Genetic Analysis and Preliminary Mapping of a Highly Male-Sterile Gene in Foxtail Millet(Setaria italica L.Beauv.) Using SSR Markers

Breeding of male-sterile lines has become the mainstream for the heterosis utilization in foxtail millet,but the genetic basis of most male-sterile lines used for the hybrid is still an area to be elucidated.In this study,a highly male-sterile line Gao146A was investigated.Genetic analysis indicated that the highly male-sterile phenotype was controlled by a single recessive gene a single recessive gene.Using F 2 population derived from cross Gao146A/K103,one gene controlling the highly male- sterility,tentatively named as ms1,which linked to SSR marker b234 with genetic distance of 16.7 cM,was mapped on the chromosome VI.These results not only laid the foundation for fine mapping of this highly male-sterile gene,but also helped to accelerate the improvement of highly male-sterile lines by using molecular marker assisted breeding method.

Genetic Diversity and Classification of Chinese Elite Foxtail Millet [Setaria italica (L.) P. Beauv.] Revealed by Acid-PAGE Prolamin

Arid and semi-arid regions of China account for more than half of the country. Because of drought resistance and high nutritive value, elite foxtail millet (Setaria Italica (L.) P. Beauv.) is one of the most important cereal crops in China. Evaluation of germplasm and genetic diversity of foxtail millet is still in its infancy, but prolamin could play an important role as a protein marker. To investigate the genetic diversity and population structure of foxtail millet from different ecological zones of China, 90 accessions of foxtail millet were collected from three major ecological areas: North, Northwest, and Northeast China. The prolamin contents were examined by acid polyacrylamide gel electrophoresis (acid-PAGE). Five to twenty-two prolamin bands appeared in tested varieties, of which were polymorphic, so prolamin patterns of foxtail millet varieties can be used in variety identification and evaluation. Structure analysis identified six groups, which matches their pedigree information but not their geographic origins. This indicated a high degree (87.78%) of consistency with a phylogenetic classification based on SSR. The results showed prolamin banding patterns were an effective method for analyzing foxtail millet genetic variability.

The NAC-like transcription factor Si NAC110 in foxtail millet(Setaria italica L.) confers tolerance to drought and high salt stress through an ABA independent signaling pathway

Foxtail millet（Setaria italica（L.）P.Beauv）is a naturally stress tolerant crop.Compared to other gramineous crops,it has relatively stronger drought and lower nutrition stress tolerance traits.To date,the scope of functional genomics research in foxtail millet（S.italic L.）has been quite limited.NAC（NAM,ATAF1/2 and CUC2）-like transcription factors are known to be involved in various biological processes,including abiotic stress responses.In our previous foxtail millet（S.italic L.）RNA seq analysis,we found that the expression of a NAC-like transcription factor,SiNAC110,could be induced by drought stress;additionally,other references have reported that SiNAC110 expression could be induced by abiotic stress.So,we here selected SiNAC110 for further characterization and functional analysis.First,the predicted SiNAC110 protein encoded indicated SiNAC110 has a conserved NAM（no apical meristem）domain between the 11–139 amino acid positions.Phylogenetic analysis then indicated that SiNAC110 belongs to subfamily III of the NAC gene family.Subcellular localization analysis revealed that the SiNAC110-GFP fusion protein was localized to the nucleus in Arabidopsis protoplasts.Gene expression profiling analysis indicated that expression of SiNAC110 was induced by dehydration,high salinity and other abiotic stresses.Gene functional analysis using SiNAC110 overexpressed Arabidopsis plants indicated that,under drought and high salt stress conditions,the seed germination rate,root length,root surface area,fresh weight,and dry weight of the SiNAC110 overexpressed lines were significantly higher than the wild type（WT）,suggesting that the SiNAC110 overexpressed lines had enhanced tolerance to drought and high salt stresses.However,overexpression of SiN AC110 did not affect the sensitivity of SiNAC110 overexpressed lines to abscisic acid（ABA）treatment.Expression analysis of genes involved in proline synthesis,Na＋/K＋transport,drought responses,and aqueous transport proteins were higher in the SiNAC110overexpressed lines than in the WT,whereas expression of ABA-dependent pathway genes did not change.These results indicated that overexpression of SiNAC110 conferred tolerance to drought and high salt stresses,likely through influencing the regulation of proline biosynthesis,ion homeostasis and osmotic balance.Therefore,SiNAC110 appears to function in the ABA-independent abiotic stress response pathway in plants.

Identification of no pollen 1 provides a candidate gene for heterosis utilization in foxtail millet(Setaria italica L.)

Male sterility is a common biological phenomenon in plant kingdom and has been used to generate male-sterile lines, which are important genetic resources for commercial hybrid seed production. Although increasing numbers of male-sterility genes have been identified in rice(Oryza sativa) and Arabidopsis(Arabidopsis thaliana), few male-sterility-related genes have been characterized in foxtail millet(Setaria italica). In this study, we isolated a male-sterile ethyl methanesulfonate-generated mutant in foxtail millet, no pollen 1(sinp1), which displayed abnormal Ubisch bodies, defective pollen exine and complete male sterility. Using bulk segregation analysis, we cloned SiNP1 and confirmed its function with CRISPR/Cas9 genome editing. SiNP1 encoded a putative glucose-methanol-choline oxidoreductase.Subcellular localization showed that the SiNP1 protein was preferentially localized to the endoplasmic reticulum and was predominantly expressed in panicle. Transcriptome analysis revealed that many genes were differentially expressed in the sinp1 mutant, some of which encoded proteins putatively involved in carbohydrate metabolism, fatty acid biosynthesis, and lipid transport and metabolism, which were closely associated with pollen wall development. Metabolome analysis revealed the disturbance of flavonoids metabolism and fatty acid biosynthesis in the mutant. In conclusion, identification of SiNP1 provides a candidate male-sterility gene for heterosis utilization in foxtail millet and gives further insight into the mechanism of pollen reproduction in plants.

增强UV-B辐射与不同水平氮素对谷子(Setaria italica(L．)Beauv．)叶片保护物质及保护酶的影响

研究了生长在1.875 mmol.L-1和15 mmol.L-1硝态氮素水平条件下的谷子(Setaria italica(L.)Beauv.)开花期间在进行强度为7.12 kJ.m-.2d-1增强UV-B辐射处理时叶片类黄酮含量、苯丙氨酸解氨酶(PAL)和保护酶活性变化差异。主要结果表明在开花期无论是否进行增强UV-B辐射处理,较低水平氮素均比较高水平氮素更有利于提高谷子叶片PAL活性;叶片类黄酮含量除在进行增强UV-B处理时较低氮素条件下生长的谷子在开花末期显著高于较高氮素条件下生长的谷子外,受氮素水平影响不甚明显。而在开花期不进行与进行增强UV-B辐射处理,氮素水平对叶片保护酶的影响有所差异:不进行增强UV-B辐射处理,整个开花期氮素水平对谷子叶片SOD活性有显著影响而对ASP活性无显著影响,对CAT和POD活性则在开花期部分阶段有显著影响。进行增强UV-B辐射处理,整个开花期氮素水平对谷子叶片SOD与CAT活性有显著影响而对ASP、POD活性影响不显著。

Effects of Fertility and Density on Biomass Production,Translocation and Lodging Resistance of Millet(Setaria italica L.)in North China

In this study, the plant biomass production, biomass translocation rates across tissues and the lodging resistant-associated traits of millet （ Setaria italica L.） in North China were investigated. Among the four summer millet cultivars, Baogu 19 exhibited improved plant biomass （PB） production at flowering and maturity stages, biomass translocation amount （BTA） from vegetative tissues to seeds during filling period, and lodging resistant-associated （LRA） traits compared with other cultivars, including enhanced stem lignin contents, increased anti-broken resistance （ABR）, anti-puncturing resistance （APR）, and stem diameter （SD） of plants. Compared with treatment regular cultivation （RC）, high fertility treatment （HF） increased the plant BP, BTA from vegetative tissue to seed at filling stage, and the plant LRA traits; whereas high density treatment （HD） decreased the plant BP at plant level, plant BTA from vegetative tissues to seeds at filling stage, and the plant LRA traits. Correlation analysis revealed that stem ABR is significantly correlated with the plant lodging resistant-associated traits including APR and SD in the summer millet cultivars examined under various cultivation treatments. Our investigation indicates that cultivar Baogu 19 together with suitable fertilization and density can promote the plant biomass production, enhance vegetative tissue biomass translocation to seeds, and improve the lodging resistance of summer millet plants in North China.

Retrotransposon-mediated DELLA transcriptional reprograming underlies semi-dominant dwarfism in foxtail millet

Retrotransposons account for a large proportion of the genome and genomic variation, and play key roles in creating novel genes and diversifying the genome in many eukaryotic species. Although retrotransposons are abundant in plants, their roles had been underestimated because of a lack of research. Here, we characterized a gibberellin Acid (GA)-insensitive dwarf mutant, 84133, in foxtail millet. Map-based cloning revealed a 5.5-kb Copia-like retrotransposon insertion in DWARF1 (D1), which encodes a DELLA protein. Transcriptional analysis showed that the Copia retrotransposon mediated the transcriptional reprogramming of D1 leading to a novel N-terminal-deleted truncated DELLA transcript that was putatively driven by Copia's LTR, namely D1-TT, and another chimeric transcript. The presence of D1-TT was confirmed by protein immunodetection analysis. Furthermore, D1-TT protein was resistant to GA3 treatment compared with the intact DELLA protein due to its inability to interact with the GA receptor, SiGID1. Overexpression of D1-TT in foxtail millet resulted in dwarf plants, confirming that it determines the dwarfism of 84133. Thus, our study documents a rare instance of long terminal repeat (LTR) retrotransposon-mediated transcriptional reprograming in the plant kingdom. These results shed light on the function of LTR retrotransposons in generating new gene functions and genetic diversity.

Genetics, Development, and Application of Cytoplasmic Herbicide Resistance in Foxtail Millet

The effect of cytoplasmic herbicide resistant gene in millet plants was studied. The heterozygous populations and isogenic lines with homocaryotic alloplasmic genes were obtained by crossing and reciprocal crossing of cytoplasmic herbicide resistant plants with susceptive plants of foxtail millet. The characters of F1, F2, backcross and composite cross groups, and the growth and development of isogenic lines were compared. The cytoplasmic herbicide resistant gene slowed the development of seedling, delayed heading, and shortened the milking stage in the foxtail millet plant. Yield capacity and main agronomic characters were all affected by the cytoplasmic herbicide resistant gene in most of the backcross, composite cross, and F2 populations. However, there was stronger hybrid vigor in F1. The backcrosses, composite crosses, and F2 populations were widely separated and some of them had good characters similar to those of susceptive groups. The plant characters and development of foxtail millet were negatively affected by the cytoplasmic herbicide resistant gene. The authors proposed a method of using hybrid vigor to obtain high yield and avoid the negative effects of herbicide resistance cytoplasm in plant growth. The expected results could be obtained by selecting individuals in separate populations of fast developed seedlings, well-developed roots, and with capacities of early heading and fast milking. Guided by the principal mentioned above, many high yield lines and hybrid crosses of foxtail millet with herbicide resistant cytoplasm were obtained.

A nucleotide substitution at the 5′ splice site of intron 1 of rice HEADING DATE 1(HD1) gene homolog in foxtail millet, broadly found in landraces from Europe and Asia

We investigated genetic variation of a rice HEADING DATE 1(HD1) homolog in foxtail millet.First, we searched for a rice HD1 homolog in a foxtail millet genome sequence and designed primers to amplify the entire coding sequence of the gene. We compared full HD1 gene sequences of 11 accessions(including Yugu 1, a Chinese cultivar used for genome sequencing) from various regions in Europe and Asia, found a nucleotide substitution at a putative splice site of intron 1, and designated the accessions with the nucleotide substitution as carrying a splicing variant. We verified by RT-PCR that this single nucleotide substitution causes aberrant splicing of intron 1. We investigated the geographical distribution of the splicing variant in 480 accessions of foxtail millet from various regions of Europe and Asia and part of Africa by d CAPS and found that the splicing variant is broadly distributed in Europe and Asia. Differences of heading times between accessions with wild type allele of the HD1 gene and those with the splicing variant allele were unclear. We also investigated variation in 13 accessions of ssp. viridis, the wild ancestor, and the results suggested that the wild type is predominant in the wild ancestor.

Plants regeneration from protoplast and their genetic variation in foxtail millet

Compact calli derived from immature spikelet of a foxtail millet variety—Jigu 11cann’t be directly used for protoplast isolation because of its firm physical structure,and must beloosened with subculturing in M1,M2 and M3 media successively and altering these media compo-sitions.The loosened calli can be selected from the regulation and used for protoplast isolationsuccessfully.Rate of protoplast division in KM8P medium was 12.3—33.5%.Calli derivedthrough protoplast division are loose and cann’t be used directly for plan regeneration because ofits soft physical structure.When they were subcultured in N6—1,N6—2,N6—3 and N6—4 media,in which the media compositions were changed,the compact calli were obtained and 129 plantletswere regenerated from them.101 plants,which grew to maturity after transplanting the plantletsinto field,exhibited sterility in some degree.Most of the subsequent lines derived from the regen-erated plants were sterile and only two lines could get normal reproduction.

The boron transporter SiBOR1 functions in cell wall integrity, cellular homeostasis, and panicle development in foxtail millet

Boron(B) is an essential micronutrient for vascular plant growth. Both B deficiency and toxicity can impair tissue development in diverse plant species, but little is known about the effect of B on reproductive panicle development and grain yield. In this study, a mutant of Setaria italica exhibiting necrotic panicle apices was identified and designated as sibor1. Sequencing revealed a candidate gene, Si BOR1, with a G-to-A alteration at the seventh exon. Knockout transgenic lines generated by clustered regularly interspaced short palindromic repeats and their associated protein-9 also had necrotic panicles, verifying the function of Si BOR1. Si BOR1 encoded a membrane-localized B efflux transporter, co-orthologous to the rice BOR1 protein. Si BOR1 was dominantly expressed in panicles and displayed a distinct expression pattern from those of its orthologs in other species. The induced mutation in Si BOR1 caused a reduction in the B content of panicle primary branches, and B deficiency-associated phenotypes such as thicker cell walls and higher cell porosity compared with Yugu 1. Transcriptome analysis indicated that differentially expressed genes involved in cell wall biogenesis, jasmonic acid synthesis, and programmed cell death response pathways were enriched in sibor1. q PCR analysis identified several key genes, including phenylalanine ammonia-lyase(Si PAL) and jasmonate-ZIM-domain(Si JAZ) genes, responsive to B-deficient conditions. These results indicate that Si BOR1 helps to regulate panicle primary branch development to maintain grain yield in S. italica. Our findings shed light on molecular mechanisms underlying the relationship between B transport and plant development in S. italica.

Innovation of Super Early-Mature Millet Germplasm with Sweet-Stem and Multi-Spikes

A new millet (Setaria italica Beauv) variety, super early-mature millet No.1, was bred by means of gene bank breedingmethod of target characters. This variety has the following outstanding characters. (1) Super early-mature. This varietyonly needs 1550C effective accumulated temperature and can normally maturate in the Bashang Region in Hebei Provinceof Chi na, which can break through the limit zone of millet cultivation and move the cultivation zone northward greatly. (2)Multi-spikes, in addition to the effect tilling at the top, the nodes in the low-middle part also can produce spikes. (3) Sweetstem have high sugar content. The contents of whole-sugar, soluable sugar and deoxidized sugar are 74.8, 200.5, 237.2%higher than the regular varieties respectively. (4) High gross protein content. The content of gross protein is higher thanthe regular varieties by 3.9-30.4%. (5)Changeable grain color. The grain color of super early-mature millet No.1 is red inShijiazhuang, but yellow in the Bashang region. In addition, this variety is characterized by good quality, high yield, andgood synthetic traits

Effects of Nitrogen Amount on the Photosynthesis Parameters of Summer Millet in North China

In this study,effects of nitrogen（ N） amount applied on photosynthesis behaviors of the summer millet in North China was investigated. Photosynthetic rates（ Pn）,chlorophyll contents（ Chl）,photosynthetic active duration（ PAD）,and chlorophyll relative steady phase（ RSP） in flag and the upper third leaves were assessed in cultivars of Baogu 19,Jigu 19,9050,and 60 D under three N treatments [i. e.,0（ control）,75,and 150 kg/hm2]. Results indicated that the photosynthesis parameters were drastically regulated by external N levels,all of them showing elevation along with the increased N input in both assayed leaves.Among the cultivars examined,behaviors of the photosynthetic parameters were much better in Baogu 19 and worse in 60 D. The plant yields in the cultivars under various N treatments were shown to be in consistent with the behavior of the photosynthesis parameters. Correlation analysis revealed that plant yield is positively correlated with Pn and Chl and significantly positively correlated with PAD and RSP,suggesting that longer effective photosynthetic duration of leaves impacts largely on plant biomass production and the yield formation potential. Our investigation indicates that suitable external N applied can increase the yield of summer millet associating with the improvement of photosynthesis behaviors in upper leaves that contribute to plant biomass at the late growth stage. Baogu 19 exhibited higher plant yield together with improved photosynthetic parameters in upper leaves,suggesting its potential as an elite cultivar in planting in the summer season of North China.

谷子苗期对不同干旱胁迫的生理响应

采用聚乙二醇(Polyethylene glycol,PEG)溶液模拟干旱环境,对谷子种质资源进行室内萌发期抗旱性鉴定评价,旨在筛选出具有抗旱特性的优质品种。以冀谷37和冀谷38抗逆谷子品种为试验材料,并以华北夏谷区域广适性品种豫谷18作为对照,采用不同浓度PEG对其进行干旱胁迫处理,并对处理7 d后幼叶的丙二醛、甜菜碱、游离脯氨酸、叶绿素含量以及超氧化物歧化酶(Superoxide dismutase,SOD)、过氧化物酶(Peroxidase,POD)活性进行检测。结果表明,与对照品种豫谷18相比,冀谷37和冀谷38在中度和重度干旱胁迫(12%PEG、18%PEG)下丙二醛含量显著降低;冀谷38在中度和重度干旱胁迫下渗透调节物质游离脯氨酸含量显著增加,冀谷37和冀谷38甜菜碱含量均显著增加;冀谷38 SOD活性显著增强,但冀谷37仅在轻度和重度干旱胁迫(6%PEG、18%PEG)SOD活性高于豫谷18;冀谷37和冀谷38的POD活性在重度干旱胁迫下显著降低;冀谷38叶绿素含量均显著增加,但冀谷37仅在轻度和中度胁迫(6%PEG、12%PEG)下叶绿素含量显著增加。综上,冀谷37和冀谷38在幼苗期具有较强的耐旱能力,耐旱性优于对照品种豫谷18,且冀谷38抗旱能力强于冀谷37。

NaCl处理谷子萌发期种子的转录组学分析

【目的】谷子具有抗旱、耐贫瘠的特性,逐渐成为研究禾本科作物的模式作物。本研究以前期筛选并获得的谷子耐盐品种和敏感品种为材料,通过RNA-Seq测序筛选鉴定谷子的盐胁迫响应基因,揭示其响应盐害机制,为培育谷子抗盐新种质提供理论依据。【方法】对14个谷子品种进行了萌发期抗盐筛选。谷子不同品种的种子经150 mmol·L-1NaCl处理萌发培养7 d后,分别统计各品种种子的萌发率、根长和芽长,综合各项指标鉴定到豫谷2为耐盐品种、安04为盐敏感品种。以这两个品种盐胁迫前、后萌发期种子为材料,应用RNA-Seq进行转录组测定,分析鉴定盐害下差异表达基因(differentially expression gene,DEG),并对DEG进行GO和KEGG功能富集分析。同时应用qRT-PCR对随机挑选的15个DEG表达模式进行验证,并与RNA-Seq结果进行相关性分析。【结果】耐盐品种豫谷2、盐敏感品种安04种子经盐胁迫处理后,分别鉴定出2786个和4413个DEG;其中,每个品种在NaCl处理前及处理后分别鉴定出1470和3826个DEG。GO和KEGG聚类分析显示,NaCl处理下参与信号转导、抗氧化、有机酸的合成及转运以及次生代谢等相关的DEG在谷子萌发期种子应对盐胁迫响应过程中具有关键性的作用,DEG主要集中在胁迫响应、离子的跨膜转运、氧化还原、次生代谢及有机酸、多胺、苯丙烷的合成等过程。qRT-PCR对15个DEG表达模式的检测结果与RNA-seq结果相关系数为0.8817。其中编码离子通道的基因(HKT)、过氧化物酶基因(POD)、黄烷酮-3-羟化酶基因(FL3H)、MYB转录因子基因等在豫谷2中表达量较高,显示其在谷子萌发期种子响应盐胁迫中可能发挥一定作用。【结论】谷子耐盐品种及盐敏感品种的种子对盐胁迫的响应程度具有一定的差异性,且品种对盐害的耐受能力主要与基因对胁迫的响应程度相关,而与DEG的数量相关性不大。

施氮量对谷子产量、氮素利用及小米品质的影响

【目的】明确不同施氮量下谷子产量、干物质分配和氮素累积转运特征,分析氮用量对小米糊化特性和有益微量元素含量的影响及其与植株氮素累积的关系,探究植株氮素营养对小米品质的影响。【方法】于2020—2021年在山西省沁县研究4个施氮水平(0、75、120和150 kg·hm^(-2))对春播谷子产量、氮素吸收与利用特征及小米品质的影响。【结果】施氮提高谷子收获穗数、穗粒数和植株的干物质生产能力,增加了氮素由营养器官向籽粒的转运率,促进了干物质及氮素向籽粒的分配,从而提高产量。施氮也提高了小米中铁、锌、钙、镁和硒的含量,其中,施氮75 kg·hm^(-2)时上述元素含量的增幅最大,氮利用率最高。与不施氮相比,施氮75 kg·hm^(-2)时谷子收获穗数、穗粒数、产量、地上部生物量、收获指数、氮素累积总量和氮素转运率增幅最高,增幅分别可达7.5%、23.3%、31.0%、21.2%、8.6%、40.3%和9.2%,小米中铁、锌、钙、镁和硒含量的增幅分别为37.2%、43.6%、56.0%、30.5%和16.9%。过量施氮(150 kg·hm^(-2))不利于谷子穗粒数和收获指数的提高及氮素由营养器官向籽粒的转运,与施氮量75 kg·hm^(-2)比较,两年氮素转运率分别降低了23.1%和28.2%;氮素施用过量也降低了小米支链淀粉含量,淀粉形成受限,抑制了小米粉最终黏度、回升值和峰谷黏度,影响糊化品质,同时氮肥利用率低至25%左右。谷子地上部氮吸收量与小米中铁、锌、钙、镁和硒含量呈极显著的正相关,但与小米中支链淀粉含量、小米粉的最终黏度和峰谷黏度呈显著的负相关。【结论】施氮量在75—120kg·hm^(-2),能促进谷子干物质及氮素向籽粒的分配,实现籽粒产量、小米糊化品质和有益微量元素含量的同步提升。

谷子显性恢复基因的AFLP分析

赤峰显性核不育谷子是在谷子中首次发现的核不育材料,该材料的育性受2对核显性基因互作控制,一对是显性核不育基因Msch,另一对是显性上位育性恢复基因Rf。两者共同存在时显性上位育性恢复基因Rf能抑制显性核不育基因Msch的表达,从而表现可育。利用已构建的不育基因Msch的上位育性恢复基因Rf的近等基因系(NILs)为材料,通过对300对AFLP引物组合进行筛选,找到了与显性上位育性恢复基因Rf紧密连锁的2个AFLP标记(E15/M52和E20/M41),与不育基因的遗传距离分别是7.0c M和12.7 c M,而且位于不育基因的同一侧,标记间相距5.7 c M。

微藻生物肥提高谷子对氮磷胁迫的耐受性

【目的】氮磷营养缺乏是作物生长发育的重要限制因素。微藻生物肥是由单细胞光合藻类生物质构成的一种新型肥料,其促进作物生长和改良土壤肥力的功效备受关注。本研究旨在解析微藻生物肥对谷子(Setaria italica L.)生长的促进作用,特别是提高谷子对氮磷缺乏的耐受性的生物学功能,为微藻生物肥在谷子等作物的化肥减量和绿色可持续生产的应用提供理论支撑。【方法】供试谷子品种为‘晋谷21’。采用水培试验,设置正常氮磷(霍格兰标准营养液中氮和磷含量分别为210和71 mg/L)、无氮磷(营养液中氮和磷含量均为0 mg/L)和1/2氮磷(营养液中氮和磷含量分别为105和35.5 mg/L)3个氮磷水平。制备小球藻(Chlorella sp.DT01)(DT)、埃氏小球藻(Chlorella emersonii)(AS)和蛋白核小球藻(Chlorella pyrenoidosa)(DB)3种微藻生物肥,分别与3个氮磷处理液等比混合,共构成9个处理,每个氮磷水平下设置1个不添加微藻微生物溶液处理为对照。在谷子幼苗生长至两叶一心时,将其移入处理液中,在温室条件下连续培养9天,期间每3天补充1次处理液。处理结束时,测定各处理谷子幼苗的生长指标、生物量、色素含量、光合活性和氮磷含量等生理生化参数,并通过qRT-PCR技术分析氮磷转运蛋白相关基因(SiNRT1.11、SiNRT2.1、SiPHT1.2和SiPHT1.1)的表达谱。【结果】与正常氮磷处理相比,无氮磷和1/2氮磷处理均显著降低了谷子幼苗生物量、光合色素(叶绿素a,b和类葫萝卜素)含量和光合作用。在3种剂量氮磷处理下,施用3种微藻生物肥处理均显著提高谷子幼苗鲜重和干重、光合色素含量(特别是类胡萝卜素)和光合作用参数[实际光化学效率Y(II),光化学淬灭系数qP和非光化学淬灭系数NPQ]。此外,微藻生物肥还显著增加了谷子幼苗全氮和全磷含量。微藻生物肥DT处理的促生效应最显著。qRT-PCR检测谷子幼苗氮磷转运相关基因表达显示,微藻生物肥DT处理显著上调谷子幼苗地上部硝酸盐转运蛋白基因SiNRT1.11和根系硝酸盐转运蛋白基因SiNRT2.1的表达,以及根系中磷酸盐转运蛋白基因SiPHT1.2和SiPHT1.1的表达。【结论】施用微藻生物肥能显著提高谷子幼苗地上部硝酸盐转运蛋白基因SiNRT1.11和根系硝酸盐转运蛋白基因SiNRT2.1,以及根系中磷酸盐转运蛋白基因SiPHT1.2和SiPHT1.1的表达,进而改善谷子幼苗生长和光合生理特性,提高谷子对氮磷胁迫的抗性。

一个谷子新抗锈基因的AFLP标记

【目的】研究谷子抗源的抗锈遗传规律,寻找和定位与谷子抗锈基因连锁的分子标记,为谷子抗锈病基因的定位、克隆和抗病育种等研究奠定基础。【方法】用谷子锈菌单胞菌系93-5接种十里香和豫谷1号及杂交后代F1、F2进行抗锈鉴定,并根据鉴定结果构建抗、感基因池;利用AFLP技术筛选128对EcoRⅠ/MseⅠ引物组合,从中寻找和定位与谷子抗锈基因连锁的分子标记;根据AFLP分析结果进行抗锈基因连锁分析并进行SCAR标记转化。【结果】根据十里香×豫谷1号杂交后代F2群体(131株)抗感谷锈病分离比例,确定十里香抗锈性由显性单基因控制。筛选获得3个与谷子抗锈基因Rusi1(暂命名)连锁的AFLP分子标记,经计算标记与该抗锈基因的遗传距离分别为7.4、9.2和27.4cM。将3个标记片段回收、克隆和测序,成功地将AFLP标记E+CTT/M+TAC-256转化为SCAR标记。初步构建了谷子抗锈基因Rusi1的遗传连锁图谱。【结论】谷子十里香抗锈性由显性单基因控制,Rusi1是一个新发现的谷子抗锈基因。

谷子类受体蛋白激酶基因SiRLK35在水稻中对盐胁迫的响应

【目的】盐胁迫影响作物的产量和品质,而作物的耐盐性受特定基因的调控。在前期获得谷子类受体蛋白激酶基因SiRLK35过表达水稻株系的基础上,拟结合植株在盐胁迫下的表型,对部分盐胁迫指标及响应基因的表达模式进行检测和验证,解析谷子SiRLK35参与盐害响应的可能机制。【方法】选取谷子SiRLK35过表达水稻株系,分别为过表达株系(over-expression,OE)-1(OE-1)、OE-2和OE-3,以野生型中花11为对照,实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测各株系中SiRLK35的表达情况;分别用含0、150和200 mmol·L-1 NaCl的MS培养液处理三叶期的对照及转基因株系幼苗,观察其表型,统计150 mmol·L-1 NaCl处理3 d后苗长及根长;用150 mmol·L-1 NaCl处理四叶期幼苗,统计处理14 d后材料干重、死亡率和死叶率,对各材料的耐盐性进行评价;进一步挑选SiRLK35过表达程度高且耐盐表型好的OE-1株系,利用3,3'-二氨基联苯胺(3,3'-Diaminobenzidine,DAB)和氮蓝四唑(nitrotetrazolium blue chloride,NBT)染色检测其胁迫下过氧化物积累及超氧化物歧化酶(superoxide dismutase,SOD)和过氧化物酶(peroxidase,POD)的活性;并对部分盐害响应基因的表达模式进行检测。【结果】谷子SiRLK35在株系OE-1中的相对表达量最高;150 mmol·L-1NaCl处理3 d幼苗后,中花11苗长和根长的生长受抑制程度均大于SiRLK35过表达株系,其中OE-1的受抑制程度最小;四叶期幼苗经150 mmol·L-1 NaCl处理14 d后,转基因株系地上部和地下部干重降低幅度均低于对照,且幼苗死亡率和死叶率均低于对照;盐胁迫下对照过氧化染色反应明显,O2-和H2O2的积累均高于过表达植株,SOD和POD抗氧化酶活性均高于对照;部分盐害响应基因在SiRLK35过表达株系中表现为上调,其中,OsLEA3在盐处理24 h的相对表达量是对照中的1.9倍。【结论】谷子SiRLK35异源转化水稻获得的过表达株系对盐胁迫具有一定的抗性,SiRLK35可通过调控抗氧化酶活性及相关信号途径,从而参与盐害响应。