SSR and SCAR mapping of a multiple-allele male-sterile gene in Chinese cabbage (Brassica rapa L.)

The genic multiple-allele inherited male-sterile gene Ms in Chinese cabbage (Brassica rapa L.) was identified as a spontaneous mutation. Applying this gene to hybrid seed production, several B. rapa cultivars have been successfully bred in China. A BC₁ population (244 plants) was constructed for mapping the Ms gene. Screening 268 simple sequence repeat (SSR) markers which cover the entire genome of Chinese cabbage was performed with bulked segregant analysis (BSA). On the basis of linkage analysis, the Ms gene was located on linkage group R07. In addition, through the amplified fragment length polymorphism (AFLP) and the sequence-characterized amplified region (SCAR) techniques combining BSA, two SCAR markers which were converted from corresponding AFLP markers flanked the Ms gene. Finally, a genetic map of the Ms gene was constructed covering a total interval of 9.0 cM. Two SCAR markers, syau_scr01 and syau_scr04, flanked the Ms gene at distances of 0.8 and 2.5 cM, respectively. All the SSR markers (cnu_m273, cnu_m030, cnu_m295, and syau_m13) were mapped on the same side of the gene as syau_scr04, the nearest one of which, syau_m13, was mapped at a distance of 3.3 cM. These SSR and SCAR markers may be useful in marker-assisted selection and map-based cloning. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The putative phytocyanin genes in Chinese cabbage (Brassica rapa L.): genome-wide identification, classification and expression analysis

Phytocyanins (PCs) are a plant-specific family of small copper-containing electron transfer proteins. PCs may bind with a single copper atom to function as electron transporters in various biological systems, such as copper trafficking and plant photosynthesis. Evidence indicates that PCs may also be involved in plant developmental processes and stress responses. Many PCs possess arabinogalactan protein-like regions and are therefore termed chimeric arabinogalactan proteins (CAGPs). Previously, 38 and 62 PC genes have been identified in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), respectively. The recent release of the Chinese cabbage genome (B. rapa ssp. Pekinensis line Chiifu-401-42) enabled us to perform a genome-wide identification and analysis. In this study we identified 84 putative PC genes in the B. rapa genome. All of the Brassica rapa phytocyanins (BrPCs) described here could be divided, based on motif constitution, into the following three main subclasses: 52 early nodulin-like proteins (ENODLs), 16 uclacyanin-like proteins (UCLs), and 11 stellacyanin-like proteins (SCLs). A structural analysis predicted that 71 BrPCs contained N-terminal secretion signals and 45 BrPCs may be glycosylphosphatidylinositol-anchored to the plasma membrane. Glycosylation prediction revealed that 48 BrPCs were CAGPs with putative arabinogalactan glycomodules, and 57 BrPCs had N-glycosylation sites. Additionally, gene duplication analysis demonstrated that almost all of the duplicated BrPC genes shared the same conserved collinear blocks and that segmental duplications play an important role in the diversification of this gene family. Surprisingly, all BrUCL genes were duplicated except for BrUCL16. Expression analyses indicated that BrENODL22/27 and BrSCL8/9 were highly expressed in reproductive organs; BrUCL6/16 was strongly expressed in roots and even more strongly expressed in stems. The genome-wide identification, classification and expression analysis of BrPCs will provide a fundamental basis for the evolution and modification of the gene family after a polyploidy event and enable the functional study of PC genes in a polyploid crop species.     

Effect of sodium chloride on gas exchange, antioxidative defense mechanism and ion accumulation in different cultivars of Indian jujube (Ziziphus mauritiana L.)

An experiment was conducted to study the effect of NaCl (electric conductivity of 0, 4, 8, 12, and 16 dS m^?1) on growth, gas exchange parameters, water status, membrane injury, chlorophyll stability index and oxidative defense mechanisms in two cultivars (Gola and Umran) of Indian jujube (Ziziphus mauritiana). Results showed that the dry mass and leaf area reduced linearly with increasing levels of salinity. Net photosynthetic rate (P _N), transpiration (E), and stomatal conductance (g _s) were comparatively lower in Umran which further declined with salinity. Leaf relative water content, chlorophyll (Chl) stability and membrane stability also decreased significantly under salt stress, with higher magnitude in Umran. Superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) activities were higher in Gola whereas hydrogen peroxide (H₂O₂) accumulation and lipid peroxidation (MDA content) were higher in control as well as salttreated plants of Umran. The Na⁺ content was higher in the roots of Gola and in the leaves of Umran, resulting in high K⁺/Na⁺ ratio in Gola leaves. Thus it is suggested that salt tolerance mechanism is more efficiently operative in cultivar Gola owing to better management of growth, physiological attributes, antioxidative defense mechanism, and restricted translocation of Na⁺ from root to leaves along with larger accumulation of K⁺ in its leaves.     

Response of photosynthetic capacity and antioxidative system of chloroplast in two wucai ( Brassica campestris L.) genotypes against chilling stress

Chilling stress during the growing season could cause a series of changes in wucai (Brassica campestris L.). WS-1 (chilling-tolerant genotype) and Ta2 (chilling-sensitive genotype) were sampled in present study to explore the chilling tolerance mechanisms. Our results indicated that photosynthetic parameters exhibited lower level in Ta2 than in WS-1 under chilling stress. The rapid chlorophyll fluorescence dynamics curve showed that chilling resulted in a greater inactivation of photosystem II reaction center in Ta2. Reactive oxygen species and malondialdehyde content of chloroplast in Ta2 were higher than WS-1. The ascorbate–glutathione cycle in chloroplast of WS-1 played a more crucial role than Ta2, which was confirmed by higher activities of antioxidant enzymes including Ascorbate peroxidase, Glutathione reductase, Monodehydroascorbate reductase and Dehydroascorbate reductase and higher content of AsA and GSH. In addition, the ultrastructure of chloroplasts in Ta2 was more severely damaged. After low temperature stress, the shape of starch granules in Ta2 changed from elliptical to round and the volume became larger than that of WS-1. The thylakoid structure of Ta2 also became dispersed from the original tight arrangement. Combined with our previous study under heat stress, WS-1 can tolerant both chilling stress and heat stress, which was partly due to a stable photosynthetic system and the higher active antioxidant system in plants, in comparison to Ta2.     

Mapping and candidate-gene screening of the novel Turnip mosaic virus resistance gene retr02 in Chinese cabbage (Brassica rapa L.)

The extreme resistance to Turnip mosaic virus observed in the Chinese cabbage (Brassica rapa) line, BP8407, is monogenic and recessive. Bulked segregant analysis was carried out to identify simple sequence repeat and Indel markers linked to this recessive resistance gene, termed recessive Turnip mosaic virus resistance 02 (retr02). Mapping of PCR-specific Indel markers on 239 individuals of a BP8407 × Ji Zao Chun F₂ population, located this resistance gene to a 0.9-cM interval between two Indel markers (BrID10694 and BrID101309) and in scaffold000060 or scaffold000104 on chromosome A04 of the B. rapa genome. Eleven eukaryotic initiation factor 4E (eIF4E) and 14 eukaryotic initiation factor 4G (eIF4G) genes are predicted in the B. rapa genome. A candidate gene, Bra035393 on scaffold000104, was predicted within the mapped resistance locus. The gene encodes the eIF(iso)4E protein. Bra035393 was sequenced in BP8407 and Ji Zao Chun. A polymorphism (A/G) was found in exon 3 between BP8407 and Ji Zao Chun. This gene was analysed in four resistant and three susceptible lines. A correlation was observed between the amino acid substitution (Gly/Asp) in the eIF(iso)4E protein and resistance/susceptibility. eIF(iso)4E has been shown previously to interact with the TuMV genome-linked protein, VPg.     

Genome-wide identification, classification and expression analysis of genes encoding putative fasciclin-like arabinogalactan proteins in Chinese cabbage (Brassica rapa L.)

Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), have both predicted AGP-like glycosylated regions and putative fasciclin (FAS) domains, which may function in cell adhesion and communication. Previous studies have identified 21, 27, and 34 FLAs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and wheat (Triticum aestivum), respectively. In this study, we identified 33 FLAs in the annotated genome of Chinese cabbage (Brassica rapa ssp. pekinensis line Chiifu-401-42). Sequence analysis indicated that FAS domains each contain two highly conserved regions, named H1 and H2, and that 17 FLAs from B. rapa (BrFLAs) possess both of these regions. Prediction of glycosylphosphatidylinositol (GPI) modification sites suggested that 15 BrFLAs were GPI-anchored to the plasma membrane. Additionally, 25 BrFLAs may have been duplicated during the processes that shaped the triplicated genome of the mesopolyploid B. rapa. Expression analyses indicated that BrFLA1, BrFLA11, BrFLA13, BrFLA28 and BrFLA32 were specifically expressed in inflorescence. Meanwhile, BrFLA9 (homologous to AtFLA12) is specifically expressed in stem, and BrFLA6/22 (homologous to AtFLA11) is also highly expressed in stem, suggesting BrFLA6/9/22 may have the same functions as AtFLA11/12 in A. thaliana. Taken together, the identification and bioinformatic analysis of FLAs in B. rapa will open the way for studying their biological functions in plant growth and development as well as evolutionary history of this gene family from A. thaliana to B. rapa.     

Salt stress regulates enzymatic and nonenzymatic antioxidative defense system in the edible part of carrot (Daucus carota L.)

Carrot (Daucus carota L.) is widely utilized all over the world due to its enriched healthy nutritional composition. However, its growth and quality is adversely affected due to saline stress. To assess salt-induced regulation in different enzymatic and nonenzymatic antioxidants in the edible part of two cultivars (T-29 and DC-4) of carrot, a greenhouse experiment was conducted. The cultivars were grown for 90 days under varying (0, 50, 100, and 150 mM NaCl) saline regimes. High accumulation of glycinebetaine (GB), malondialdehyde (MDA), and ascorbic acid (AsA) contents was found in the roots of both carrot lines under varying saline regimes. However, total soluble proteins and activities of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) declined in the edible part of both carrot cultivars. Alpha-tocopherol (α-Toco) contents remained almost unaffected at all saline regimes except at 150 mM NaCl, where tocopherol content increased markedly. Of both carrot cultivars, cv. DC-4 accumulated relatively higher amount of GB, soluble protein and α-Toco contents than cv. T-29. The cv. T-29 had considerably higher amounts of AsA and MDA and activities of POD, SOD, and CAT than those in cv. DC-4 both under saline and nonsaline conditions. Overall, GB, AsA, and MDA contents increased while enzymatic antioxidant activities decreased in both carrot cultivars under different saline regimes which indicated that the enzymatic antioxidant metabolism was negatively influenced in the edible part of carrot due to salinity stress. So it can be suggested that the carrot is salt sensitive and its nutritional value in terms of antioxidants declines under salt stress.     

Effect of 24-epibrassinolide on growth, protein content and antioxidative defense system of Brassica juncea L. subjected to cobalt ion toxicity

Brassica juncea L. plants were subjected to cobalt (Co) ion (0, 5?×?10^?4, 10^?3, 1.5?×?10^?3 and 2?×?10^?3?M) toxicity and were sprayed with different concentrations of 24-epibrassinolide (24-EBL) (0, 10^?10, 10^?8 and 10^?6?M) at 15-day stage after sowing. They were sampled at 30 and 60?days after sowing and analyzed for growth parameters in terms of shoot length and number of leaves. Thereafter, leaves were excised and content of proteins and the activities of antioxidative enzymes (superoxide dismutase (SOD) (EC 1.15.1.1) catalase (CAT) (EC 1.11.1.6), ascorbate peroxidase (APOX) (EC 1.11.1.11), guaiacol peroxidase (POD) (EC 1.11.1.7) glutathione reductase (GR) (EC 1.6.4.2), monodehydroascorbate reductase (MDHAR) (EC 1.1.5.4) and dehydroascorbate reductase (DHAR) (EC 1.8.5.1)) were analyzed. The plants exposed to cobalt ion exhibited a significant decline in growth in terms of shoot length and number of leaves. However, foliar spray treatment with 24-EBL was able to alleviate the stress generated by cobalt ion and significantly improved the above parameters. The activities of antioxidative enzymes (SOD, CAT, POD, GR, APOX, MDHAR and DHAR) and protein content were also regulated considerably in leaves of plants treated with 24-EBL alone, 10^?8?M concentration being the most effective. The activities of antioxidative enzymes also increased in leaves of B. juncea plants by the application of cobalt ion to soil and consequently sprayed with 24-EBL. Similarly, the protein content was also regulated in leaves of B. juncea plants treated with 24-EBL as compared to untreated control plants, thereby revealing stress-protective properties of 24-EBL.     

Comparative study of nitrogen fixation and carbon metabolism in two chick-pea (Cicer arietinum L.) cultivars under salt stress

Two cultivars of Cicer arietinum with differentialtolerance to salinity have been compared by analysing growth,photosynthesis, nodulation, nitrogenase activity, and carbon metabolism inthe nodule cytosol. The aim was to help elucidate the relationshipsbetween, on the one hand, sucrose and malate metabolism in nodules and, onthe other, the inhibition of nitrogen fixation under salt stress. Chick-peacultivars Pedrosillano (sensitive) and ILC1919 (tolerant) inoculated withMesorhizobium ciceri strain Ch-191 were grown in acontrolled environmental chamber and were treated with salt (0, 50, 75, and100 mM NaCl) from sowing to harvest time (28 d). Plant growth andphotosynthesis were more affected by salt in Pedrosillano than in ILC1919.Also the effect of salt on nodulation and nitrogen fixation was much morepronounced in Pedrosillano. The increase in nodular mass in ILC1919 canpartially counteract the inhibition of nitrogenase activity. The enzymes ofsucrose breakdown were inhibited by NaCl, but in ILC1919 a rise in alkalineinvertase was observed with salinity, which could compensate for the lackof the sucrose synthase hydrolytic activity. The activity of PEPC wasstimulated by salt in ILC1919. Also, this cultivar showed higher malateconcentrations in root nodules.     

Mapping QTLs for mineral accumulation and shoot dry biomass under different Zn nutritional conditions in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the most important vegetables in China. Genetic dissection of leaf mineral accumulation and tolerance to Zn stress is important for the improvement of the nutritional quality of Chinese cabbage by breeding. A mapping population with 183 doubled haploid (DH) lines was used to study the genetics of mineral accumulation and the growth response to Zn. The genetic map was constructed based on 203 AFLPs, 58 SSRs, 22 SRAPs and four ESTPs. The concentration of 11 minerals was determined in leaves for 142 DH lines grown in an open field. In addition shoot dry biomass (SDB) under normal, deficient and excessive Zn nutritional conditions were investigated in hydroponics experiments. Ten QTLs, each explaining 11.1–17.1% of the Na, Mg, P, Al, Fe, Mn, Zn and Sr concentration variance, were identified by multiple-QTL model (MQM) mapping. One common QTL was found affecting SDB under normal, deficient and excessive Zn nutritional conditions. An additional QTL was detected for SDB under Zn excess stress only. These results offer insights into the genetic basis of leaf mineral accumulation and plant growth under Zn stress conditions in Chinese cabbage.     

Interactive effects of gibberellic acid (GA3) and salt stress on growth, ion accumulation and photosynthetic capacity of two spring wheat (Triticum aestivum L.) cultivars differing in salt tolerance

A sand culture experiment assessed whether gibberellic acid(GA₃) could alleviate the adverse effects of salt stress on thegrowth, ion accumulation and photosynthetic capacity of two spring wheatcultivars, Barani-83 (salt sensitive) and SARC-I (salt tolerant).Three-week-oldplants of both cultivars were exposed to 0, 100 and 200 molm^–3 NaCl in Hoagland's nutrient solution. Threeweeks after the initiation of salt treatments, half of the plants of eachcultivar were sprayed overall with 100 mg L^–1GA₃ solution. Plants were harvested 3 weeks after theapplication of GA₃. Fresh and dry weights of shoots and roots, plantheight and leaf area were decreased with increasing supply of salt, butgibberellic acid treatment caused a significant ameliorative effect on both thecultivars with respect to these growth attributes. However, GA₃caused no significant change in grain yields but increased grain size in boththe cultivars. Saline growth medium caused a marked increase in theconcentrations of Na⁺ and Cl^– in shoots androots of both the lines. However, with the application of GA₃accumulation of Na⁺ and Cl^– was enhanced inboth shoots and roots of both wheat lines, but more ions accumulated in saltsensitive Barani-83 than in salt tolerant SARC-1. Net CO₂assimilation rate (A) of both wheat lines decreased consistently withincreasingsupply of NaCl, but application of GA₃ alleviated the effect of saltstress on this variable in both the cultivars. However, the ameliorative effectof the hormone was more pronounced in Barani-83 than in SARC-1. Althoughwater-use efficiency (A/E=CO₂assimilation/transpiration) and intrinsic water use efficiency(A/g_s=CO₂ assimilation/stomatalconductance) decreased significantly with increasing salt concentration of thegrowth medium in both the cultivars, GA₃ was more effective inenhancing both the water-use attributes in Barani-83 than in SARC-1. Overall,GA₃ treatment stimulated the vegetative growth of both cultivars ofwheat under salt stress, but it caused a slight reduction in grain yield.GA₃ treatment enhanced the accumulation of Na⁺ andCl^– in both shoots and roots of wheat plants under saltstress.It also caused a significant increase in photosynthetic capacity in both linesat the vegetative stage under both saline and non-saline media.     

Transformation of Brassica juncea (L.) Czern with bacterial codA gene enhances its tolerance to salt stress

The codA gene for biosynthesis of glycinebetaine from Arthrobacter globiformis was used for transforming Brassica juncea cv. Pusa Jaikisan (which lack any means to synthesize glycinebetaine) through Agrobacterium mediated transformation. The stable insertion of the codA gene in the shoots obtained on medium with kanamycin and hygromycin was confirmed by PCR analysis of the nptII gene. Southern hybridization with a codA probe further demonstrated its successful integration. Immunoblot analysis revealed the presence of choline oxidase demonstrating that the bacterial codA gene had been successfully transcribed and translated. The seeds of transgenic lines showed enhanced capacity to germinate under salt stress as compared to that of the wild type. Further, the seedlings of transgenic plants that expressed codA gene showed significantly higher growth than that of the wild type under salt stress conditions. These results demonstrated that the introduction of a biosynthetic pathway for glycinebetaine into Brassica juncea significantly enhanced their salt tolerance. Hence, homozygous genotypes of selected transformed lines can be exploited for improving the salt tolerance of the desirable cultivars of Brassica juncea through breeding programmes.     

Photosynthetic activity, pigment composition and antioxidative response of two mustard (Brassica juncea) cultivars differing in photosynthetic capacity subjected to cadmium stress

Photosynthetic performance, contents of chlorophyll and associated pigments, cellular damage and activities of antioxidative enzymes were investigated in two mustard (Brassica juncea L.) cultivars differing in photosynthetic capacity subjected to cadmium (Cd) stress. Exposure to Cd severely restricted the net photosynthetic rate (P(N)) of RH-30 compared to Varuna. This corresponded to the reductions in the activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase (Rubisco) in both the cultivars. Decline in chlorophyll (Chl) (a+b) and Chl a content was observed but decrease in Chl b was more conspicuous in Varuna under Cd treatments, which was responsible for higher Chl a:b ratio. Additionally, the relative amount of anthocyanin remained higher in Varuna compared to RH-30 even in the presence of high Cd concentration, while percent pheophytin content increased in RH-30 at low Cd concentration. A higher concentration of Cd (100 mg Cd kg(-1) soil) resulted in elevated hydrogen peroxide (H(2)O(2)) content in both the cultivars. However, Varuna exhibited lower content of H(2)O(2) in comparison to RH-30. This was reflected in the increased cellular damage in RH-30, expressed by greater thiobarbituric acid reactive substances (TBARS) content and electrolyte leakage. The enhanced activities of antioxidative enzymes, ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) and also lower activity of superoxide dismutase (SOD) in Varuna alleviated Cd stress and protected the photosynthetic activity.     

Effects of different light quality on growth, chlorophyll concentration and chlorophyll biosynthesis precursors of non-heading Chinese cabbage (Brassica campestris L.)

The aim of this study was to evaluate the effects of different light quality of light emitting diode (LED) on the growth, concentration of chlorophyll and chlorophyll biosynthesis precursors of non-heading Chinese cabbage (Brassica campestris L.). Seedlings of the cultivar Te Ai Qing were cultured for 28 days under 6 treatments: red light (R), blue light (B), green light (G), yellow light (Y), red plus blue light (RB) and dysprosium lamp (CK). Lighting experiments were performed under controlled conditions (photon flux density 150 μmol m^?2 s^?1; 12 h photoperiod; 18–20 °C). The fresh and dry mass were the greatest under RB, which were significantly higher than other light treatments. The fresh mass under RB was almost twice higher compared to other light treatments. Plant height was highest under R treatment and was lowest under B. RB treatment also lowered the plant height significantly. The highest soluble sugar concentration was observed under B. The soluble protein concentration was the greatest under RB. The R treatment was adverse to pigment accumulation. The concentration of photosynthetic pigments and chlorophyll biosynthesis precursors were higher under RB. The RB treatment was beneficial to pigment accumulation.