Characterization of flower-bud transcriptome and development of genic SSR markers in Asian lotus (Nelumbo nucifera Gaertn.).

BACKGROUND: Asian lotus (Nelumbo nucifera Gaertn.) is the national flower of India, Vietnam, and one of the top ten traditional Chinese flowers. Although lotus is highly valued for its ornamental, economic and cultural uses, genomic information, particularly the expressed sequence based (genic) markers is limited. High-throughput transcriptome sequencing provides large amounts of transcriptome data for promoting gene discovery and development of molecular markers. RESULTS: In this study, 68,593 unigenes were assembled from 1.34 million 454 GS-FLX sequence reads of a mixed flower-bud cDNA pool derived from three accessions of N. nucifera. A total of 5,226 SSR loci were identified, and 3,059 primer pairs were designed for marker development. Di-nucleotide repeat motifs were the most abundant type identified with a frequency of 65.2%, followed by tri- (31.7%), tetra- (2.1%), penta- (0.5%) and hexa-nucleotide repeats (0.5%). A total of 575 primer pairs were synthesized, of which 514 (89.4%) yielded PCR amplification products. In eight Nelumbo accessions, 109 markers were polymorphic. They were used to genotype a sample of 44 accessions representing diverse wild and cultivated genotypes of Nelumbo. The number of alleles per locus varied from 2 to 9 alleles and the polymorphism information content values ranged from 0.6 to 0.9. We performed genetic diversity analysis using 109 polymorphic markers. A UPGMA dendrogram was constructed based on Jaccard's similarity coefficients revealing distinct clusters among the 44 accessions. CONCLUSIONS: Deep transcriptome sequencing of lotus flower buds developed 3,059 genic SSRs, making a significant addition to the existing SSR markers in lotus. Among them, 109 polymorphic markers were successfully validated in 44 accessions of Nelumbo. This comprehensive set of genic SSR markers developed in our study will facilitate analyses of genetic diversity, construction of linkage maps, gene mapping, and marker-assisted selection breeding for lotus.

[Effects of NaCl stress on the seedling growth and K(+)- and Na(+) -allocation of four leguminous tree species].

Taking the pot-cultured seedlings of four leguminous tree species (Albizia julibrissin, Robinia pseudoacacia, Sophora japonica, and Gleditsia sinensis) as test materials, this paper studied their growth indices, critical salt concentration (C50), and K+ and Na+ allocation under different levels of NaCl stress, aimed to understand the difference of test tree species in salt tolerance. NaCl stress inhibited the seedling growth of the tree species. Under NaCl stress, the dry matter accumulation decreased, while the root/shoot ratio increased, especially for A. julibrissin and G. sinensis. Quadratic regression analysis showed that the C50 of A. julibrissin, R. pseudoacacia, S. japonica, and G. sinensis was 3.0 per thousand, 5.0 per thousand, 4.5 per thousand, and 3.9 per thousand, respectively, i.e., the salt tolerance of the four tree species was in the order of R. pseudoacacia > S. japonica > G. sinensis > A. julibrissin. In the root, stem, and leaf of the four tree species seedlings, the Na+ content increased with the increase of NaCl stress, while the K+ content (except in the root of A. julibrissin) decreased after an initial increase, resulting in a larger difference in the K+/Na+ ratio in the organs. Under the same NaCl stress, the allocation of Na+ in different organs of the four tree species seedlings decreased in the order of root>stem>leaf, while that of K+ differed with tree species and NaCl stress, and leaf was the main storage organ for K+. The K+/Na+ ratio in different organs decreased in the sequence of leaf>stem>root. R. pseudoacacia under NaCl stress accumulated more K+ and less Na+ in stem and leaf, and had higher K+/Na+ ratio in all organs and higher dry mass, being assessed to be more salt-tolerant. In contrast, A. julibrissin under high NaCl stress accumulated more Na+ in stem and leaf, and had a lower K+/Na+ ratio in all organs and lower dry mass, being evaluated to be lesser salt-tolerant. The K+ accumulation in seedling stem and leaf and the Na+ retention in seedling root could be the main reasons for the salt tolerance of leguminous tree species under NaCl stress.