Genus Caragana Research Articles

Chloroplast Genomes Evolution and Phylogenetic Relationships of Caragana species.

Caragana sensu lato (s.l.) includes approximately 100 species that are mainly distributed in arid and semi-arid regions. Caragana species are ecologically valuable for their roles in windbreaking and sand fixation. However, the taxonomy and phylogenetic relationships of the genus Caragana are still unclear. In this study, we sequenced and assembled the chloroplast genomes of representative species of Caragana and reconstructed robust phylogenetic relationships at the section level. The Caragana chloroplast genome has lost the inverted repeat region and wascategorized in the inverted repeat loss clade (IRLC). The chloroplast genomes of the eight species ranged from 128,458 bp to 135,401 bp and contained 110 unique genes. All the Caragana chloroplast genomes have a highly conserved structure and gene order. The number of long repeats and simple sequence repeats (SSRs) showed significant variation among the eight species, indicating heterogeneous evolution in Caragana. Selective pressure analysis of the genes revealed that most of the protein-coding genes evolved under purifying selection. The phylogenetic analyses indicated that each section forms a clade, except the section Spinosae, which was divided into two clades. This study elucidated the evolution of the chloroplast genome within the widely distributed genus Caragana. The detailed information obtained from this study can serve as a valuable resource for understanding the molecular dynamics and phylogenetic relationships within Caragana.

Chloroplast genomes of Caragana tibetica and Caragana turkestanica: structures and comparative analysis

BackgroundThe genus Caragana encompasses multiple plant species that possess medicinal and ecological value. However, some species of Caragana are quite similar in morphology, so identifying species in this genus based on their morphological characteristics is considerably complex. In our research, illumina paired-end sequencing was employed to investigate the genetic organization and structure of Caragana tibetica and Caragana turkestanica, including the previously published chloroplast genome sequence of 7 Caragana plants.ResultsThe lengths of C. tibetica and C. turkestanica chloroplast genomes were 128,433 bp and 129,453 bp, respectively. The absence of inverted repeat sequences in these two species categorizes them under the inverted repeat loss clade (IRLC). They encode 110 and 111 genes (4 /4 rRNA genes, 30 /31tRNA genes, and 76 /76 protein-coding genes), respectively. Comparison of the chloroplast genomes of C. tibetica and C. turkestanica with 7 other Caragana species revealed a high overall sequence similarity. However, some divergence was observed between certain intergenic regions (matK-rbcL, psbD-psbM, atpA-psbI, and etc.). Nucleotide diversity (π) analysis revealed the detection of five highly likely variable regions, namely rps2-atpI, accD-psaI-ycf4, cemA-petA, psbN-psbH and rpoA-rps11. Phylogenetic analysis revealed that C. tibetica’s sister species is Caragana jubata, whereas C. turkestanica’s closest relative is Caragana arborescens.ConclusionsThe present study provides worthwhile information about the chloroplast genomes of C. tibetica and C. turkestanica, which aids in the identification and classification of Caragana species.

Chloroplast genome analyses of Caragana arborescens and Caragana opulens

BackgroundNumerous species within the genus Caragana have high ecological and medicinal value. However, species identification based on morphological characteristics is quite complicated in the genus. To address this issue, we analyzed complete plastid genome data for the genus.ResultsWe obtained chloroplast genomes of two species, Caragana arborescens and Caragana opulens, using Illumina sequencing technology, with lengths of 129,473 bp and 132,815 bp, respectively. The absence of inverted repeat sequences in the two species indicated that they could be assigned to the inverted repeat-lacking clade (IRLC). The genomes included 111 distinct genes (4 rRNA genes, 31 tRNA genes, and 76 protein-coding genes). In addition, 16 genes containing introns were identified in the two genomes, the majority of which contained a single intron. Repeat analyses revealed 129 and 229 repeats in C. arborescens and C. opulens, respectively. C. arborescens and C. opulens genomes contained 277 and 265 simple sequence repeats, respectively. The two Caragana species exhibited similar codon usage patterns. rpl20-clpP, rps19-rpl2, and rpl23-ycf2 showed the highest nucleotide diversity (pi). In an analysis of sequence divergence, certain intergenic regions (matK-rbcL, psbM-petN, atpA-psbI, petA-psbL, psbE-petL, and rps7-rps12) were highly variable. A phylogenetic analysis showed that C. arborescens and C. opulens were related and clustered together with four other Caragana species. The genera Astragalus and Caragana were relatively closely related.ConclusionsThe present study provides valuable information about the chloroplast genomes of C. arborescens and C. opulens and lays a foundation for future phylogenetic research and molecular marker development.

Multiple configurations of the plastid and mitochondrial genomes of Caragana spinosa.

In this study, we assembled the complete plastome and mitogenome of Caragana spinosa and explored the multiple configurations of the organelle genomes. Caragana spinosa belongs to the Papilionoidea subfamily and has significant pharmaceutical value. To explore the possible interaction between the organelle genomes, we assembled and analyzed the plastome and mitogenome of C. spinosa using the Illumina and Nanopore DNA sequencing data. The plastome of C. spinosa was 129,995bp belonging to the inverted repeat lacking clade (IRLC), which contained 77 protein-coding genes, 29 tRNA genes, and four rRNA genes. The mitogenome was 378,373bp long and encoded 54 unique genes, including 33 protein-coding, three ribosomal RNA (rRNA), and 18 transfer RNA (tRNA) genes. In addition to the single circular conformation, alternative conformations mediated by one and four repetitive sequences in the plastome and mitogenome were identified and validated, respectively. The inverted repeat (PDR12, the 12th dispersed repeat sequence in C. spinosa plastome) of plastome mediating recombinant was conserved in the genus Caragana. Furthermore, we identified 14 homologous fragments by comparing the sequences of mitogenome and plastome, including eight complete tRNA genes. A phylogenetic analysis of protein-coding genes extracted from the plastid and mitochondrial genomes revealed congruent topologies. Analyses of sequence divergence found one intergenic region, trnN-GUU-ycf1, exhibiting a high degree of variation, which can be used to develop novel molecular markers to distinguish the nine Caragana species accurately. This plastome and mitogenome of C. spinosa could provide critical information for the molecular breeding of C. spinosa and be used as a reference genome for other species of Caragana. In this study, we assembled the complete plastome and mitogenome of Caragana spinosa and explored the multiple configurations of the organelle genomes.

DNA barcoding of recently diverging legume genera: Assessing the temperate Asian Caragana (Fabaceae: Papilionoideae)

AbstractObtaining reliable species identification of the legume genus Caragana has been challenging. Until now, species identification was mostly carried out utilizing diagnostic morphological characteristics, in addition to some successful applications of secondary chemical compounds. This study was designed to establish a DNA barcoding protocol enabling unambiguous identification of 238 accessions belonging to 67 species of Caragana. The performance of four DNA barcoding regions nrITS, trnH‐psbA, matK, and rbcL was explored using three analytical approaches, Pairwise Genetic Distance, Sequence Similarity and Phylogenetic Tree method. The chloroplast regions rbcL and matK showed lower discriminatory power compared with the nuclear region internal transcribed spacer (ITS) and the chloroplast region trnH‐psbA. The nrITS outperformed the other regions in the resolution rate. The present study brings forth an efficient barcode locus for Caragana. A barcode based either on a single‐locus nrITS or the combination of nrITS and trnH‐psbA was found to be most suitable for species discrimination with distinctive barcoding gaps. An attempt has also been made to resolve taxonomic issues in the Caragana opulens complex. DNA barcoding tools when complemented with alpha taxonomic evidence can aid in solving complex systematic problems, especially when taxa are characterized by overlapping traits, such as species belonging to the Fabaceae family.

Determining the chemical profile of Caragana jubata (Pall.) Poir. by UPLC-QTOF-MS analysis and evaluating its anti-ischemic stroke effects.

Caragana jubata, belonging to the Leguminosae family, is a shrubby medicinal plant distributed in high-altitude areas of China. The red heartwood of C. jubata is the original source of 'zuomuxing', a Tibetan medicine that promotes blood circulation and removes blood stasis to treat different diseases associated with the blood. To date, research on the chemical constituents of C. jubata remains very limited. The anti-ischemic stroke (anti-IS) effects of this plant have not been studied. The aim of the present study was to analyze the chemical profile of C. jubata, establish various anti-IS models to comprehensively evaluate the anti-IS effects of C. jubata, and explore the mechanism of action. Ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was chosen to analyze the chemical profile. A middle cerebral artery occlusion reperfusion (MCAO/R) model, zebrafish cerebral thrombosis model, and oxygen-glucose deprivation/reperfusion (OGD/R) model in PC12/BV2 cells were used to thoroughly evaluate the anti-IS effects of C. jubata. Additionally, western blotting and immunofluorescence staining were used to detect the mechanism of action. Fifty-three compounds were identified from a 95% ethanol extract of C. jubata (ECJ) by UPLC-QTOF-MS analysis. 17 and 7 compounds were identified from C. jubata and the genus Caragana for the first time. ECJ was found to attenuate infarct size and reduce brain edema and neurological scores in MCAO/R rats. ECJ notably reduced the zebrafish cerebral thrombosis incidence in a dose-dependent manner compared with that in the model group. Surprisingly, compared to the positive control drug aspirin, 50μg/ml ECJ exhibited a better therapeutic effect than aspirin at 30μg/ml. Additionally, ECJ significantly increased the viability of PC12/BV2 cells injured by OGD/R. Moreover, ECJ inhibited the protein expression of M1 markers (TNF-α, iNOS, and IL-1β) and increased that of M2 markers (Arg-1 and CD206) in OGD/R-injured BV2 cells. ECJ significantly decreased the immunofluorescence intensity of CD16 and increased that of CD206. The results from UPLC-QTOF-MS analysis showed that ECJ was rich in flavonoids. The results from pharmacological experiments verified the anti-IS effects of C. jubata in vivo and in vitro for the first time. In addition, ECJ could regulate the polarization of microglia. The present study highlights the medicinal value of C. jubata, thus providing a theoretical basis for the further development of new drugs from C. jubata to treat IS.

Study on Components with Anti-Neuroinflammatory Activities from Caragana microphylla Lam.

Caragana microphylla Lam., is a perennial herb in the genus Caragana in the Fabaceae family. Two undescribed triterpenoid saponins (1-2), along with thirty-five known components (3-37) were obtained from the roots of C. microphylla Lam. These compounds were identified using physicochemical analyses and various spectroscopic methods. The anti-neuroinflammatory activities were evaluated by measuring the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 microglial cells. Compared with positive control minocycline, compounds 10, 19, and 28 exhibited significant effects with IC50 values of 14.04, 19.35 and 10.20 μM, respectively.

The chloroplasts genomic analyses of four specific Caragana species.

Many species of the genus Caragana have been used as wind prevention and sand fixation plants. They are also important traditional Chinese medicine, and ethnic medicine resource plant. Thus, chloroplast genomes (cp-genome) of some of these important species must be studied. In this study, we analyzed the chloroplast genomes of C. jubata, C. erinacea, C. opulens, and C. bicolor, including their structure, repeat sequences, mutation sites, and phylogeny. The size of the chloroplast genomes was between 127,862 and 132,780 bp, and such genomes contained 112 genes (30 tRNA, 4 rRNA, and 78 protein-coding genes), 43 of which were photosynthesis-related genes. The total guanine + cytosine (G+C) content of four Caragana species was between 34.49% and 35.15%. The four Caragana species all lacked inverted repeats and can be classified as inverted repeat-lacking clade (IRLC). Of the anticipated genes of the four chloroplast genomes, introns were discovered in 17 genes, most of which were inserted by one intron. A total of 50 interspersed repeated sequences (IRSs) were found among them, 58, 29, 61, and 74 simple sequences repeats were found in C. jubata, C. bicolor, C. opulens, and C. erinacea, respectively. Analyses of sequence divergence showed that some intergenic regions (between trnK-UUU and rbcl; trnF-GAA and ndhJ; trnL-CAA and trnT-UGU; rpoB and trnC-GCA; petA and psbL; psbE and pebL; and sequences of rpoC, ycf1, and ycf2) exhibited a high degree of variations. A phylogenetic tree of eight Caragana species and another 10 legume species was reconstructed using full sequences of the chloroplast genome. (1) Chloroplast genomes can be used for the identification and classification of Caragana species. (2) The four Caragana species have highly similar cpDNA G+C content. (3) IRS analysis of the chloroplast genomes showed that these four species, similar to the chloroplast genome of most legumes, lost IRLC regions. (4) Comparative cp-genomic analysis suggested that the cp genome structure of the Caragana genus was well conserved in highly variable regions, which can be used to exploit markers for the identification of Caragana species and further phylogenetic study. (5) Results of phylogenetic analyses were in accordance with the current taxonomic status of Caragana. The phylogenetic relationship of Caragana species was partially consistent with elevation and geographical distribution.

Mapping Shrub Coverage in Xilin Gol Grassland with Multi-Temporal Sentinel-2 Imagery

In recent decades, shrubs dominated by the genus Caragana have expanded in a large area in Xilin Gol grassland, Inner Mongolia, China. This study comprehensively evaluated the performances of multiple factors for mapping shrub coverage across the Xilin Gol grassland based on the spectral and temporal signatures of Sentinel-2 imagery, and for the first time produced a large-scale shrub coverage mapping result in this region. Considering the regional differences and gradients in the types and sizes of shrub in the study area, the study area was divided into three subregions based on precipitation data, i.e., west, middle and east regions. The shrub coverage estimation accuracy from dry- and wet-year data, different types of vegetation indices (VIs) and multiple regression methods were compared in each subregion, and the key phenological periods were selected. We also compared the accuracy of four mapping strategies, which were pairwise combinations of zoning (i.e., subregions divided by precipitation) and non-zoning, and full time series of VIs and key phenological period. Results show that the mapping accuracy in a dry year (2017) is higher than that in a wet year (2018). The optimal VIs and key phenological periods show high spatial variability. In terms of mapping strategies, the accuracy of zoning is higher than that of non-zoning. The root mean square error (RMSE), overall accuracy (OA) and recall for ‘zoning + full time series (or + key phenological period)’ strategy were 0.052 (0.055), 76.4% (79.7%) and 91.7% (94.6%), respectively, while for ‘non-zoning + full time series (or + key phenological period)’ strategy were 0.057 (0.060), 75.5% (74.6%) and 91.7% (88.6%), respectively. The mapping using VIs in key phenological periods is better than that of using full time series in the low-value prediction of shrub cover. Based on the strategy of ‘zoning + key phenological period’, the shrub coverage map of the whole region was generated with a RMSE of 0.055, OA of 80% and recall of 95%. This study not only provides the first large-scale mapping data of shrub coverage, but also provides reference for shrub dynamic monitoring in this area.

<p><strong>Molecular phylogeny and systematic evaluation of the <em>Caragana opulens</em> species complex (Fabaceae, Papilionoideae) based on the molecular and morphological data</strong></p>

In this study, we explored the evolutionary history and taxonomic treatment of the Caragana opulens complex taking information from morphological and molecular data. The complex consists of three species, C. opulens, C. licentiana and C. kansuensis. Moreover, the morphological characters currently used to differentiate the species present in the complex have been found insignificant and inconsistent and do not help diagnose the species. For the present study, we investigated its range and sampled 139 accessions from the different populations of the genus Caragana and 17 accessions of the complex. DNA sequence data from one nrDNA ITS and one cpDNA trnH-psbA loci were sequenced and analyzed using Maximum Likelihood and Bayesian methods. The resulting phylogenies were congruent in topologies. Based on morphological and molecular data, it is concluded that all three species of the complex are one of the same with significant morphological variations. Hence C. opulens is accepted as the correct name along with C. licentiana and C. kansuensis as synonyms.

Chemical constituents from the roots of Caragana grandiflora (M. B.) DC. and their chemotaxonomic importance

A comprehensive phytochemical research on roots of Caragana grandiflora, a native plant to Iran, resulted in isolation of ten compounds including four phenolic compounds (2, 4, 5, 8), two fatty alcohols (1, 6), one fatty acid (9), one triterpene (3), one glyceride derivatives (7) and one fatty acid methyl ester (10), from which eight compounds (1, 2, 4–6 and 8–10) were isolated from the genus Caragana and two compounds (5 and 10) from the family Fabaceae, for the first time. All compounds (1–10) were described from Caragana grandiflora for the first time. Chemical structures of the purified compounds were identified through FT-IR, NMR and MSS, and spectral data comparison with literature reported evidences.Our findings provide valuable information in reporting the rare existence of natural fatty acid methyl ester (10) in the Fabaceae family. Moreover, the chemotaxonomic significance of these compounds was discussed.

Determination of ureid content in shoots of russian pea shrub and siberian pea tree

Among natural ureides, allantoin (5-ureidohydantoin or 2,5-dioxoimidazolidin-4-yl urea) is the most famous compound which have been found in various organisms: bacteria, plants, and animals. Allantoin is a product of purine catabolism in mammals (except for primates and humans) and insect larvae. It is found in many plants: comfrey (Symphytum officinale L.), soybean (Glycine hispida (Moench) Maxim), alfalfa (Medicago sativa L.), purslane (Portulaca oleracea L.).
 Allantoin has a reparative, antimicrobial, and anti-inflammatory effect, that substantiate it use in the composition of numerous cosmetic and therapeutic dermatological agents. It can be used also used for the treatment of gastroduodenal ulcers and chronic gastritis. In most species of the legume family allantoin is a transport form of nitrogen, besides, its role in the adaptation of plants to stress factors is known.
 The plants of the genus Caragana (Caragana) of the legume family (Fabaceae), due to nitrogen-fixing bacteria, receive nitrogen from the soil and convert it into transport forms, namely allantoin and allantoic acid. These substances were previously isolated from the shoots of russian pea shrub (Caragana frutex (L.) C. Koch) and siberian pea tree (Caragana arborescens Lam.) and identified, but the quantitative content of ureids in these raw materials has not been studied.
 The aim of the work was to determine the content of ureides in the shoots of russian pea shrub tree and siberian pea tree.
 The objects of study were shoots of russian pea shrub tree and siberian pea tree harvested in Kharkov in May 2019 (during flowering period). The total ureide content was determined by spectrophotometric method based on the reaction of chemical decomposition of allantoin to glyoxylic acid with subsequent formation of coloured phenylhydrazone, which had absorption maximum at 530 nm.
 The study results have shown that the content of ureides (expressed as allantoin) is slightly lower in the shoots of russian pea shrub than in the shoots of siberian pea tree and equals 0.34 ± 0.03% and 0.41 ± 0.02% respectively.
 The content of total ureids in the shoots of russian pea shrub and siberian pea tree was determined. The flowering shoots of these plants can be used as herbal raw material for the development of therapeutic and prophylactic dermatological and antiulcer drugs possessing reparative, anti-inflammatory, and astringent effects.

Chemical constituents from roots of Caragana stenophylla and their anti-tumor activities

Fourteen compounds were isolated from the 80% ethanol extract of Caragana stenophylla root, by using a combination of various chromatographic approaches, including silica gel sephadex LH-20 column chromatography, and preparative HPLC. On the basis of their physical and chemical properties and spectroscopic data, their structures were elucidated as 2-(4-hydroxy-3-methoxy lphenyl)-3-methoxyl benzofuran-6-ol (1), mucodianin C (2), isopterofuran (3), formononetin (4), afromosin (5), calycosin (6), acacetin (7), 3-O-methylkaempferol (8), liquiritigenin (9), isoliquiritigenin (10), variabilin (11), resveratrol (12), zhebeiresinol (13), and 2, 3-dicarboxy-6, 7-dihydroxy-1-(3', 4'-dihydroxy)-phenyl-1, 2-dihydronaphthalen (14). Compound 1 is a new benzofuran derivative, named as mucodianin S; compounds 2, 3, 11, 13, 14 were isolated from the genus Caragana for the first time, and compounds 4-10 were firstly isolated from Caragana stenophylla. MTT assay was used to determine their cytotoxicity of the isolated compounds against human tumor cell lines, and 2 showed cytotoxicity against human hepato cellular cancer (HepG2) and human cervical (HeLa) lines, with IC₅₀ values of (16.18±0.95), (3.75±0.08) μmol•L ⁻¹, respectively.

Isoflavones and pterocarpans from Caragana changduensis

Phytochemical investigation of the heartwoods of the stems of Caragana changduensis Lion f. led to the isolation and characterization of six isoflavones (1–6), four pterocarpans (7–10), three lignans (13–15), one chalcone (11), and one isoflavan (12). Among them, compounds 3, 5, 9, 10 and 12–14 were firstly obtained from genus Caragana. The chemotaxonomic significance of these compounds was summarized.

New flavonol glycosides from the leaves of Caragana brachyantha

Two new flavonol glycosides, brachysides C and D, together with three known flavonol glycosides, were isolated from the leaves of Caragana brachyantha. The structures of brachysides C and D were elucidated on the basis of detailed spectroscopic analysis as quercetin 5-O-[α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside]-7-O-[α-l-rhamnopyranoside] and quercetin 5-O-[α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside]-7-O-[α-l-rhamnopyranoside]-4′-O-[α-l-rhamnopyranoside], respectively. The presence of flavonol tetra- and triglycosides bearing a sugar moiety at position 5 was the first report from this genus Caragana.

Brachysides A and B, New Lignan Glucosides from Caragana Brachyantha

Brachysides A and B, new lignan glucosides, have been isolated from the n-butanol soluble fraction of Caragana brachyantha, along with four known lignan glucosides, acanthoside B, tortoside B, lariciresinol 4'- O-β-D-glucopyranoside and pinoresinol 4,4'-di-O-β-D-glucopyranoside, reported for the first time from the genus Caragana. Antioxidant activities were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay.

Chemical constituents from Caragana tangutica

Phytochemical investigation of Caragana tangutica Maxim. resulted in the isolation of ten flavonoids, melilotocarpan A (1), medicarpin (2), maackiain (3), 2-(2′4′-dihydroxyphenyl)-3-methyl-6-methoxy benzofuran (4), cajanin (5), formononetin (6), 7,3′-dihydroxy-5-methoxy isoflavone (7), texasin (8), 2′,4,4′-trihydroxy chalone (9) and bolusanthin III (10), as well as one aromatic acid, p-ethoxy benzoic acid (11). Compounds 1, 4, 9, 10 and 11 were isolated for the first time from the genus Caragana. The chemotaxonomic significance of these compounds was summarized.

Effects of climatic gradients on genetic differentiation of Caragana on the Ordos Plateau, China

The genus Caragana (Fabr.) in the Ordos Plateau of Inner Mongolia, China, provides a strong opportunity to investigate patterns of genetic differentiation along steep climatic gradients, and to identify the environmental factors most likely to be responsible for driving the radiation. This study used a factorial, multi-model approach to evaluate alternative hypotheses and identify the combination of environmental factors that appear to drive genetic divergence of Caragana in the Ordos Plateau. We had three specific hypotheses. First, we expected that gradients of changing climate would act as resistant factors limiting gene flow, and would provide stronger prediction of genetic differentiation than isolation by distance. Second, we expected that variation in precipitation would be a stronger predictor of genetic differentiation among populations than variation in temperature. Third, we expected that the pattern of phylogenetic differences, in terms of derived versus ancestral states of rachis and leaf shape, would be highly correlated with these gradients of changing precipitation, reflecting adaptive radiation along gradients of changing precipitation driven by reduced gene flow and differential patterns of directional selection. As we expected, variation in precipitation was a much stronger predictor of genetic differentiation than were other climatic variables or isolation by distance. The pattern of phylogenetic differentiation among Caragana species is also closely associated with gradients of changing patterns of precipitation, suggesting that differential precipitation plays a major role in driving the genetic differentiation and adaptive radiation of the Caragana genus in the region of the Ordos Plateau.

The Genus Caragana Fabr. and Its Mongolian Ecological Culture

The genus Caragana has very important cultural value in addition to its significance in both ecology landscape. The ethnoecological study revealed that Mongolian ecological culture can accurately reflect the cultural, ecological resource value of Caragana. Firstly,except for the original Mongolian nameHaraganafor this genus,the blending Altaganafrom the Mongolian adjective Altan ( meaning golden) and Mongolian noun Haraganaalso contributes a lot to the development of modern plant science. Secondly,different thicketization-grasslands in either the steppe zone or the desert zone constructed or dominated by Caragana microphylla,C. liouana,C. tibetica,C. stenophylla or C. korshinskii are the very pastures for the Mongolian nationality to herd with sea- sons or with different livestock in accordance with the geomorphic characteristics,grassland characteristics or feeding habits of livestock. This has also become the cultural scientific foundation for the establishment of the grazing system unit centralized by rotational grazing. Thirdly,the folk songs of Mongolian nationality which speak highly of sand-fixing wind-preventing function of Caragana can reflect the Mongolian’s correct understanding of the ecological principles of biological sand fixation. They are also the authentic cultural materials of the construction of the ecological civilization in the steppe zone or the desert zone in China.

Reference Gene Selection for Quantitative Real-time PCR Normalization in Caragana intermedia under Different Abiotic Stress Conditions

Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1α would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1α were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana.