Association Analysis and Identification of SNP Markers for Stemphylium Leaf Spot (Stemphylium botryosum f. sp. spinacia) Resistance in Spinach (Spinacia oleracea)

Stemphylium leaf spot, caused by Stemphylium botryosum f. sp. spinacia, is an important fungal disease of spinach (Spinacia oleracea L.). The aim of this study was to conduct association analysis to identify single nucleotide polymorphism (SNP) markers associated with Stemphylium leaf spot resistance in spinach. A total of 273 spinach genotypes, including 265 accessions from the USDA spinach germplasm collection and eight commercial cultivars, were used in this study. Phenotyping for Stemphylium leaf spot resistance was evaluated in greenhouse;genotyping was conducted using genotyping by sequencing (GBS) with 787 SNPs;and single marker regression, general linear model, and mixed linear model were used for association analysis of Stemphylium leaf spot. Spinach genotypes showed a skewed distribution for Stemphylium leaf spot resistance, with a range from 0.2% to 23.5% disease severity, suggesting that Stemphylium leaf spot resistance in spinach is a complex, quantitative trait. Association analysis indicated that eight SNP markers, AYZV02052595_115, AYZV02052595_122, AYZV02057770_10404, AYZV02129827_205, AYZV0-2152692_182, AYZV02180153_337, AYZV02225889_197, and AYZV02258563_213 were strongly associated with Stemphylium leaf spot resistance, with a Log of the Odds (LOD) of 2.5 or above. The SNP markers may provide a tool to select for Stemphylium leaf spot resistance in spinach breeding programs through marker-assisted selection (MAS).

Identification of a male-specific region(MSR)in Spinacia oleracea

Dioecy,the presence of male and female individual,has evolved independently from hermaphroditism in multiple flowering plant lineages.Spinach,an important leafy vegetable crop worldwide,is a dioecious species with an XY sex-determination system.Although some markers that are fully linked to male-determination locus were identified,the male-specific region(MSR)remained unclear.In this research,five male individuals and five female lines were resequenced to identify the male-specific region.We identified a region(∼21 kb)on chromosome 4(putative sex chromosome)where the five females had a low reads coverage,while the five males had high coverage.A KASP marker,SponR,developed from a single nucleotide polymorphism(SNP)closely linked to the MSR was co-segregating with the sex determination gene in the population of 958 individuals,suggesting that the MSR might be specifically present in male spinach plants.

Physiological Response of Halophyte (<i>Suaeda altissima</i>(L.) Pall.) and Glycophyte (<i>Spinacia oleracea</i>L.) to Salinity

We have done a comparative study of ion status, growth and biochemical parameters in shoots and roots of seablite (Suaeda altissima (L.) Pall.) and spinach (Spinacia oleracea L.) grown with different salinity levels in the medium (0.5 - 750 mМ). A distinctive feature of the halophyte was a high Na+ content in tissues at its low concentration in the medium (0.5 mM). In these conditions, Na+ accumulation in seablite roots was four-fold higher than in spinach roots, and Na+ content in seablite leaves was almost 20-fold higher than in spinach. Together with an increase in sodium concentration in the medium, K+ content decreased six-fold in seablite leaves, while in spinach it did not decrease so drastically. We can suppose that in the halophyte, some processes occur only in the presence of sodium, and these functions of sodium cannot be fully fulfilled by potassium. Analysis of protein and total nitrogen content in tissues shows that at high salinity, the ability to synthesize non-protein nitrogen-containing compounds increases in the halophyte and decreases in the glycophyte. Data on proline content dynamics show that its increase in tissues of spinach (salinity levels 150 and 250 mМ) and seablite (salinity levels 0.5 and 750 mМ) is an indicator of plant injury. In seablite and spinach, proline is not a major osmoregulator. Its concentration both in roots and leaves was no more than 2.5 μmol/g fresh weight. The data presented in this work concern the accumulation and distribution of Na+, Cl?, K+ and ions, as well as growth and biochemical parameters. Our data show that the development of adaptation reactions in the whole plants in the conditions of high salinity is determined by morphofunctional systems and their interaction.

Optimization of micropropagation and <i>Agrobacterium</i>-mediated gene transformation to spinach (<i>Spinacia oleracea</i>L.)

Spinach is one of the dioecious plant which is considered as a model plant in genetic and molecular studies of sex determination because of its special characteristics such as low chromosome number and short life cycle. An efficient protocol for Spinacia oleracea Agrobacterium-mediated gene transformation was developed. The leaf disks, roots, hypocotyls and cotyledons of this plant were inoculated with LBA4404. LBA4404 carrying pCAMBIA3301 binary vector with 35SCaMV gusint and 35SCaMV bar cassettes. Effects of two preparation condition (induction of vir genes and noninduction) were considered. Also effects of different number days of co-cultivation and pre-culture of explants were examined. After co-cultivation, the explants were transferred to regeneration medium containing 250 mg·L-1 Carbeniciline. Transient expression efficiency was calculated based on the number of blue spots per explants one week after inoculation. Based on the results of transient expression, stable transformation was carried out. After formation of callus the histochemical GUS assay was carried out on some parts of them and other parts were leaved for being regenerated.

Isolation and Expression Profiling of a CONSTANS-Like Gene and Two FLOWERING LOCUS T-Like Genes from Spinacia oleracea L.

Spinach (Spinacia oleracea L.) develops leaf rosettes under short-day conditions, and starts reproductive growth including bolting and flowering under long-day conditions. Japanese people prefer Oriental spinach that bolts easily with a shorter photoperiod than European spinach. This is one of the main reasons that Oriental spinach is difficult to grow year-round. In order to understand spinach flowering mechanisms and obtain knowledge for spinach breeding, we isolated one CONSTANS-like (COL) and two FLOWERING LOCUS T (FT) homologs, which are key components of photoperiodic regulation of flowering time, from a Japanese cultivar. The expression of SoCOL1 showed diurnal rhythm with the highest expression at the end of the dark cycle. This diurnal rhythm is similar to the expression of BvCOL1 from sugar beet (Beta vulgaris), whose flower-promoting effect was observed when overexpressed in Arabidopsis. Phylogenetic analysis showed that SoCOL1 is the closest homolog of BvCOL1, suggesting that SoCOL1 is an ortholog of BvCOL1. SoFT1 and SoFT2 are closely related to BvFT1 and BvFT2, respectively. The expression of SoFT1 and SoFT2 were induced in advance of flower bud formation after changing the photoperiod, but the expression level of SoFT1 was much lower than SoFT2. Currently, we are speculating that SoFT2 is a flower-promoting factor of spinach, and that SoFT1 has a role in light signaling because the expression of SoFT1 showed a diurnal rhythm.

An in vitro Anti-urolithiasis Activity of a Herbal Formulation:Spinacia oleracea L.and Coriandrum sativum L.

Background:Hard mineral and acid salt deposits that clump together in concentrated urine are known as kidney stones.A complicated chain of physicochemical events,including hyper saturation,nucleation,growth,aggrega-tion,and retention in the urinary tract,leads to urolithiasis,the third most prevalent urinary tract issue.Numerous traditional medications and therapies are less successful in treating urolithiasis and frequently cause recurrences.Objective:The present study is focused on evaluating the anti-urolithiasis activities of the polyherbal formulation prepared from Spinacia oleracea L.and Coriandrum sativum L.seeds.Methods:To evaluating the anti-urolithiasis activities of the polyherbal formulation prepared from S.oleracea and C.sativum seeds against oxalate crystals by employing an in vitro inhibition,aggregation,and nucleation assay,employing a spectrophotometer followed by microscopic observation,and further monitored by egg semiperme-able membrane model,using cystone as a standard drug.Results:In the present work,the alkaloids,glycosides,flavonoids,glycosides,phenols,saponins,steroids,tannins,terpenoids,and triterpenoids were found in the extracts of S.oleracea and C.sativum.The in vitro assays in the present study clearly indicated that all the mixture extracts readily prevented crystal nucleation,growth and ag-gregation.The aqueous extract of the mixture inhibited significant inhibitory potential against the crystallization process.Conclusion:It can be said that the phyto-constituents enriched homemade preparation possibly will be used as a medication for the management of kidney stones by dissolving the oxalate stone in the kidney.

Understanding mechanisms for differential salinity tissue tolerance between quinoa and spinach:Zooming on ROS-inducible ion channels

Soil salinity is a wo rldwide issue and a major threat to global food security.Salinity tolerance is a complex mechanism that is conferred by numerous molecular,physiological,and biochemical traits.Of critical importance are plant's ability to regulate redox balance without compromising reactive oxygen species(ROS)signalling and maintain cytosolic ion homeostasis.In this study,the mechanistic basis of H^(+) retention ability in leaf mesophyll(an important but highly sensitive plant tissue)was compared between halophytic quinoa and glycophytic spinach.Phenotypic data showed quinoa outperformed spinach under 100 to 500 mmol L^(-1) NaCl salinity.The major difference behind this differential salinity sensitivity was a differential H^(+) uptake in leaf mesophyll.Electrophysiological and molecular experiments revealed that a superior ability of mesophyll H^(+) retention in quinoa was conferred by three complementary mechanisms:(ⅰ)an intrinsically lower H^(+)-ATPase activity in quinoa(potentially as an energy saving strategy);(ⅱ)reduced sensitivity of H^(+) transporters to ROS;and(ⅲ)increased sensitivity of ROS-inducible Ca^(2+)-permeable channels,Moreover,the sensitivity of H^(+)-transport systems to ROS was further examined in NaCl-acclimated quinoa and spinach plants.The key factors differentiating between H^(+) retention in acclimated leaf mesophyll was associated with the reduced sensitivity and gene expression of H^(+)-permeable outward rectifying channel(GORK),Arabidopsis potassium transporter 1(AKT1),and high affinity potassium transporter 5(HAK5)to additional NaCl and ROS stress,along with the upregulation of ROS scavenging system.Taken together,our results showed that the tissue-specific and ROS-specific regulation of H^(+) retention are important for conferring salinity tolerant at least in the halophyte quinoa.

菠菜HD-ZIP Ⅲ基因家族鉴定与表达分析

植物叶片在生长发育过程中,近-远轴极性的建立与维持是叶片正常行使光合效应、呼吸作用、蒸腾作用的结构基础.HD-ZIPⅢ是重要的叶片近轴极性基因,本研究在菠菜(Spinacia oleracea)基因组中鉴定到3个HD-ZIPⅢ基因家族成员,依次命名为SoHB1,SoHB2和SoHB3,并对其蛋白理化性质、保守结构域和进化关系进行了分析.研究结果表明:3个HD-ZIPⅢ蛋白都具有4个保守结构域.共线性分析表明:菠菜SoHB1,SoHB2和SoHB3分别与拟南芥ATHB8,ATREV和ATHB15存在共线性关系.启动子顺式作用元件预测结果表明:菠菜HD-ZIPⅢ家族基因启动子上游包含光响应、逆境胁迫反应、激素响应等顺式作用元件.在不同叶型菠菜种质中的表达模式分析结果表明:单戟型叶片的SoHBs表达量最高;此外,经干旱胁迫处理后,SoHB1,SoHB2和SoHB3的基因表达量普遍下降;对菠菜包括根、茎、薹叶、功能叶、薹叶柄、功能叶柄等部位进行表达分析,发现SoHB1在根部表达最为丰富,SoHB2和SoHB3在薹叶处表达最为丰富.

崇明小菠菜轮作模式的效益及品质分析

为筛选适合崇明小菠菜(Spinacia oleracea)的特色蔬菜轮作模式,以叶菜为对照,比较了崇明小菠菜与香酥芋、红皮土豆、崇明金瓜、崇明白扁豆、崇明香芋和崇明山药为主要轮作作物的6种轮作模式的经济效益,并分析了轮作对崇明小菠菜产量和品质的影响.结果表明:崇明金瓜+鲜食玉米+崇明小菠菜(T2)、崇明白扁豆+鲜食玉米+崇明小菠菜(T3)轮作模式的经济效益在轮作2年周期内均最高,增幅均在对照的20%以上.崇明小菠菜产量增幅在轮作第2年较高,产量最高的轮作模式是崇明红皮土豆+鲜食玉米+崇明小菠菜(T6).各轮作模式下,崇明小菠菜的维生素C、可溶性蛋白质及草酸含量均无显著差异;崇明香芋+崇明小菠菜(T1)轮作模式的硝态氮含量显著高于其他模式;可溶性糖含量在T6轮作模式下含量最高.综合考量经济效益、产量和品质,T2,T3和T6模式可优先应用于崇明小菠菜高效轮作生产.

负离子处理对菠菜贮藏品质的影响研究

目的研究负离子处理时长对菠菜(Spinacia oleracea L.)贮藏品质的影响。方法本实验利用负离子发生器使之在密闭空间中产生负离子,用于菠菜贮藏前的预处理,通过测定贮藏期间菠菜的菌落总数、呼吸速率、质量损失率、色差(ΔE)、丙二醛、总酚含量等品质指标,研究了4种处理时长(0、30、60和90 min)对菠菜贮藏品质的影响。结果负离子处理能抑制菠菜表面微生物的生长,降低微生物对机体造成的侵害,延缓其氧化衰老,同时降低了菠菜呼吸速率,减少营养物质的消耗和叶绿素的分解,并维持较低的质量损失,随着负离子处理时长的增加,对菠菜贮藏过程中的品质保护效果越明显,负离子预处理90 min对菠菜的贮藏品质维护效果最好。可直观体现在菠菜的外观的变化以及色泽的维持。结论负离子合适的处理时长可以有效延长菠菜的货架期,为负离子预处理应用于果蔬保鲜提供了重要依据。

菠菜农药残留风险评估探究

为给农药的合理使用和最大残留限量标准的制定提供依据,随机采集多份菠菜样品,对其中的68种农药残留进行检测并得出详细实检测数据。在此基础上,运用慢性、急性膳食摄入风险评估模型,对农药残留的风险进行了全面分析;运用风险矩阵对LD50值、ADI值、该农药残留的食品占总膳食的比例、农药使用频率、是否存在高暴露人群、检测中值分别赋分的方式进行风险排序。结果显示,急性和慢性膳食摄入风险均在可接受范围内。建议对菠菜种植中对农药使用加强监控和指导,加快农药对高危人员多残留的研究,作好菠菜中农药合理使用的指导。

Characterization of Energy_Transduction in Thermal Pretreated Chloroplast from Spinach

Characterization of energy-transduction on die chloroplast thylakoid membranes from spinach (Spinacia oleracca L.) after thermal pretreatment was investigated. The related reactions of energy-transduction in chloroplasts were seriously affected by thermal pretreatment. The results were obtained as following: (1) The rate of cyclic photophosphorylation declined when the pretreatment temperature increased in the range of 25 to 45 degreesC. (2) The thermal pretreatment led to a decrease of the activity of thylakoid membrane-bounded ATPase. (3) Proton uptake of chloroplasts acid the fluorescence quenching of 9-aminoacridine (9-AA) in thylakoid membrane decreased after the thermal pretreatment, but addition of dicyclohexylcarbodiimide (DCCD) could partially restore the fluorescence quenching of 9-AA. (4) Both the rates of fast phase in electrochroism absorption change at 515 nm and the millisecond delayed light emission (ms-DLE) of chloroplast showed a progressive decrease upon raising the temperature of pretreatment. (5) Immunbloting analysis showed that the thermal pretreatment caused the changes of protein content and the electrophoresis mobility of thylakoid membrane-bound ATPase and its alpha -subunit. (6) If the temperature of pretreatment were higher than 33 degreesC, oxygen uptake of PS I -mediated in the samples was rapidly inhibited, but addition of sinapine into the reaction medium could partially restore the ability of oxygen uptake in the samples. These results are briefly discussed in relation to the change of permeability of thylakoid membranes, the dissociation of coupling factor complex as well as accumulation of the radicals in the thylakoid membranes after thermal pretreatment.

NaCl胁迫对菠菜萌发和苗期生理特性的影响

以3个菠菜(Spinaciaoleracea L.)自交系材料S26,S77和S4为研究对象,研究了不同浓度的NaCl溶液对菠菜萌发期和幼苗期生理特性的影响.结果表明:供试浓度范围内,低浓度(50 mmol∙L^(-1))NaCl处理可以促进菠菜种子的萌发,但高浓度处理则抑制萌发;与S4相比,S77和S26在NaCl胁迫下具有较高的相对发芽率、相对发芽势和相对萌发指数,且幼苗根长、叶片长、根重和地上部分鲜重受NaCl抑制程度较小.苗期内,在NaCl胁迫下,S77,S26的株高、叶宽、根重、地上部分鲜重高于S4,且S77和S26的可溶性糖含量、脯氨酸含量,及超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性要高于S4,而S4的电解质渗透率(EC)和丙二醛(MDA)含量高于S77和S26.综上,供试材料S77,S26在萌发期和苗期均表现出较高的耐盐性,S4为NaCl敏感材料;菠菜萌发期与苗期的NaCl耐性评价结果一致,但NaCl筛选浓度差异较大,低浓度(50 mmol∙L^(-1))NaCl处理能促进菠菜种子萌发,但对幼根尤其是子叶生长的NaCl胁迫无缓解作用.

菠菜转录因子MYB基因家族的鉴定及表达分析

利用生物信息学手段鉴定了76个具有典型R结构的菠菜转录因子(Spinacia oleracea)MYB,其中包括72条R2R3-MYB基因(2R-MYB)和4条R1R2R3-MYB基因(3R-MYB)。通过生物信息学对菠菜MYB转录因子家族成员的理化性质、染色体定位、结构域序列保守性和系统进化关系进行了分析,结果表明:菠菜MYB家族有32个基因位于染色体正链,另外44个基因位于染色体反链;MYB的DNA结合域中的保守域主要位于两个R重复序列的第二和第三螺旋之间,结合域中每个R重复的第一和第二色氨酸之间的氨基酸序列相对不保守;根据菠菜、拟南芥及甜菜的MYB家族系统进化关系可以推测,菠菜MYB家族中56个成员可以按功能划分为4类,在菠菜的生长发育过程中可能起着重要的调节作用,其余成员中有7个MYB基因可能参与菠菜响应氮素浓度的氮素利用及生长发育进程。

菠菜叶酸合成代谢途径基因鉴定及表达谱分析

从菠菜(Spinacia oleracea L.)基因组数据库中筛选鉴定了与菠菜叶酸合成转运,及与C1代谢相关的25个基因,并对其编码的蛋白做进化树和保守域分析,发现叶酸蛋白在进化上表现出保守型和复杂性。用实时荧光定量聚合酶链式反应(qRT-PCR)分析了叶酸含量不同的菠菜的叶酸相关基因表达量,发现在转录水平上只有少数叶酸基因表达量与菠菜叶酸含量有关。

菠菜SpNHX1基因的克隆及生物信息学分析

从菠菜(Spinacia oleracea L.)栽培种中克隆得到1个NHX1基因,命名为SpNHX1。利用生物信息学软件对获得的基因核苷酸序列及编码的蛋白质序列进行分析,结果发现SpNHX1属于Vac亚家族,序列长度为6090 bp,开放阅读框为1662 bp,编码553个氨基酸残基。利用相关软件或在线工具对SpNHX1进行生物信息学分析,结果表明其编码蛋白分子式为C2818H4363N697O772S25,属于稳定的疏水性蛋白;进化树分析表明SpNHX1蛋白与拟南芥AtNHX1、AtNHX2亲缘关系较近,属于定位在液泡膜上的蛋白,SpNHX1蛋白与双子叶植物的NHX蛋白亲缘关系较近。

高温胁迫对菠菜叶片保护酶活性和膜透性的影响

对两个耐热性不同的菠菜 (SpinaciaoleraceaL .)品种进行了高温 (昼 35℃ /夜 2 5℃ )处理 ,以2 0℃ / 15℃为对照 ,测定了高温胁迫对菠菜叶片中保护性酶类活性和膜透性的影响。结果表明 ,高温处理后 ,耐热品种叶片中超氧化物歧化酶 (SOD)、过氧化氢酶 (CAT)和过氧化物酶 (POD)活性的增幅大于不耐热品种 ,而不耐热品种叶片中丙二醛 (MDA)

菠菜甜菜碱醛脱氢酶基因在棉花中的过量表达和抗冻耐逆性分析

分离出菠菜甜菜碱醛脱氢酶基因(SoBADH)构建成由CaMV35S驱动的双元植物表达载体pBSB,农杆菌菌株LBA4404携带该载体转化棉花,获得转基因棉花植株。65株转基因植株经过PCR筛选、Southern blotting分析证明有45株为成功的转化株,外源基因已经被整合到棉花的染色体组中,并以单拷贝插入居多。对部分株系的SoBADH基因的表达进行分析表明均有较高的mRNA和蛋白的表达。经测定这些株系中的甜菜碱脱氢酶活性显著提高,达到0.66～1.70nmol/min/mg水平。同时这些转基因株系在盐胁迫下比对照长势强,株高和地上部分的鲜重显著高于非转基因对照;在低温胁迫下,这些转基因株系表现出显著的抗冻性能。结果表明菠菜甜菜碱醛脱氢酶能够在异源植物棉花中过量表达,并具有较高的酶活性,转基因棉花可作为抗逆育种的种质材料。

供氮水平对菠菜营养品质和体内抗氧化酶活性的影响

通过水培实验 ,研究了供氮水平对菠菜营养品质和抗氧化酶活性的影响 .结果表明 ,供氮水平由4mmol·L- 1增加到 8mmol·L- 1,菠菜产量显著增加 ,叶片中的维生素C(Vc)含量随着供氮浓度由 4mmol·L- 1提高到 8mmol·L- 1,再提高供氮水平 ,Vc含量则明显下降 .叶片硝酸盐含量随着氮浓度的提高而增加 .供氮浓度从 4mmol·L- 1增加到 8mmol·L- 1,叶片可溶态草酸含量略有下降 ,再提高供氮水平则明显上升 ,而草酸总量随供氮水平提高 ,先显著升高然后略有降低 .SOD和POD酶的活性随供氮水平由 4mmol·L- 1提高到 8mmol·L- 1而增加 ,再提高供氮水平 ,酶活性显著下降 ;CAT活性随供氮水平的增加而降低 ,叶片MDA含量先降低后显著升高 ,而游离脯氨酸含量随氮水平的升高而增加 .可见供氮水平为 8mmol·L- 1时 ,菠菜叶片具有较高的生物量、Vc含量和抗氧化酶活性 ,较低的硝酸盐和草酸含量以及较低的MDA和游离脯氨酸含量 ,表明供氮浓度 8mmol·L- 1有利于提高菠菜的产量、营养品质和抗逆能力 ,是菠菜生长较适宜的供氮水平 .

海水胁迫对菠菜叶绿素代谢的影响

以耐海水品种‘荷兰3号’和海水敏感品种‘圆叶菠菜’为试验材料,采用营养液栽培,研究海水胁迫对菠菜叶绿素代谢的影响。结果表明,海水胁迫下,2个菠菜品种叶片的叶绿素a(Chl a)、叶绿素b(Chl b)和总叶绿素含量以及叶绿素合成前体———原叶绿素酸(Pchl)、镁原卟啉IX(Mg-proto IX)、原卟啉IX(Proto IX)和尿卟啉原III(UroIII)含量均明显降低,而胆色素原(PBG)和δ-氨基酮戊酸(ALA)积累,‘圆叶菠菜’的变化幅度大于‘荷兰3号’。海水胁迫下,‘荷兰3号’叶片的叶绿素酶(Chlase)活性无显著变化,胆色素原脱氨酶(PBGD)和尿卟啉原III合酶(UROS)活性在胁迫第3天显著下降,而‘圆叶菠菜’Chlase活性显著上升,PBGD和UROS活性显著下降。研究发现,在海水胁迫条件下,菠菜叶片的叶绿素合成代谢受阻,受阻位点位于PBG→UroIII的转化过程,其中‘圆叶菠菜’的受阻程度大于‘荷兰3号’;耐海水品种‘荷兰3号’叶片叶绿素含量降低主要由叶绿素合成代谢受阻引起,而海水敏感品种‘圆叶菠菜’叶绿素含量的降低则是由叶绿素合成受阻和叶绿素降解共同作用的结果。