Multi-locus DNA barcoding identifies matK as a suitable marker for species identification in Hibiscus L.

The genus Hibiscus L. includes several taxa of medicinal value and species used for the extraction of natural dyes. These applications require the use of authentic plant materials. DNA barcoding is a molecular method for species identification, which helps in reliable authentication by using one or more DNA barcode marker. In this study, we have collected 44 accessions, representing 16 species of Hibiscus, distributed in the southern peninsular India, to evaluate the discriminatory power of the two core barcodes rbcLa and matK together with the suggested additional regions trnH-psbA and ITS2. No intraspecies divergence was observed among the accessions studied. Interspecies divergence was 0%-9.6% with individual markers, which increased to 0%-12.5% and 0.8%-20.3% when using two- and three-marker combinations, respectively. Differentiation of all the species of Hibiscus was possible with the matK DNA barcode marker. Also, in two-marker combinations, only those combinations with matK differentiated all the species. Though all the three-marker combinations showed 100% species differentiation, species resolution was consistently better when the matK marker formed part of the combination. These results clearly showed that matK is more suitable when compared to rbcLa, trnH-psbA, and ITS2 for species identification in Hibiscus.

Physiological responses and tolerance of kenaf (Hibiscus cannabinus L.) exposed to chromium.

Selection of kenaf species with chromium (Cr) tolerance and exploring the physiological mechanisms involved in Cr tolerance are crucial for application of these species to phyto-remediation. In the present study, a hydroponic experiment was conducted to investigate the variation in two kenaf cultivars, K39-2 and Zhe50-3 under Cr stress. At the same Cr concentration, the tolerance index (TI) of K39-2 was higher than that of Zhe50-3, indicating that K39-2 may be more tolerant to Cr than Zhe50-3. It was also observed that high concentration of chromium was accumulated both in the shoots and the roots of Hibiscus cannabinus L. The leaves of K39-2 accumulated 4760.28mgkg(-1) of dry weight under 1.50mM Cr stress, and the roots accumulated 11,958.33mgkg(-1). Physiological response shows that the antioxidant enzymes' superoxide dismutase (SOD), catalase activity (CAT) and peroxidase (POD) activities increased in the leaves and decreased in roots of the Cr-stressed plants nearly compared to the control. Moreover, the variation of antioxidant enzymes activities indicated Zhe50-3 was more vulnerable than K39-2, and the contents of the non-protein thiol pool (GSH, NPT and PCs) were higher in K39-2 than Zhe50-3 with the increased Cr concentration. Based on the observations above, it can be concluded that the well-coordinated physiological changes confer a greater Cr tolerance to K39-2 than Zhe50-3 under Cr exposure, and Hibiscus cannabinus L. has a great accumulation capacity for chromium.

Produtividade de vinagreira, pimenta rosa e carobinha cultivadas em sucessão a mucuna preta e feijão de porco / Productivity of roselle, Brazilian peppertree, and carobinha grown in succession to black velvet bean and jack bean

RESUMO O uso de adubos verdes e/ou de plantas de cobertura é uma técnica que vem sendo difundida para o cultivo de espécies medicinais, por garantir a sustentabilidade do solo agrícola, ser flexível em seu uso e por melhorar a atividade biológica do solo. O objetivo do trabalho foi avaliar o uso de culturas de cobertura para incrementar a produção de massa de plantas de vinagreira, pimenta rosa e carobinha. Foram avaliadas duas espécies de leguminosas tropicais (mucuna preta e feijão de porco) como cobertura do solo e uma testemunha (sem cultura de cobertura) e três espécies medicinais (vinagreira, pimenta rosa e carobinha). O experimento foi arranjado em esquema de parcelas subdivididas, tendo nas parcelas as coberturas vegetais e nas subparcelas, as plantas medicinais. As plantas para a cobertura vegetal foram propagadas por semeadura direta no local de cultivo, enquanto as medicinais foram transplantadas diretamente entre a palhada da cobertura vegetal. A produção da mucuna preta foi de 8,37 t ha-1 de massa seca e do feijão de porco, de 14,37 t ha-1 de massa seca. O uso das culturas de cobertura contribuiu para uma maior biomassa microbiana do solo. As produções de massas frescas de folhas, caules e frutos de vinagreira e de frutos da pimenta rosa foram maiores quando cultivadas em sucessão às coberturas vegetais do solo, independente da espécie. As produções da carobinha foram semelhantes quando cultivadas em áreas com ou sem a cobertura. Visando à maior produtividade da vinagreira e pimenta rosa, são indicadas as culturas antecessoras feijão de porco e mucuna preta.

ABSTRACT The use of green manure and/or cover crops is a technique that has been widespread for the cultivation of medicinal species, since it ensures the sustainability of agricultural soil, is flexible in its use, and improves biological activity in the soil. The purpose of this study was to evaluate the use of cover crops to increase biomass production of roselle, Brazilian peppertree, and carobinha plants. Two species of tropical legumes (black velvet bean and jack bean) were evaluated as ground covers, and also assessed were one control plot (without cover crops) and three medicinal species (roselle, Brazilian peppertree, and carobinha). The experiment was arranged in a split-plot design, where the plots had cover crops and the subplots contained the medicinal plants. The plants for the cover crop were propagated by direct sowing in the growing site, while the medicinal ones were transplanted directly between the cover crop. The dry mass production of the velvet beans and jack beans were 8.37 t ha-1 and 14.37 t, respectively. The use of cover crops contributed to the increased microbial biomass of the soil. The production of dry mass of leaves, stems, and fruits of roselle and fruits of Brazilian peppertree were higher when grown in succession to the crop cover for the soil, regardless of the species. The production of carobinha was similar when grown in areas with or without the cover. To increase the yield of roselle and Brazilian peppertree, preceding crops of jack bean and black velvet bean are recommended.

Flower color changes in three Japanese hibiscus species: further quantitative variation of anthocyanin and flavonols.

One anthocyanin and four flavonols were detected from the petals of Hibiscus hamabo, H. tiliaceus and H. glaber. They were identified as cyanidin 3-0- sambubioside, gossypetin 3-O-glucuronide-8-O-glucoside, quercetin 7-O-rutinoside, gossypetin 3-O-glucoside and gossypetin 8-O-glucuronide by UV spectra, LC-MS, acid hydrolysis and HPLC. The flavonoid composition was essentially the same among the petals ofH. hamabo, H. tiliaceus and H. glaber, and there was little quantitative variation, except for cyanidin 3-O-sambubioside, the content of which in the petals ofH. tiliaceus and H. glaber was much higher than in that of H. hamabo. Flower colors of H. tiliaceus and H. glaber change from yellow to red, and that of H. hamabo changes from yellow to orange. These changes were caused by contents of anthocyanin and flavonols, which increased after flowering of H. hamabo, H. tiliaceus and H. glaber.

[Molecular identification of Hibiscus syriacus and its adulterants using ITS2 barcode].

OBJECTIVE: To identify Hibiscus syriacus and its adulterants using DNA barcoding technique. METHODS: Nine samples of five species were PCR amplified and sequenced, and twelve samples were downloaded from the GenBank. The intra-specific and interspecific K2P distances were calculated, and neighbor-joining( NJ) tree was constructed by MEGA 5.0. RESULTS: The results showed the intra-specific genetic distances of Hibiscus syriacus were ranged from 0.009 to 0.056, which were far lower than inter-specific genetic distances between Hibiscus syriacus and its adulterants (0.236 - 0.301). Variable sites within Hibiscus syriacus ranged from 2 to 9 which were far less than the adulterants (45 - 52); Different samples of Hibiscus syriacus were gathered together and could be distinguished from its adulterants by NJ tree. CONCLUSION: ITS2 can discriminate Hibiscus syriacus from its adulterants correctly. The ITS2 region is an efficient barcode for authentication of Hibiscus syriacus and its adulterants.

Genetic diversity of kenaf (Hibiscus cannabinus) evaluated by inter-simple sequence repeat (ISSR).

Understanding genetic diversity is very useful for scientific utilization for breeding. In this study, we estimated the genetic distances in a panel of 84 kenaf accessions collected from 26 countries and regions using ISSR markers. The results of UPGMA analysis showed that kenaf germplasm had abundant genetic variation, with genetic dissimilarity coefficients ranging from 0.01 to 0.62. The in-group dissimilarity coefficient (0.29) was observed in 84 kenaf accessions, and all the accessions could be divided into three groups: cultivars (L1-1), relatively wild species (L1-2 and L1-3), and wild species (the others). Further in-group analysis in group L1-1 (0.19) revealed that the kenaf cultivars could be divided into five subgroups with distinct regional characteristics. It is imperative that genes be exchanged among all kinds of tested varieties from different origins. The results provide a useful basis for kenaf germplasm research and breeding.

[Effects of treating with concentrated sulfuric acid on the seed germination of ten Hibiscus hamabo provenance families].

The seeds from ten Hibiscus hamabo provenance families were treated with concentrated sulfuric acid for different durations (0, 10, 15, and 20 min) , and the seed germination rate, germination energy, and germination index, as well as the seed relative water adsorption rate, soluble sugar and starch contents, and alpha-amylase activity during the germination, were determined, aimed to study the effects of treating with concentrated sulfuric acid on the seed germination of H. hamabo and the differences of the seed germination among different H. hamabo provenance families. After treated with concentrated sulfuric acid, the seed germination rate, germination energy, and germination index increased significantly, and the germination time shortened remarkably. Treating with concentrated sulfuric acid for 15 minutes had the best effect, i. e., the germination rate, germination energy, and germination index were up to 95.7%, 91.3%, and 13.28, respectively, and the germination time was the shortest. The seed germination rate, germination energy, and germination index differed significantly with different provenance families, the highest germination index (15.13) being 2.12 times of the lowest germination index (7.15), and the highest germination energy (98.0%) being 1.77 times of the lowest one (55.5%). Treating with concentrated sulfuric acid accelerated the physiological and biochemical processes of seed germination, and the relative water absorption rate, soluble sugar content, and a-amylase activity were decreased after an initial increase, with the maximum at the early stage of germination. There was a significant negative relationship between the seed starch content and the seed germination index.

Identification and genetic variation among Hibiscus species (Malvaceae) using RAPD markers.

Germplasm identification and characterization is an important link between the conservation and utilization of plant genetic resources. Traditionally, species or cultivars identification has relied on morphological characters like growth habit or floral morphology like flower colour and other characteristics of the plant. Studies were undertaken for identification and determination of genetic variation within the two species of Hibiscus and 16 varieties of Hibiscus rosa-sinensis L. through random amplified polymorphic (RAPD) markers. Primer screening was made by using the DNA of variety "Prolific". Genetic analysis was made by using ten selected decamer primers. A total of 79 distinct DNA fragments ranging from 0.3 to 2.5 kb were amplified by using ten selected random decamer primers. The genetic similarity was evaluated on the basis of presence or absence of bands. The cluster analysis indicated that the 16 varieties and two species formed one cluster. The first major cluster consisted of three varieties and a second major cluster consisted of two species and 13 varieties. The genetic distance was very close within the varieties and also among the species. Thus, these RAPD markers have the potential for identification of species/varieties and characterization of genetic variation within the varieties. This is also helpful in Hibiscus breeding programs and provides a major input into conservation biology.

Characterization of Ty3-gypsy-like elements in Hibiscus syriacus.

Southern blot analysis revealed a ubiquitous distribu-tion and high copy number of Ty3-gypsy-like elements in the genome of Hibiscus syriacus. Comparative phylogenetic analysis of the large subunit of Rubisco and the integrase region of Ty3-gypsy elements in various plant species indicated that the retrotransposon-like sequences have different evolutionary histories and their own unique polymorphism in the H. syriacus population. Sequence-tagged site-restriction fragment length polymorphisms (STS-RFLP) analysis also indicated great variability in the numbers and sequences of Ty3-gypsy-like elements within H. syriacus varieties. Ty3-gypsy-like elements may still be active within H. syriacus, since Northern analysis of wounded leaves of H. syriacus variety Saehan with a probe for the integrase domain gave strong hybridization signals. The sequence heterogeneity and ubiquity of the Ty3-gypsy-like elements in H. syriacus genomes could provide reliable DNA markers for line identification as well for the analysis of genetic diversity in H. syriacus.

Paralogy and orthology in the MALVACEAE rpb2 gene family: investigation of gene duplication in hibiscus.

A sample of the second largest subunit of low-copy nuclear RNA polymerase II (rpb2) sequences from Malvaceae subfamily Malvoideae suggests that rpb2 has been duplicated early in the subfamily's history. Hibiscus and related taxa possess two rpb2 genes, both of which produce congruent phylogenetic patterns that are largely concordant with cpDNA topologies. No evidence of functional divergence or disruption was found among duplicated copies, suggesting that long-term maintenance of duplicated copies of rpb2 is usual in this lineage. Therefore, this gene may be suitable for the potential diagnosis of relatively old polyploid events. One probable pseudogene was found in Radyera farragei and a single chimeric sequence was recovered from Howittia trilocularis, suggesting that the rpb2 locus is not as prone to evolutionary processes that can confound phylogenetic inferences based on nDNA sequences. The pattern of relationships among rpb2 sequences, coupled with chromosome number information and Southern hybridization data, suggests that an early polyploid event was not the cause of the duplication, despite independent evidence of paleopolyploidy in some members of Malvoideae. Rpb2 exons and introns together are suitable for phylogenetic analysis, producing well-resolved and well-supported results that were robust to model permutation and congruent with previous studies of subfamily Malvoideae using cpDNA characters.

Genetic diversity of Hibiscus tiliaceus (Malvaceae) in China assessed using AFLP markers.

Amplified fragment length polymorphism (AFLP) markers were used to investigate the genetic variations within and among nine natural populations of Hibiscus tiliaceus in China. DNA from 145 individuals was amplified with eight primer pairs. No polymorphisms were found among the 20 samples of a marginal population of recent origin probably due to a founder effect. Across the other 125 individuals, 501 of 566 bands (88.5%) were polymorphic, and 125 unique AFLP phenotypes were observed. Estimates of genetic diversity agreed with life history traits of H. tiliaceus and geographical distribution. AMOVA analysis revealed that most genetic diversity resided within populations (84.8%), which corresponded to results reported for outcrossing plants. The indirect estimate of gene flow based on phiST was moderate (Nm=1.395). Long-distance dispersal of floating seeds and local environments may play an important role in shaping the genetic diversity of the population and the genetic structure of this species.

Diversity and varietal classification of Hibiscus syriacus L. with the heterogeneity within retrotransposon-like elements.

Retrotransposons are present in multi-copy numbers that are integrated into plant genomes with considerable heterogeneous sequences within a single plant and between plant species, which allows the use of retrotransposons as additional sources of DNA polymorphism. A primer design for the sequence-tagged specific site and cleaved amplified polymorphic sequences (STS-CAPs) that are derived from retrotransposon-like sequences was developed for the molecular marker analysis in Hibiscus syriacus. This method was applied for the detection of sequence variations of intact retrotransposons that exist in plant genomes, which resulted in higher polymorphisms than in the amplified fragment length polymorphism (AFLP). Through STS-CAPs, specific fingerprinting data among H. syriacus varieties can be easily distinguished and generated with reproducible results. It could also be adapted to any species that possess multi-copy retrotransposons for varietal identification as well as the assessment of genetic relationships.