Characterization of UDP-glucosyltransferase from Indigofera tinctoria.

Indican is a secondary metabolite in Indigofera tinctoria; its synthesis from indoxyl and UDP-glucose is catalyzed by a UDP-glucosyltransferase (UGT). In this study, we partially purified UGT extracted from I. tinctoria leaves and analyzed the protein by peptide mass fingerprinting. We identified two fragments that were homologous to UGT after comparison with the transcriptomic data of I. tinctoria leaves. The fragments were named itUgt1 and itUgt2 and were amplified using rapid amplification of cDNA ends polymerase chain reaction to obtain full-length cDNAs. The resultant nucleotide sequences of itUgt1 and itUgt2 encoded peptides of 477 and 475 amino acids, respectively. The primary structure of itUGT1 was 89% identical to that of itUGT2 and contained an important plant secondary product glycosyltransferase (PSPG) box sequence and a UGT motif. The recombinant proteins expressed in Escherichia coli were found to possess high indican synthesis activity. Although the properties of the two proteins itUGT1 and itUGT2 were very similar, itUGT2 was more stable at high temperatures than itUGT1. Expression levels of itUGT mRNA and protein in plant tissues were examined by UGT assay, immunoblotting, and semi-quantitative reverse transcription polymerase chain reaction. So far, we presume that itUGT1, but not itUGT2, primarily catalyzes indican synthesis in I. tinctoria leaves.

Analysis of Genetic Diversity and Structure Pattern of Indigofera Pseudotinctoria in Karst Habitats of the Wushan Mountains Using AFLP Markers.

Indigofera pseudotinctoria Mats is an agronomically and economically important perennial legume shrub with a high forage yield, protein content and strong adaptability, which is subject to natural habitat fragmentation and serious human disturbance. Until now, our knowledge of the genetic relationships and intraspecific genetic diversity for its wild collections is still poor, especially at small spatial scales. Here amplified fragment length polymorphism (AFLP) technology was employed for analysis of genetic diversity, differentiation, and structure of 364 genotypes of I. pseudotinctoria from 15 natural locations in Wushan Montain, a highly structured mountain with typical karst landforms in Southwest China. We also tested whether eco-climate factors has affected genetic structure by correlating genetic diversity with habitat features. A total of 515 distinctly scoreable bands were generated, and 324 of them were polymorphic. The polymorphic information content (PIC) ranged from 0.694 to 0.890 with an average of 0.789 per primer pair. On species level, Nei's gene diversity (Hj), the Bayesian genetic diversity index (HB) and the Shannon information index (I) were 0.2465, 0.2363 and 0.3772, respectively. The high differentiation among all sampling sites was detected (FST = 0.2217, GST = 0.1746, G'ST = 0.2060, θB = 0.1844), and instead, gene flow among accessions (Nm = 1.1819) was restricted. The population genetic structure resolved by the UPGMA tree, principal coordinate analysis, and Bayesian-based cluster analyses irrefutably grouped all accessions into two distinct clusters, i.e., lowland and highland groups. The population genetic structure resolved by the UPGMA tree, principal coordinate analysis, and Bayesian-based cluster analyses irrefutably grouped all accessions into two distinct clusters, i.e., lowland and highland groups. This structure pattern may indicate joint effects by the neutral evolution and natural selection. Restricted Nm was observed across all accessions, and genetic barriers were detected between adjacent accessions due to specifically geographical landform.

Genetic diversity and population structure of Indigofera szechuensis complex (Fabaceae) based on EST-SSR markers.

Indigofera, the third largest genus of Fabaceae, comprises approximately 750 species worldwide with a pantropical distribution. Eight Indigofera species, namely I. calcicola, I. delavayi, I. franchetii, I. hancockii, I. lenticellata, I. pendula, I. rigioclada, and I. szechuensis, are considered a species complex because of their morphological similarities and the phylogenetic analysis based on Internal Transcribed Spacer (ITS). Small populations of these species are allopatrically distributed in Hengduan Mountains in China. Although considerable EST-SSR markers have been developed from the transcriptome of I. szechuensis, no codominant markers have been applied to study population genetic structure of the complex. In this study, we selected 66 EST-SSR markers which were transferable in Indigofera szechuensis complex for estimating polymorphism, of which 44 EST-SSRs (66.67%) were polymorphic. Amplification with selected 23 polymorphic EST-SSRs revealed a moderately high genetic diversity in this complex. The mean value of Observed number of alleles, Expected heterozygosity, Polymorphism information content and Shannon's information index was 10.4783, 0.4335, 0.6228 and 1.4369 respectively. A total of 758 genotypes were detected, with an average of 32.9565 genotypes per locus. The Mantel test showed a significant correlation between genetic and geographic distance (r=0.0748, P=0.0100). High differentiation and limited gene flow were detected among all populations (Fst=0.3589, Nm=0.5168). The PCoA and structure analysis grouped 31 populations of Indigofera szechuensis complex into five main species. I. delavayi was obviously separated from other species, and the result was in accordance with that of morphology and phylogeny. I. pendula was a separate species, and had two distinct phenotypes. Four I. szechuensis populations (Pop23, Pop24, Pop27 and Pop30) were the same species. I. calcicola, I. hancockii, I. rigioclada, I. franchetii, I. lenticellata, and I. szechuensis, distributed in southwest of Jinshajiang, could be considered as the same species; the species of I. franchetii, I. lenticellata, and I. szechuensis distributed in northeast of Jinshajiang could be considered as another separate species. Given the above information, the morphological classification of I. calcicola, I. franchetii, I. hancockii, I. lenticellata, I. rigioclada, and I. szechuensis was incredible.

The demographic response of a deciduous shrub (the Indigofera bungeana complex, Fabaceae) to the Pleistocene climate changes in East Asia.

East Asia harbors the highest level of floristic diversity among the world's temperate regions. Despite the increase in phylogeographic studies of temperate plants in East Asia, far less attention has been paid to widely distributed deciduous shrubs that widespread across several floral regions. We sequenced two chloroplast DNA (cpDNA) fragments (ndhJ-trnF and trnD-trnT) and one nuclear DNA (Pgk1) of 472 individuals from 51 populations of such a group, the Indigofera bungeana complex. We used population genetic data as well as ecological niche modelling to examine the evolutionary history and glacial refugia during the Last Glacial Maximum (LGM) of this group. We recovered 133 cpDNA and 68 nuclear haplotypes. The star-phylogeny of the recovered cpDNA and nuclear haplotypes and demographic analyses suggested distinct range expansion of I. bungeana complex have occurred during the early and middle Pleistocene. The climate change of the LGM might have affected little on the distribution of this complex based on the niche modelling. However, these climate changes and geographic isolation probably resulted in fixtures of the private haplotypes and genetic differentiations between regions. Our results suggested that this arid-tolerant species complex may have different responses to the Quaternary climate changes with those climate-sensitive species.