in the intron region of DjNAC3 and that of (GCA)₅ was in the first exon of DjNAC4. SSR primers were designed according to the flanking sequences of SSR loci in DjNAC3 and DjNAC4. Genomic DNAs of D. jenkinsiana and other 20 different rattan samples were selected as templates for amplification. PAGE electrophoresis analysis showed that the primers had universality and polymorphism in the samples. The sequencing result of amplification products from six different templates further confirmed the polymorphism such as variation of SSR type, the number change of repetitions, and SSR locus missing, which were found in the sequences generated by the primer pair designed based on DjNAC3. The main difference in the sequences generated by the primer pair designed according to DjNAC4 was the number change of repetitions.[Conclusion]There are significant differences between DjNAC3 and DjNAC4, such as gene structures, gene expression patterns, and SSR locus, which means that DjNAC3 and DjNAC4 might play different roles in the growth and development of D. jenkinsiana. The universality and polymorphism of SSR markers developed from DjNAC3 and DjNAC4 indicate that they can serve as molecular markers for rattan classification and molecular-assisted breeding.

Key words: Daemonorops jenkinsiana, NAC, gene expression, SSR development