Comparative study of interspecific genetic divergence and phylogenic analysis of genus <Emphasis Type="Italic">Jatropha</Emphasis> by RAPD and AFLP

Genus Jatropha with 172 species having significant economic importance belongs to the family Euphorbiaceae. There are no reports on molecular characterization and phylogenetic relationship among the species of Jatropha. Hence, the present study was undertaken to assess the extent of genetic variability that exist and also to establish phylogenetic relationship among Jatropha curcas, J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica and J. tanjorensis using RAPD and AFLP. The percentage of loci that are polymorphic among the species studied was found to be 97.74% by RAPD and 97.25% by AFLP. The mean percentage of polymorphism (PP) was found to be 68.48 by RAPD and 71.33 by AFLP. The phylogram generated with RAPD and AFLP data showed maximum similarity. With the generated data maximum relatedness was found between J. curcas and J. integerrima this may be the reason for the success of inter hybrid crosses between these two species. Neither RAPD nor AFLP data generated in this study supports the view of J. tanjorensis, a natural interspecific hybrid between J. curcas and J. gossypifolia. The present study concludes that both RAPD and AFLP techniques are comparable in divergence studies of Jatropha species. The markers generated by RAPD and AFLP can be employed efficiently for interspecific hybrids identification, marker assisted selection and genetic resource management.     

Cross species amplification ability of novel microsatellites isolated from <Emphasis Type="Italic">Jatropha curcas</Emphasis> and genetic relationship with sister taxa

The present investigation was undertaken with an aim to check the ability of cross species amplification of microsatellite markers isolated from Jatropha curcas—a renewable source of biodiesel to deduce the generic relationship with its six sister taxa (J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica, and J. tanjorensis). Out of the 49 markers checked 31 markers showed cross species amplification in all the species studied. JCDS-30, JCDS-69, JCDS-26, JCMS-13 and JCMS-21 amplified in J. curcas. However, these markers did not show any cross species amplification. Overall percentage of polymorphism (PP) among the species studied was 38% and the mean genetic similarity (GS) was found to be 0.86. The highest PP (24) and least GS (0.76) was found between J. curcas/J. podagrica and J. curcas/J. multifida and least PP (4.44) and highest GS (0.96) was found between J. integerrima/J. tanjorensis. Dendrogram analysis showed good congruence to RAPD and AFLP than nrDNA ITS data reported earlier. The characterized microsatellites will pave way for intraspecies molecular characterization which can be further utilized in species differentiation, molecular identification, characterization of interspecific hybrids, exploitation of genetic resource management and genetic improvement of the species through marker assisted breeding for economically important traits.     

Molecular phylogenetic analysis of key <Emphasis Type="Italic">Jatropha</Emphasis> species inferred from nrDNA ITS and chloroplast (<Emphasis Type="Italic">trn</Emphasis>L-F and <Emphasis Type="Italic">rbc</Emphasis>L) sequences

The genus Jatropha (Euphorbiaceae) contains species that are of significant economic and ornamental value. However, Jatropha breeding material is rather limited due to incomplete information regarding phylogenetic relationships among germplasm resources. Phylogenetic analyses were performed based on the internal transcribed spacer of nuclear ribosomal DNA (nrDNA ITS), two chloroplast regions (trnL-F and rbcL), and the combined (ITS+trnL-F+rbcL) dataset among twenty-five specimens representing six key Jatropha species. Phylogenetic relationships of Jatropha were well resolved between subgenus Curcas and subgenus Jatropha, and demonstrated the intermediate position of section Polymorphae among sections of both subgenera. Jatropha curcas and J. integerrima demonstrated a close phylogenetic relationship. The molecular data agreed with the morphological classification that recognized J. multifida and J. podagrica in sec. Peltatae. The distinct intraspecific divergence that occurred in J. curcas could be attributed to restricted gene flow caused by geographical isolation and different ecological conditions. Phylograms produced with trnL-F and rbcL sequence data suggested slow rates of sequence divergence among Jatropha spp., while the ITS gene tree had good resolution suggesting high genetic variation of ITS among Jatropha species.     

Genetic Diversity Among Jatropha and Jatropha-Related Species Based on ISSR Markers

Jatropha curcas (jatropha) is a potential biodiesel crop. A major limitation in production is that jatropha remains wild with low genetic variation. Related species/genera in the Euphorbiaceae can potentially be used for its genetic improvement. In this study, we employed inter-simple sequence repeats (ISSRs) to assess genetic variation among 30 accessions of jatropha, two accessions of bellyache bush (Jatropha gossypifolia), two accessions of spicy jatropha (Jatropha integerrima), two accessions of bottleplant shrub (Jatropha podagrica), and three accessions of castor bean hybrids. Genetic relationships were evaluated using 27 of 86 ISSR markers, yielding 307 polymorphic bands with polymorphism contents ranging from 0.76 to 0.95 for IMPN 1 and UBC 807 markers, respectively. Dice’s genetic similarity coefficient ranged from 0.39 to 0.99, which clearly separated the plant samples into seven groups at the coefficient of 0.48. The first group comprised J. curcas from Mexico, the second group comprised J. curcas from China and Vietnam, the third group comprised J. curcas from Thailand, the fourth group was J. integerrima, the fifth group was J. gossypifolia, the sixth group was J. podagrica, and the last and most distinct group was Ricinus communis. Analysis of molecular variance revealed that 63% of the variability was attributable to variation among groups, while 37% was due to variation within groups. Based on Nei’s genetic distance, the population from G2 (J. curcas from China) and G4 (J. curcas from Vietnam) had the least ISSR variability (0.0668), whereas G8 (R. communis) and Jatropha spp. displayed the highest distance (0.6005–0.7211).     

Molecular characterization of Jatropha genetic resources through inter-simple sequence repeat (ISSR) markers

The genetic diversity among eight Jatropha species and three Jatropha curcas accessions were analyzed using ISSR-PCR. Nine ISSR primers generated reproducible amplification banding pattern of 61 polymorphic bands out of 64 scored accounting for 98.14% polymorphism across the species. The ISSR primers viz., I1, I2, I3, I4, I5, I6, I7 and I10 generated 100% polymorphic patterns. Jaccard’s coefficient of similarity varied from 0.346 to 0.807, indicative of high level of genetic variation among the genotypes studied. The UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L (TNMJ1, TNMJ 22 and TNMJ 23), while second included four species viz., J. tanjorensis J. L. Ellis et Saroja., J. gossypifolia L., J. podagrica Hook and J. maheshwarii Subrum and M.P. Nayer and the third cluster included another four species viz., J. villosa Wight J. multifida L., J. integerrima Jacq and J. glandulifera Roxb. The overall grouping pattern of clustering corresponds well with principal component analysis (PCA) confirming patterns of genetic diversity observed among the species. So far, there are no reports on the molecular diversity of the Jatropha species through ISSR marker. This study provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources and also for further breeding programme towards biodiesel production.     

Isolation of 55 microsatellite markers for Jatropha curcas and its closely related species

Jatropha curcas L. (physic nut) is native to Central America and now naturalized widely in many tropical and subtropical areas. Microsatellite markers were isolated and characterized. Eleven out of 55 markers showed polymorphisms, and the allelic variation was investigated using 26 accessions of J. curcas collected from several provinces in Thailand. Each marker showed 2 to 5 alleles and the average polymorphic information content (PIC) was 0.49. Thirty four markers (62 %) were also successfully amplified in J. integerrima, J. gossypifolia and J. podagrica.     

Development of SCAR Marker Specific to Non-Toxic <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. and Designing a Novel Multiplexing PCR Along with nrDNA ITS Primers to Circumvent the False Negative Detection

Jatropha curcas L., a multipurpose shrub, has acquired significant economic importance for its seed oil which can be converted to biodiesel an emerging alternative to petro-diesel. In addition to the commercial value, it is also having medicinal and even high nutritional value to use as animal fodder which is limited due to the toxicity. Development of molecular marker will enable to differentiate non-toxic from toxic variety of J. curcas in a mixed population and also for quality control since the toxic components of J. curcas has deleterious effect on animals. In the present study, the efforts were made to generate the specific SCAR marker for toxic and/or non-toxic J. curcas from RAPD markers. Among the markers specific for toxic and non-toxic varieties, four were selected, purified, cloned, sequenced, and designed primers out of which one set of primers NT-JC/SCAR I/OPQ15-F and R could able to discriminate the non-toxic with toxic Jatropha by giving expected 430 bp size amplification in non-toxic variety. Furthermore, novel multiplex PCR was designed using the nrDNA ITS primers to overcome the false negatives. Present work also demonstrates utility of the conserved regions of nrDNA coding genes in ruling out the artifacts in PCR-like false negatives frequently occur in SCAR due to various reasons. The specific SCAR markers generated in the present investigation will help to distinguish non-toxic from toxic varieties of J. curcas or vice versa, and isolated marker along with designed multiplex protocol has applications in quality control for selective cultivation of non-toxic variety and will also assist in breeding and molecular mapping studies.     

Development and characterization of EST-SSR markers from Jatropha curcas EST database and their transferability across jatropha-related species/genus

Jatropha curcas (jatropha) is a multipurpose plant with potential as a raw material for biofuel. In the present study, a total of 43,349 expressed sequence tags (ESTs) from J. curcas were searched for type and frequency of simple sequence repeat (SSR) markers. Five thousand one hundred and seventy-five sequences were indentified to contain 6,108 SSRs with 90.8% simple and 9.2% compound repeat motifs. One hundred and sixty-three EST-SSRs were developed and used to evaluate the transferability and genetic relatedness among 4 accessions of J. curcas from China, Mexico, Thailand and Vietnam; 5 accessions of congeneric species, viz. J. gossypiifolia, dwarf J. integerrima, normal J. integerrima, J. multifida, J. podagrica; and Ricinus communis. The polymorphic markers showed 75.56–85.19% transferability among four species of Jatropha and 26.67% transferability across genera in Ricinus communis. Investigation of genetic relatedness showed that J. curcas and J. integerrima are closely related. EST-SSRs used in this study demonstrate a high efficiency of cross species/genera amplification and are useful for identifying genetic diversity of jatropha and its close taxa and to choose the desired related species for wide crossing to improve new varieties of jatropha. The markers can also be further exploited for genetic resource management and genetic improvement of related species/genera through marker-assisted breeding programs.     

Irritant phorbol derivatives from four Jatropha species

Four Jatropha species used in folk medicine were screened for irritant constituents. By chromatographic and countercurrent distribution procedures, highly irritant factors were isolated from each species. They represent new polyunsaturated esters of the tigliane-type diterpenoids 16-hydroxyphorbol (J. podagrica, J. multifida) and 12-deoxy-16-hydroxyphorbol (J. curcas, J. gossypifolia).     

Morphology,anatomy, and histochemistry of three species of Jatropha: a contribution to plant recognition and selection

• Jatropha, a traditional medicinal plant known for its anti-inflammatory, antimicrobial, anticancer, antiviral, antidiabetic, and anticoagulant properties, was the subject of our study.
• We examined the morphology and chemical composition of three Jatropha species using cross- and longitudinal sections of fresh samples, observed with light microscopy. Histochemical analysis was conducted using various reagents to reveal the metabolites present.
• Anatomically, the distinguishing feature among the three Jatropha species was the presence of secretory cavities. These structures were identified in the petiole and stem bark of J. multifida, while in J. gossypiifolia and J. curcas they were present in roots. The stem bark cells of J. gossypiifolia were roundish in shape, whereas the others were rectangular. Laticifers were detected in the leaves, petioles, and stem bark of all three Jatropha species, while idioblasts were present in almost all organs. Histochemical tests revealed that excretory idioblasts and laticifers in Jatropha species contained alkaloids, phenolics, lipophilic compounds, and terpenoids. The cuticle of non-glandular trichomes contained terpenoids, while phenolic compounds were found within the secretory cavities.
• These findings contribute to the identification of Jatropha species and provide valuable insights for the selection and collection of specific plant organs containing bioactive compounds.

     

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacer of the nuclear ribosomal DNA

The internal transcribed spacer (ITS1, 5.8S rDNA, and ITS2) region of nuclear ribosomal DNA (nrDNA) was sequenced from 53 species, which represent most of the living species diversity in the genus Phalaenopsis (Orchidaceae). A phylogeny was developed for the genus based on the neighbor-joining and maximum parsimony analyses of molecular data. Results of these analyses provided support for the monophyly of the genus Phalaenopsis and concurred in that the genera Doritis and Kingidium should be treated as being parts of the genus Phalaenopsis as suggested by Christenson (2001). Within the genus Phalaenopsis, neither subgenera Aphyllae nor Parishianae were monophyletic, and they were highly clustered with subgenus Proboscidioides plus sections Esmeralda and Deliciosae of subgenus Phalaenopsis based on ITS data. Those species also have the same characters of morphology of four pollinia and similar biogeographies. Furthermore, neither subgenus Phalaenopsis nor Polychilos was monophyletic. Within the subgenus Phalaenopsis, only section Phalaenopsis was highly supported as being monophyletic. As for the subgenus Polychilos, only section Polychilos was moderately supported as being monophyletic. In conclusion, the present molecular data obtained from the ITS sequence of nrDNA of the genus Phalaenopsis provide valuable information for elucidating the phylogeny of this genus.     

Growth,reproductive phenology and yield responses of a potential biofuel plant,Jatropha curcas grown under projected 2050 levels of elevated CO2

Jatropha (Jatropha curcas) is a non‐edible oil producing plant which is being advocated as an alternative biofuel energy resource. Its ability to grow in diverse soil conditions and minimal requirements of essential agronomical inputs compared with other oilseed crops makes it viable for cost‐effective advanced biofuel production. We designed a study to investigate the effects of elevated carbon dioxide concentration ([CO₂]) (550 ppm) on the growth, reproductive development, source‐sink relationships, fruit and seed yield of J. curcas. We report, for the first time that elevated CO₂ significantly influences reproductive characteristics of Jatropha and improve its fruit and seed yields. Net photosynthetic rate of Jatropha was 50% higher in plants grown in elevated CO₂ compared with field and ambient CO₂‐grown plants. The study also revealed that elevated CO₂ atmosphere significantly increased female to male flower ratio, above ground biomass and carbon sequestration potential in Jatropha (24 kg carbon per tree) after 1 year. Our data demonstrate that J. curcas was able to sustain enhanced rate of photosynthesis in elevated CO₂ conditions as it had sufficient sink strength to balance the increased biomass yields. Our study also elucidates that the economically important traits including fruit and seed yield in elevated CO₂ conditions were significantly high in J. curcas that holds great promise as a potential biofuel tree species for the future high CO₂ world.     

Phylogeography and molecular diversity analysis of Jatropha curcas L. and the dispersal route revealed by RAPD,AFLP and nrDNA-ITS analysis

Jatropha curcas L. (Euphorbiaceae) has acquired a great importance as a renewable source of energy with a number of environmental benefits. Very few attempts were made to understand the extent of genetic diversity and its distribution. This study was aimed to study the diversity and deduce the phylogeography of Jatropha curcas L. which is said to be the most primitive species of the genus Jatropha. Here we studied the intraspecific genetic diversity of the species distributed in different parts of the globe. The study also focused to understand the molecular diversity at reported probable center of origin (Mexico), and to reveal the dispersal route to other regions based on random amplified polymorphic DNA, amplified fragment length polymorphism and nrDNA-ITS sequences data. The overall genetic diversity of J. curcas found in the present study was narrow. The highest genetic diversity was observed in the germplasm collected from Mexico and supports the earlier hypothesis based on morphological data and natural distribution, it is the center for origin of the species. Least genetic diversity found in the Indian germplasm and clustering results revealed that the species was introduced simultaneously by two distinct germplasm and subsequently distributed in different parts of India. The present molecular data further revealed that J. curcas might have spread from the center of the origin to Cape Verde, than to Spain, Portuguese to other neighboring countries and simultaneously to Africa. The molecular evidence supports the Burkill et al. (A dictionary of the economic products of the Malay Peninsula, Governments of Malaysia and Singapore by the Ministry of Agriculture and Co-operatives. Kuala Lumpur, Malaysia, 1966) view of Portuguese might have introduced the species to India. The clustering pattern suggests that the distribution was interfered by human activity.     

Intraspecific groups of Umbelopsis ramanniana inferred from nucleotide sequences of nuclear rDNA internal transcribed spacer regions and sporangiospore morphology

Umbelopsis ramanniana is a well-known species in this genus. A characteristic morphological feature of this fungus is the remarkable variation in the sporangiospore shape, which implies the genetic variations occur in the nucleotide sequences of the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (nrDNA) in the U. ramanniana isolates. The relationship between the variations of the sequences of the nrDNA ITS regions and those of the sporangiospore morphology was investigated for 12 isolates of U. ramanniana collected in Europe. Neighbor-joining and parsimony analyses on the sequences suggested that these isolates split into three groups. Precise examination of the morphology showed that the isolates of those respective groups were different from each other in their sporangiospore shape. The present study implies at least three intraspecific groups exist in U. ramanniana and that the variations in the nucleotide sequences of the nrDNA ITS regions correlate well with those in the sporangiospore shape in these intraspecific groups.     

Effect of fertilizer application on photosynthesis and oil yield of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

The use of Jatropha curcas oil as a source of biofuel has been well-explored. However, the physiological and growth studies of J. curcas have received considerably lesser attention. In this study, leaf gas exchange measurements and leaf nitrogen content were determined for four varieties of J. curcas, grown in the field or in pots. Based on stable carbon isotope analysis (δ¹³C) and gas-exchange studies, J. curcas is a C₃ sun plant and the range of leaf photosynthetic rates (or CO₂ assimilation rates, P _Nmax) were typically between 7 and 25 μmol(CO₂) m⁻² s⁻¹ and light saturation generally occurred beyond 800 μmol(quanta) m⁻² s⁻¹. Higher rates of leaf photosynthesis were generally obtained with the mature leaves. In addition, increased foliar P _Nmax were recorded in potted J. curcas variety Indiana with increasing nitrogen (N) nutrition levels. These plants also showed greater growth, increased leaf N content, higher maximum CO₂ assimilation capacity (P _NhighCO2) and chlorophyll (Chl) content, indicating the potential of optimizing the growth of Jatropha by varying fertilizer nutrient levels. A rapid assessment for leaf N using a nondestructive and portable Chl meter had been established for J. curcas. This approach will allow repeated sampling of the same plant over time and thus enable the monitoring of the appropriate levels of soil fertility to achieve good Jatropha plantation productivity. High N nutrition improved the overall plant oil yield by increasing the total number of fruits/seeds produced per plant, while not affecting the intrinsic seed oil content.     

Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia,Asteraceae): Additional information from internal transcribed spacer sequences of nuclear ribosomal DNA

The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.     

四照花亚属的nrDNA ITS致同进化不完全

四照花亚属(Cornus subg.Syncarpea)隶属于山茱萸科山茱萸属(Cornus),我国该亚属共有5种8亚种。为探讨四照花亚属nrDNA ITS序列的致同进化不完全现象及假基因产生的可能原因,分析了该亚属4种(每种1~2个居群)共21个个体的nrDNA ITS序列。结果表明,这些类群的nrDNA ITS存在多态性,通过分析这些nrDNA ITS克隆序列的G+C含量、5.8S保守基序和二级结构最小自由能,推测其可能存在假基因。系统发育研究结果显示所有nrDNA ITS序列分成5个分支,同一个体的不同拷贝被分别置于两个甚至多个分支中,且不同分支显示了不同种间关系。四照花亚属物种个体内部存在nrDNA ITS不完全致同进化,可能归咎于不完全的世系分选(incomplete lineage sorting)、种间杂交或多倍化等进化事件,从而导致基因组内nrITS区序列出现多态性,同时也导致难以通过外部形态来划分亚属内种间界限。     

Algal filtrate: a low cost substitute to synthetic growth regulators for direct organogenesis of embryo culture in <Emphasis Type="Italic">Jatropha curcas</Emphasis> (Ratanjyot)

Jatropha curcas, the energy plant has attained great attention in recent years because of its biodiesel production potential and medicinal value. This makes it imperative to search for techniques for its rapid propagation. Our research communication has shown for the first time direct organogenesis without callus formation from embryo culture of Jatropha. All previous reports embody callusing before further propagation and use of whole seeds. We also report the very economical protocol for J. curcas using cyanobacterial culture filtrate (Aulosira fertilissima) in place of chemical hormones giving this paper a cutting edge to in vitro propagation of J. curcas. The result showed that the number of days taken for shoots and root induction was quicker by adding the cyanobacterial filtrate and shoot and root length was comparatively higher than the other treatment with synthetic plant growth regulator. The same trend was found for chlorophyll a and b. No such report previously has ever focused on the use of cyanobacterial filtrate on in vitro germination of J. curcas embryo to regenerate plants at a faster rate. Ex vitro rooting is a new approach, which will reduce the time for regeneration still further, is an area that is being presently tried out.     

衣藻属的系统发育分析——基于形态形状和nrDNA ITS序列

通过实验分析莱茵衣藻 ( Chlamydomonas reinhardtii) 1个种和互连网获得衣藻属 1 5个种及丝藻属 1个种 ( Ulothrix zonata) ,共 1 7个种的 nr DNA ITS序列 ,并以 U.zonata为外类群 ,采用计算机分析软件包对其进行分析及构建分子系统发育树图。同时以 1 2个传统分类性状 ,对此 1 6种衣藻构建数据矩阵 ;以 U.zonata动孢子的相应性状为外类群原始性状 ,用Wagner法在计算机上对其进行分枝分析 ;然后比较并分析分子系统树和表征性状分支分析树的异同。初步尝试以 ITS分子序列系统发育分析作为传统性状分析的补充来研究衣藻种间的亲缘关系。