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Abiotic stressors, such as drought and high salinity, seriously affect plant growth, productivity, and quality. Maintaining reactive oxygen species (ROS) homeostasis and osmotic balance plays a crucial role in abiotic stress tolerance. β-amylase (BAM) hydrolyzes α-1,4-glycosidic bonds by releasing maltose from starch in the regulation of soluble sugars. However, the function and mechanism of BAMs related to abiotic stress resistance remain unclear in sweetpotato (Ipomoea batatas (L.) Lam.). In this study, we isolated a novel β-amylase gene IbBAM1.1, which was strongly induced by PEG6000, NaCl, and maltose treatments in sweetpotato variety Yanshu25. Overexpression of IbBAM1.1 conferred enhanced tolerance to the drought and high salinity stressors in Arabidopsis thaliana. The activity of β-amylase and the degradation of starch were promoted under drought or salt stress. Accordingly, the contents of osmoprotectants, including maltose and proline were significantly higher in the transgenic lines than those in wild type (WT) plants. Less ROS, such as H2O2 and O2−, accumulated in the overexpressing lines than in WT plants. Superoxide dismutase activity was strongly enhanced and the level of malondialdehyde was lower under the drought or salt treatment in transgenic plants. Taken together, these results demonstrate that IbBAM1.1 acted as a positive regulator, at least in part, by regulating the level of osmoprotectants to balance the osmotic pressure and activate the scavenging system to maintain ROS homeostasis in the plants.

To study the molecular mechanism of salt stress response of peanut small GTP binding protein gene

Peanut is an important oilseed crop whose production is threatened by various abiotic and biotic stresses. Study of the molecular mechanism of salt tolerance could provide important information for the salt tolerance of this crop. WRKY transcription factors (TFs) are one of the largest TF families in plants and are involved in growth and development, defense regulation and the stress response. Here, we cloned a novel WRKY transcription factor gene belonging to the WRKY IIc subfamily, AhWRKY75, from the salt-tolerant mutant M34. The expression of AhWRKY75 was induced by NaCl stress treatment. After salt treatment, AhWRKY75-overexpressing peanuts grew better than wild-type plants. Furthermore, several genes related to the reactive oxygen species (ROS) scavenging system were up-regulated; the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were significantly higher in transgenic lines than in non-transgenic control plants; and the malondialdehyde (MDA) and superoxide anion contents were significantly lower in transgenic lines than in control plants. The net photosynthetic rate (Pn), stomatal conductance (GS) and transpiration rate (Tr) of transgenic lines were significantly higher in transgenic plants than in control plants, and the intercellular CO2 concentration (Ci) was significantly lower in transgenic plants than in control plants. These results demonstrated that the AhWRKY75 gene conferred salt tolerance in transgenic peanut lines by improving the efficiency of the ROS scavenging system and photosynthesis under stress treatment. This study identifies a novel WRKY gene for enhancing the tolerance of peanut and other plants to salt stress.

Peanut (Arachis hypogaea L.) is an important oil crop globally and high oil content is one of the major targets in peanut breeding programs. Previous studies indicated that the osmotic pressure (OP) of the leaves of peanut plants subjected to drought stress was negatively correlated with kernel oil content. Based on this knowledge, we established a practical and reliable method for creating new peanut varieties with high oil content using in vitro mutagenesis and directional OP-based selection. Using embryonic leaflets of peanut variety Huayu 20 as explants, pingyangmycin (PYM) as the mutagen, and hydroxyproline (HYP) as the OP regulator, we developed 15 HYP-tolerant regenerated plants. For each regenerated plant, we selected offspring with oil content>55% (relative to 49.5% for Huayu 20). We developed and released three new peanut varieties with high yield and high oil content from the offspring of the HYP-tolerant regenerated plants. The three new varieties were named as Yuhua 4, Yuhua 9 and Yuhua 14 and their oil contents were 57.7, 61.1 and 59.3%, respectively. The results indicate that in vitro mutagenesis with PYM followed by directed screening with HYP is a useful approach for breeding peanut varieties with high oil contents.

Auxin response factors (ARFs) are transcription factors that regulate the transcription of auxin-responsive genes during plant growth and development. In this study, 29 and 30 ARF members were identified from the two wild peanut species, A. duranensis and A. ipaensis, respectively. The ARFs, including their classifications, conserved domains and evolutionary relationships were characterized. RNA-seq analyses revealed that some of the ARF genes were responsive to abiotic stress, particularly high salinity. In addition to abiotic stress, the expression of 2 ARF members was also regulated by biotic stress, specifically Bradyrhizobium infection in A. duranensis. The ARF gene Arahy.7DXUOK was predicted to be a potential target of miR160. Overexpression of miR160 could cause degradation of the Arahy.7DXUOK target gene transcript and increased salt tolerance in miR160OX transgenic plants. Therefore, these molecular characterization and expression profile analyses provide comprehensive information on ARF family members and will help to elucidate their functions to facilitate further research on peanuts.

Peanut (Arachis hypogaea L.) is an allotetraploid oilseed crop worldwide due to its abundant high-quality oil production. Peanut oil stability and quality are determined by the relative proportions of saturated fatty acids (SFAs) and unsaturated fatty acids (UFAs). The principle approach to minimize the content of SFAs in peanut is to reduce the content of palmitic acid, which is linked to cardiovascular disease. Acyl-acyl carrier protein thioesterases (FATs) determine the types and levels of fatty acids that are exported them from the plastids. Two different classes of FAT have been classified into two families in plants, FatA and FatB. Among them, AhFatB has become the primary objective to genetically reduce the content of palmitic acid in peanut. Here, we identified 18 AhFatB genes in A. hypogaea genome and grouped into four major subfamilies through gene structures and phylogenetic relationships. Expression profiling of AhFatB genes was assessed using the publicly available RNA-seq data and qRT-PCR in 22 tissues. Using the CRISPR/Cas9 system, we designed two sgRNAs to edit the homologs AhFatB genes Arahy.4E7QKU and Arahy.L4EP3N, and identified different types of mutations. Additionally, we discovered mutations at Arahy.4E7QKU exhibited low palmitic acid and high oleic acid phenotypes. The obtained peanut mutants with altered SFAs content have great potential for improving peanut oil quality for human health.

Background: Non-specific lipid transfer proteins (nsLTPs) are known to transfer various lipid molecules between lipid bilayers in plants. In Arachis duranensis, however, little is known about the nsLTPs and their responses to biotic and abiotic stresses. Results: In this study, we identified 64 nsLTP family members (AdLTPs) in A. duranensis. These AdLTPs were classified into six subfamilies (Types 1, 2, C, D, E, and G) and were randomly distributed along nine chromosomes. The Ks value and Ka/Ks value significantly differed between Type 1 and Type D subfamilies. Among paralogs, eight AdLTPs were under positive selection, indicating that these AdLTPs might have different functions in the evolutionary history of A. duranensis. qRT-PCR results showed that the expression of AdLTPs changed in response to abiotic stresses, including salinity, PEG, low temperature, and ABA. Using RNA-seq data, we also found three AdLTPs (AdLTP1.14, AdLTPd8, and AdLTPe2) that were possibly associated with resistance to nematode infection. Among them, AdLTP1.14, which belongs to the Type 1 subfamily, was up-regulated at three time points after nematode infection. Co-expression analysis indicated that DOF and WRI1 transcription factors may regulate the AdLTP response to nematode infection. Conclusions: We identified AdLTPs in A. duranensis. Based on both RNA-seq and qRT-PCR datasets, we found that AdLTPs are involved in responses to biotic and abiotic stresses. Our results could provide valuable genomic information for the breeding of peanut cultivars that are resistant to biotic and abiotic stresses.

Small GTP-binding proteins, Rab7, are known to be responsive to abiotic stresses in plants, but the molecular mechanism is poorly understood. To investigate how AhRab7 increases resistance to salinity stress in peanut, this study compared a transgenic genotype (S5) that overexpressed the AhRab7 gene and that had high salinity resistance with a non-transgenic genotype (S7) that had low salinity resistance. Digital gene expression (DGE) sequencing was performed with leaves of S5 and S7 before and after salinity-stress treatment. In total, 2697 differentially expressed genes (DEGs) were identified between S5 and S7, and KEGG enrichment analyses showed that the DEGs are involved pathways including endocytosis, lysosome, hormone signaling, phosphatidylinositol signaling, calcium, and others. Among them, 164 were differentially regulated after salinity-stress treatment. Of 164 DEGs, 110 were responsive to salinity stress in S5 and/or S7. The 110 DEGs included genes that encode the following kinds of transcription factors and proteins known to be involved in resistance to salinity stress: WRKY, NAC, MYM-type zinc finger, late embryogenesis abundant proteins, lipid transfer protein, 1-cys peroxiredoxin, aquaporin, oleosin, and others. AhRab7 gene might mediate signaling pathways including phosphatidylinositol, calcium, abscisic acid, etc., and then regulate the expression of transcription factors and downstream genes for ROS scavenging in peanut. The results of this study will be useful for further investigations of the mechanism underlying the role of the AhRab7 gene in resistance to salinity stress in peanut.

Yuhua91 is a new peanut variety with high oleic acid content bred by Qingdao Agricultural University. The crossing was conducted with Luhua11 as female parent and with Kainong1715, an F435-type variety with high oleic acid content as male parent. The real F1 hybrids were screened by sequencing on PCR amplification products, and those homozygotes with bb genotype in F2 populations were screened by the same sequencing method as above. The content of oleic and linoleic acid was measured on the kernels harvested from F2 single plants by near infrared ray method, and those kernels whose content of oleic was above 80%, oleic and linoleic acid ratio was above 10.0 were obtained and planted into a row, with pedigree method for subsequent selection breeding. Yuhua91 has some characters of small pod, light and obvious pod texture, 148.06 g per 100 pods, 63.31 g per 100 kernels, 75.15% shelling percentage, long elliptic seed kernel, pink seed coat, without crack, white endotesta. Its content of protein, oil, oleic acid, linoleic acid and palmitic acid was 26.57%, 52.72%, 80.40%, 2.50% and 5.57% respectively. Yuhua91 has other characters of strong seedlings, compact pod areas, and moderate resistance to leaf spot disease and bacterial wilt. Average pod yield is 215.79 kg per Mu, 15.27% higher than the control variety Huayu20. Average seed kernels yield is 157.33 kg per Mu, 21.64% higher than the control variety Huayu20. Yuhua 91 has been registered on department of agriculture in 2018, and the registration No. is GPD peanut (2018) 370210, fit for growing in Shandong Province.宇花91 是青岛农业大学选育的高油酸花生新品种。以普通油酸含量品种鲁花11 号为母本，F435 型高油酸花生品种开农1715 为父本配置杂交组合。利用PCR 产物测序法筛选获得F1 代真杂种，对F2 代单株提取叶片基因组DNA，利用PCR 产物测序法筛选基因型纯合的单株个体。对当代收获的单株籽粒利用近红外法多粒模型测定油酸、亚油酸含量，筛选油酸含量在80%以上且油酸亚油酸比值在10.0 以上的单株种植成株行，随后利用系谱法进行选择育种。宇花91 荚果为普通型小果，网纹较细、较明显，百果重148.06 g，百仁重63.31 g，果皮薄，出米率75.15%。籽仁长椭圆形，种皮粉红色、无裂纹，内种皮白色。籽仁蛋白质含量26.57%，脂肪含量52.72%，油酸含量80.40%，亚油酸含量2.50%，棕榈酸含量5.57%，油酸亚油酸比值32.16。苗期生长旺盛，封垄早，结果集中，中抗叶斑病和青枯病。2017 年参加山东省夏播多点试验，平均荚果产量215.79 kg/667 m²，比对照花育20 号增产15.27%；平均籽仁产量157.33 kg/667 ² 号增产21.64%。2018 年通过国家花生品种登记，登记号：GPD 花生 (2018) 370210，适于在山东花生产区种植。.

Induced mutations have played an important role in the development of new plant varieties. In this study, we investigated the effects of fast neutron irradiation on somatic embryogenesis combined with plant regeneration in embryonic leaflet culture to develop new peanut (Arachis hypogaea L.) germplasm for breeding. The dry seeds of the elite cultivar Luhua 11 were irradiated with fast neutrons at dosages of 9.7, 14.0 and 18.0 Gy. The embryonic leaflets were separated and incubated in a medium with 10.0-mg/l 2,4-D to induce somatic embryogenesis. Next, they were incubated in a medium with 4.0-mg/l BAP for plant regeneration. As the irradiation dosage increased, the frequency of both somatic embryo formation and plantlet regeneration decreased. The regenerated plantlets were grafted onto rootstocks and were transplanted into the field. Later, the mature seeds of the regenerated plants were harvested. The M2 generation plants from most of the regenerated cultivars exhibited variations and segregation in vigor, plant height, branch and pod number, pod size, and pod shape. To determine whether the phenotypes were associated with genomic modification, we compared the DNA polymorphisms between the wild-type plants and 19 M3-generation individuals from different regenerated plants. We used 20 pairs of simple sequence repeat (SSR) primers and detected polymorphisms between most of the mutants and the wild-type plants (Luhua 11). Our results indicate that using a combination of fast neutron irradiation and tissue culture is an effective approach for creating new peanut germplasm.

The embryonic leaflets of peanut (Arachis hypogaea) variety Huayu 20 were used as explants and pingyangmycin as a mutagen to induce somatic embryos. Four weeks after the inoculation, the survived explants were transferred to somatic embryo germination medium containing screening reagent hydroxyproline, and finally 15 regenerated plants were obtained. Pedigree breeding method was used during the following selection breeding, and three lines with significantly increased yield and 23 lines with high oil content were obtained from these mutant offsprings. The line with both high yield and high oil content has passed peanut variety multi-location in Anhui province and was named "Yuhua 4". Its yield was 16.63% higher than that of the control variety Baisha 1016, ranking the first in all the testing varieties. Yuhua 4 showed the characteristics of early maturity, small pod and high oil content. The oil content of kernels was 56.10%, higher than that of original parent Huayu 20 with 49.50% oil content, tested by the Ministry of Agriculture of Oil and Products Quality Supervision, Inspection and Test Center (Wuhan), and the yield was 15% higher than that of Huayu 20. It was concluded that in vitro mutagenesis and target screening was an effective way on creating new germplasm and breeding new variety in peanut.为了丰富花生遗传资源，开拓新的育种方法，本研究对离体诱变创造花生新种质、培育花生新品种进行了研究。利用花生品种花育20 号胚小叶作为外植体，平阳霉素 (PYM) 作为诱变剂进行离体诱变培养，然后在含有羟脯氨酸 (HYP) 的培养基上进行定向筛选，最终获得了15 个再生小苗。再生小苗经嫁接移栽田间，从后代中获得了23 个高油株系，3 个产量显著提高的品系，其中一个高产高油品系2015 年通过了安徽省新品种登记鉴定，定名为宇花4 号，在参试的品种中名列第一，比对照白沙1016 增产16.63%。宇花4 号为早熟、小粒、高油花生品种，经农业部油料及制品质量监督检验测试中心 (武汉) 化验，籽仁含油率达56.10%，达到高油标准，比诱变亲本花育20 号 (含油率49.50%) 高6.6 个百分点，荚果产量比花育20 号增产15%以上。本研究结果表明，离体诱变结合离体定向筛选是创造花生新种质、培育新品种的有效途径。.

To expand the salt-tolerant gene resources of peanut, we conducted in vitro mutagenesis with pingyangmycin (PYM) as the mutagen and directed screening with medium containing NaCl. After embryonic leaflets from mature peanut seeds (variety Huayu 22) were cultured on somatic embryogenesis-induction medium containing 4 mg/L PYM for 4 weeks, the surviving somatic embryos were sequentially transferred to a germination medium containing 15 and then 20 g/L NaCl. The 30 NaCl-tolerant plantlets obtained were grafted and transplanted in the field in 2011, and the mature seeds of 26 regenerated plants were harvested. In 2012, all seeds from each plant were sown in the field. The offspring (M2 generation) of 23 of 26 NaCl-tolerant, regenerated plants differed from their mutagenic parent in vigor, growth habit, flowering habit, pod shape, and seed coat color, and they also exhibited trait segregation from the same NaCl-tolerant, regenerated plant. In a germination test with a 0.7 % NaCl solution and the M3-generation seeds from 18 of the NaCl-tolerant, regenerated plants, the germination rate was substantially higher for the seeds from 6 plants than for seeds from the mutagenic parent (Huayu 22). To determine whether the changes in plant traits might be associated with gene mutations, DNA polymorphisms between the mutagenic parent and 19 M3 generation individuals from different NaCl-tolerant, regenerated plants were analyzed with 39 pairs of SSR primers, and all mutants differed from the mutagenic parent in >2 loci. The results indicate that the use of PYM-based mutagenesis in combination with directed in vitro screening with NaCl is effective for creating and identifying salt-tolerant mutants of peanut.

Soil salinity seriously limits plant growth and yield. Strategies have been developed for plants to cope with various environmental stresses during evolution. To screen for the broad-spectrum genes and the molecular mechanism about a hydroxyproline-tolerant mutant of peanut with enhanced salinity resistance under salinity stress, digital gene expression (DGE) sequencing was performed in the leaves of salinity-resistant mutant (S2) and Huayu20 as control (S4) under salt stress. The results indicate that major transcription factor families linked to salinity stress responses (NAC, bHLH, WRKY, AP2/ERF) are differentially expressed in the leaves of peanut under salinity stress. In addition, genes related to cell wall loosening and stiffening (xyloglucan endotransglucosylase/hydrolases, peroxidases, lipid transfer protein, expansin, extension), late embryogenesis abundant protein family, fatty acid biosynthesis and metabolism (13-lipoxygenase omega-6 fatty acid desaturase, omega-3 fatty acid desaturase) and some previously reported stress-related genes encoding proteins such as defensin, universal stress protein, metallothionein, peroxidase etc, and some other known or unknown function stress related genes, have been identified. The information from this study will be useful for further research on the mechanism of salinity resistance and will provide a useful genomic resource for the breeding of salinity resistance variety in peanut.

A high quality cDNA library was constructed from the brown alga Laminaria japonica, with the titer of 1.2×105 pfu/ml. The average insert size of the cDNA library is about 1.6 kb. From the cDNA library, 591 cDNA clones were randomly selected and sequenced. As a result, 574 EST (expressed sequence tag) sequences were generated. All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198. The cDNA library was screened with a α-32p labeled 453 bp TPS gene probe, which is a partial sequence yielded from Porphyra yezoensis. Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L. japonica TPS cDNA sequence. After PCR amplification, sequencing and assembling, the entire ORF (open reading frame) sequence of the TPS gene was obtained, which was named LjTPS. LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons. The LjTPS gene was successfully expressed in E. coli and rice. The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the TPS gene function and mechanism to stress tolerance.

Rab7 is a member of small GTP-binding protein family involved in intracellular vesicle trafficking from late endosome to vacuole, some of them are found to be responsive by different environmental stresses. Here we report the isolation of a Rab7 (RabG3b) cDNA from peanut (Arachis hypogaea). Overexpression of the open reading frame (ORF) of Rab7 cDNA encoding 205 amino acids in E. coli conferred the cells tolerance to various abiotic stresses. Transcription of AhRabG3b was differentially up-regulated by different environmental stimuli such as cold, dehydration, NaCl and the plant hormone ABA. Overexpression of the AhRabG3b exhibited enhanced tolerance to drought and salinity stresses in transgenic peanut. These results suggested that the AhRabG3b be a potential candidate gene responsible for both salinity and dehydration tolerance in cultivated peanut.

A semidwarf line of Indica rice, Xinguiai, was derived from the progeny of a cross between the double dwarf mutant Xinguiaishuangai and the wild-type variety Nanjing 6. The semidwarf phenotype was controlled by the semidwarf gene, sdg. The second sheath and shoot elongation responses of the dwarf mutant to exogenous gibberellin (GA3) showed that sdg was insensitive to gibberellin (GA), and its endogenous GAs content was higher than that in wild-type cultivars. The SDG gene was cloned by a map-based cloning method and sequencing analysis revealed that the coding region of sdg had a single nucleotide substitution resulting in a single amino acid change from alanine to threonine. A cleaved amplified polymorphic sequence marker was designed according to sequences from mutant and wild-type materials. This sequence marker could be used to distinguish wild types and mutants, and thus, could be used for molecular marker-assisted selection. The dwarf phenotype of the sdg mutant was restored to a normal phenotype by introducing the wild-type SDG gene. Rice transformation experiments and GUS staining demonstrated that the SDG gene was predominantly expressed in vegetative organs.

Restriction site amplified polymorphism (RSAP) is a molecular marker technique which just requires a simple polymerase chain reaction to amplify fragments around restriction sites. The RSAP analytic system was set up and applied to Porphyra genetic variation analysis in this study for the first time. Fourteen Porphyra lines were screened by the RSAP analytic system with 30 primer combinations, 12 of which produced stable and reproducible amplification patterns in three repeated experiments. The 12 primer combinations produced 408 amplified fragments, 402 of which (98.53%) were polymorphic, with an average of 33.5 polymorphic fragments for each primer combination, ranging in size from 50 to 500 bp. The 408 fragments were scored one by one and then used to develop a dendrogram of the 14 Porphyra lines with unweighted pair-group method arithmetic average. The genetic distance among these Porphyra lines ranged from 0.10 to 0.50. These Porphyra lines were divided into two major groups at the 0.71 similarity level: one group contained only Porphyra haitanensis lines and the other group contained Porphyra yezoensis lines. In addition, some specific RSAP markers were acquired from each Porphyra line apart from P. yezoensis Yqd-2-1, and five of them were sequenced. One of the specific markers, R1/R3-8119 from P. yezoensis Y-9101, was successfully converted into sequence characterized amplification region marker. The result suggested that TRAP was a simple, stable, polymorphic, and reproducible molecular marker technique for the classification and resource protection of Porphyra lines.

In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM) as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP). After being extracted from mature seeds (cv. Huayu 20), the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation). The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS) and nine individuals with higher protein content (>30%) and 21 individuals with higher oil content (>58%) were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut.

This paper puts forward an improved ampere-hour integral method to estimate SOC for Lithium-ion Battery Pack in Electric Vehicle. According to the low estimation accuracy of traditional ampere-hour integral method, based on a large number of experiments, this paper analysed the parameters which affect the estimation accuracy of integral method, then used the least square method to establish the mathematical model and realized the correction factor of the parameters. The experiments show that this method can improve the accuracy of the battery SOC, and meet the application requirements for the safe operation of the electric vehicle.