Development of organelle single nucleotide polymorphism (SNP) markers and their application for the identification of cytoplasmic inheritance patterns in Pyropia yezoensis (Bangiales,Rhodophyta)

The genus Pyropia contains several important cultivated species.Genetic research in nori species has mainly focused on the cell nucleus,with few studies on organelles(chloroplast and mitochondria).Due to the high copy numbers of organelles in cells,which influence the development and traits of algae,it is necessary to study their genetic mechanism.In this study,the marine red alga Pyropia yezoensis,an important economic macroalga,was selected as the study object.To investigate organelle(chloroplast and mitochondria)inheritance in P.yezoensis,the wild type RZ(maternal strain)was crossed with the red mutant HT(paternal strain)and 30 color-sectors from 11 F1 gametophytic blades were examined.The complete chloroplast and mitochondrial genomes of the red mutant(HT)were assembled for the first time.One reliable and stable single nucleotide polymorphism(SNP)loci filtrated by bioinformatics analysis was used as a molecular marker for chloroplast and mitochondrial DNA,respectively,in subsequent experiments.PCR amplification and sequence analysis showed that the haplotypes of color-sectors detected were consistent with those of the maternal parent,confirming that both chloroplast and mitochondrial genomes were inherited maternally in P.yezoensis.The inheritance pattern of organelles in P.yezoensis can be used to guide the hybridization and breeding of nori.Additionally,the organelle SNP markers developed in this study can be applied in subsequent genetic research.

Molecular Marker Techniques Using Single Primers and Their Advances

Molecular marker techniques have been widely applied in the fields of genetic diversity analysis,germplasm resources identification,molecular fingerprint and genetic linkage map construction,QTL mapping and molecular assisted breeding.On the basis of stating the concept of molecular marker techniques based on single primer amplification reactions,this study focused on the sorting and induction of single-primer molecular marker techniques,and expounded their derivative development.Finally,the application prospect and future expectation of single-primer molecular marker techniques were described in detail.The purpose of this study was to clarify the types of molecular marker techniques based on single primer amplification reactions,so that researchers can quickly and conveniently select molecular marker techniques according to their own specific scientific research conditions.

Quality assessment of Chrysanthemum indicum Flower by simultaneous quantification of six major ingredients using a single reference standard combined with HPLC fingerprint analysis

Chrysanthemum indicum Flower is usually consumed as functional food. This paper described an improved total quality assessment method for Chrysanthemum indicum Flower by simultaneous quantitation using a single standard to determine multi-components method combined with high performance liquid chromatography fingerprint analysis. Six main components of Chrysanthemum indicum Flower including two flavonoids and four phenolic acids were simultaneously quantified using linarin as the internal reference standard. The method was fully validated with respect to linearity, precision, accuracy, robustness and stability.The validated method was successfully applied to the analysis of thirty three batches of Chrysanthemum indicum Flower samples. Under the same chromatographic conditions, fingerprint analysis in combination with Similarity analysis and principal component analysis was performed to identify the samples from different regions. In general, an effective assessment using a single standard to determinate multi-components method combined with fingerprint analysis make the reliable qualitation and quantitation analysis of Chrysanthemum indicum Flower available.

Single-nucleotide polymorphisms,mapping and association analysis of 1-FFT-A1 gene in wheat

Fructans are major nonstructural carbohydrates in wheat （Triticum aestivum L.）. Fructan 1-fructosyltransferase （1-FFT） is the key enzyme in fructan biosynthesis. In the present study, 96 sequence variants were detected in the 1-FFT-A 1 gene among 26 wheat accessions including UR208, and 15 of them result in amino acid substitutions, forming four haplotypes. Two markers M39 and M2164 were developed based on the InDe121-39 and SNP-2164 polymorphisms to distinguish the three haplotypes in the 1-FFT-AI. 1-FFT-A1 was located on chromosome 4A using marker M2164 and was flanked by markers Xcwm27 and 6-SFT-A 1. By association analysis using a natural wheat population consisted of 154 accessions, the results showed that the two markers were significantly associated with water-soluble carbohydrate （WSC） content in the lower internode stem and total stem at the early and middle grain filling stages, 1 000-grain weight （TGW） at different grain filling stages and peduncle length （PLE）. Comparison of the effects of three haplotypes on agronomic traits indicated that TGW, PLE and total number of spikelets per spike （TNSS）were significantly influenced by haplotypes. Haplll showed a significant positive effect on TGW, PLE and TNSS.

Single-cell RNA sequencing in cornea research:Insights into limbal stem cells and their niche regulation

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye,which acts as a protective barrier and is critical for clear and stable vision.Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells(LSCs),a cell population that resides at the limbus in a highly regulated niche.Dysfunction of LSCs or their niche can cause limbal stem cell deficiency,a disease that is manifested by failed epithelial wound healing or even blindness.Nevertheless,compared to stem cells in other tissues,little is known about the LSCs and their niche.With the advent of single-cell RNA sequencing,our understanding of LSC characteristics and their microenvironment has grown considerably.In this review,we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology,including the heterogeneity of the LSC population,novel LSC markers and regulation of the LSC niche,which will provide a reference for clinical issues such as corneal epithelial wound healing,ocular surface reconstruction and interventions for related diseases.

Identification of Multiple Alleles at the Wx Locus and Development of Single Segment Substitution Lines for the Alleles in Rice

The microsatellite markers 484/485 and 484/W2R were used to identify the multiple alleles at the Wx locus in rice germplasm. Fifteen alleles were identified in 278 accessions by using microsatellite class and G-T polymorphism. Among these alleles, （CT）12-G, （CT）15-G, （CT）16-G, （CT）17-G, （CT）18-G and （CT）21-G have not been reported. Seventy-two single-segment substitution lines （SSSLs） carrying different alleles at the Wx locus were developed by using Huajingxian 74 with the （CT）11-G allele as a recipient and 20 accessions containing 12 different alleles at the Wx locus as donors. The estimated length of the substituted segments ranged from 2.2 to 77.3 cM with an average of 17.4 cM.

南美白对虾多密度高通量液相分型技术芯片设计与评估

针对南美白对虾育种研究中开展大规模全基因组重测序成本非常昂贵的问题,本研究对来自两个人工选择品种和四个市场领先公司的共180只南美白对虾进行全基因组重测序及全基因组单核苷酸多态性(Single nucleotide polymorphisms,SNPs)标记开发,并设计了12k、46k和92k三种密度规格的高通量液相分型技术芯片。对三款芯片性能进行评估的结果显示,三款芯片分别覆盖了11618、20055和20056个基因,基因覆盖度分别为43.50%、75.08%和75.09%;位于基因区位点分别为11618、35071和65138,占芯片总位点93.15%、76.24%和70.80%。次等位基因频率(Minor allele frequency,MAF)分别为0.27±0.11、0.34±0.12和0.27±0.12。三款芯片的位点在染色体上均匀分布,其中51.11%、76.63%和93.87%的位点平均间距范围为1~50 kB,均覆盖了MAPK、mTOR、Wnt和NOD-like受体等多个参与生长免疫功能的信号通路。本研究为南美白对虾HD-Marker体系建立提供了基础,从而为全基因组选择育种、种质资源评估、数量性状位点(Quantitative trait locus,QTL)定位和全基因组关联分析(Genome-wide association study,GAWS)提供技术支持。

单细胞转录组测序技术在脊髓损伤研究中的应用

背景:近年来,单细胞转录组测序技术在脊髓损伤的研究为创伤后中枢神经系统的细胞和分子异质性以及结构变化提供了新的见解。目的:就单细胞转录组测序技术在脊髓损伤的研究进展进行综述,更加全面、深入地阐述单细胞转录组测序技术在脊髓损伤领域的应用。方法:应用计算机系统检索PubMed、Web of Science、中国知网和万方数据库中2009-2023年出版的文献,英文检索词为“single-cell RNA sequencing,spinal cord injury,sequencing technology”;中文检索词为“单细胞转录组测序,脊髓损伤,测序技术”。排除质量较差、内容重复及不相关的文献,最终纳入57篇文献进行综述分析。结果与结论:目前,单细胞转录组测序技术在脊髓损伤的研究可归纳为以下几点:①鉴定了小胶质细胞、星形胶质细胞、少突胶质细胞、巨噬细胞、B细胞、神经元和神经干细胞等细胞亚群,并识别了这几种亚群的特异性标记基因。②小胶质细胞在脊髓损伤后保持永久活跃,通过增殖、免疫和稳态功能来协调脊髓损伤后的早期阶段。星形胶质细胞以激活的方式在脊髓损伤中发挥许多重要功能,包括维持微环境平衡、清除坏死组织、形成保护屏障以及胶质瘢痕等。巨噬细胞和小胶质细胞均在脊髓损伤后的慢性神经炎症中起重要作用。③神经干细胞和神经元亚群能够在脊髓损伤后进行自我更新,新发现的SCV^(sx2::Hoxa7:Zfhx3→lumbar)和SCV^(sx2::Hoxa10)等神经元亚群可再生到自然靶区,有利于运动功能的恢复。④发现细胞亚群的动态变化提高了研究者们对脊髓损伤病变进程的理解,并为脊髓损伤在不同时间点的治疗提供了新见解。截至目前,这些研究结果均还需要更多的基础研究和足够的临床试验来验证。在未来,单细胞转录组测序技术通过与生物信息学、计算机科学、组织工程学和临床医学等跨学科合作,有望为脊髓损伤的诊疗打开新的一扇窗户。

Genetic Diversity of Tropical Hybrid Rice Germplasm Measured by Molecular Markers

Investigation of genetic diversity and relationships among breeding lines is of great importance to facilitate parent selection in hybrid rice breeding programs.In this study,we characterized 168 hybrid rice parents from International Rice Research Institute with 207 simple sequence repeat （SSR） and 353 single nucleotide polymorphism （SNP） markers.A total of 1 267 SSR and 706 SNP alleles were detected with the averages of 6.1 （SSR） and 2.0 （SNP） alleles per locus respectively across all lines.Based on the genetic distances estimated from the SSR and SNP markers separately and combined,the unrooted neighbor-joining cluster and STRUCTURE analyses consistently separated the 168 hybrid rice parents into two major groups：B-line and R-line,which is consistent with known parent pedigree information.The genetic distance matrices derived from the SSR and SNP genotyping were highly correlated （r=0.81,P 0.001）,indicating that both of the SSR and SNP markers have distinguishable power to detect polymorphism and are appropriate for genetic diversity analysis among tropical hybrid rice parents.A subset of 60 SSR markers were also chosen by the Core Hunter with 368 alleles,and the cluster analysis based on the total and subset of SSR markers highly corresponded at r=0.91 （P 0.001）,suggesting that fewer SSR markers can be used to classify and evaluate genetic diversity among parental lines.

InDel and SNP Markers and Their Applications in Map-based Cloning of Rice Genes

High-density markers are necessary for map-based cloning of rice genes, but the currently available markers are not satisfactory enough. InDel (insertion-deletion length polymorphism) and SNP (single nucleotide polymorphism) are the new generation of molecular markers and can basically meet the need of fine mapping. InDel and SNP markers can be developed through bioinformatics. These markers are valuable markers with the characters of low cost, high specificity and stability. This article introduced the methods for designing InDel and SNP markers, taking the mapping of a rice rolled leaf gene as an example. In addition, some key factors in improving the design efficiency were also discussed.

Transcriptome analysis of the endangered Notopterygium incisum: Coldtolerance gene discovery and identification of EST-SSR and SNP markers

Notopterygium incisum C. C. Ting ex H. T. Chang(Apiaceae) is an endangered perennial herb in China. The lack of transcriptomic and genomic resources for N. incisum greatly hinders studies of its population genetics and conservation. In this study, we employed RNA-seq technology to characterize transcriptomes for the flowers, leaves, and stems of this endangered herb. A total of 56 million clean reads were assembled into 120,716 unigenes with an N50 length of 850 bp. Among these unigenes, 70,245(58.19%) were successfully annotated and 65,965(54.64%) were identified as coding sequences based on their similarities with sequences in public databases. We identified 21 unigenes that had significant relationships with cold tolerance in N. incisum according to gene ontology(GO) annotation analysis. In addition, 13,149 simple sequence repeats(SSRs) and 85,681 single nucleotide polymorphisms were detected as potential molecular genetic markers. Ninety-six primer pairs of SSRs were randomly selected to validate their amplification efficiency and polymorphism. Nineteen SSR loci exhibited polymorphism in three natural populations of N. incisum. These results provide valuable resources to facilitate future functional genomics and conservation genetics studies of N. incisum.

dCAPS markers developed for nitrate transporter genes TaNRT2L12s associating with 1000-grain weight in wheat

Nitrate transporters(NRTs)are regulators of nitrate assimilation and transport.The genome sequences of TaNRT2L12-A,-B and-D were cloned from wheat(Triticum aestivum L.),and polymorphisms were analyzed by sequencing.TaNRT2L12-D in a germplasm population was highly conserved.However,38 single nucleotide polymorphisms(SNPs)in TaNRT2L12-A coding region and 11 SNPs in TaNRT2L12-B coding region were detected.Two derived cleaved amplified polymorphic sequences(dCAPS)markers A-CSNP1 and A-CSNP2 were developed for TaNRT2L12-A based on SNP-351 and SNP-729,and three haplotypes were identified in the germplasm population.B-CSNP1 and B-CSNP2 were developed for TaNRT2L12-B based on SNP-237 and SNP-1227,and three haplotypes were detected in the germplasm population.Association analyses between the markers and agronomic traits in 30 environments and phenotypic comparisons revealed that A-CSNP2-A is a superior allele of shorter plant height(PH),length of penultimate internode(LPI)and peduncle length(PL),B-CSNP2-G is a superior allele of higher grain number per spike(GNS).Hap-6B-1 containing both superior alleles B-CSNP1-C and B-CSNP2-A is a superior haplotype of 1 OOO-grain weight(TGW).Expression analysis showed that TaNRT2L12-B is mainly expressed in the root base and regulated by nitrate.Therefore,TaNRT2L12 may be involved in nitrate transport and signaling to regulate TGW in wheat.The superior alleles and dCAPS markers of TaNRT2L12-A/B are beneficial to genetic improvement and germplasm enhancement with molecular markers-assisted selection.

Molecular Markers for Tm-2 Alleles of Tomato Mosaic Virus Resistance in Tomato

Tomato mosaic virus (ToMV) is one of the most infectious virus diseases in tomato (Solanum lycopersicum L). The practical and effective method of controlling this disease is through genetic control by using major resistance genes. So far, three genes Tm-1, Tm-2 and Tm-22 conferring resistance to ToMV have been reported and utilized in tomato culti-var development. Marker assisted selection (MAS) has become very important and useful tool in selection of ToMV re-sistant tomato lines or hybrids. The objective of this research was to identify allele-specific PCR-based, cleaved ampli-fied polymorphic sequence (CAPS), and allele-derived single nucleotide polymorphism (SNP) markers for Tm-2 loci. Four allele-specific PCR-based markers were identified: one for Tm-2, one for Tm-22, and two for the susceptible allele tm-2. Three allele-derived CAPS markers were identified, which can identify and distinguish three alleles, tm-2, Tm-2 and Tm-22 in tomato germplasm. Three SNP markers were developed specific for Tm-2 locus. These markers will pro-vide breeders with a tool in selection of Tm-2 and Tm-22 resistance genes in tomato breeding program.

一测多评法测定美洲大蠊药材5种核苷类成分

目的建立高效液相色谱(HPLC)一测多评法同时测定美洲大蠊中尿嘧啶、尿苷、次黄嘌呤、肌苷及鸟苷5种成分的含量。方法采用Agilent ZORBAX SB-Aq色谱柱(250 mm×4.6 mm,5μm);以甲醇-0.01 mol·L^(-1)磷酸二氢钾溶液为流动相,梯度洗脱;流速:1.0 mL·min^(-1);柱温:20℃;检测波长:260 nm;进样量:10μL。计算内参物尿苷与其他成分的相对校正因子(fa/b)。一测多评法测定10批美洲大蠊中5种成分含量,并与外标法比较。结果5种核苷类在各自范围内线性良好(r>0.9995),平均加样回收率97.0%~100.8%。尿嘧啶、次黄嘌呤、肌苷、鸟苷的fa/b分别是0.9080、1.0053、1.9695、1.3034。结论所建立的方法准确稳定,可为美洲大蠊药材及提取物的质量控制提供理论参考。

Microarray-assisted Marker Development for Molecular Breeding

The advantage of being able to investigate the abundance and composition of millions of sequences in parallel has established microarrays as an attractive technology for genome wide transcription and sequence variation pro-

天子蓝盘丽鱼皮肤组织的单细胞转录组测序

【目的】通过单细胞转录组测序比较天子蓝盘丽鱼和野生阿莲卡盘丽鱼皮肤组织中与体色形成相关的基因表达模式,为深入研究鱼类体色及良种选育提供数据基础,也为盘丽鱼或其他水产鱼类进行单细胞转录组研究提供技术借鉴。【方法】利用单细胞转录组测序技术对天子蓝(人工选育)和阿莲卡(野生型)2种盘丽鱼的皮肤组织进行转录组测序,经fastp质控过滤后,通过Cell Ranger比对参考基因组生成单细胞表达矩阵文件,使用Seurat进行降维聚类后构建转录组图谱,筛选出细胞类型识别与鉴定的标记基因;同时以阿莲卡盘丽鱼为对照,通过DEGseq筛选出2个样本间的差异表达基因(DEGs),并进行GO功能注释分析和KEGG信号通路富集分析。【结果】经单细胞转录组测序,从天子蓝、阿莲卡盘丽鱼样本中获得的原始序列(Raw reads)分别为815237286和728886552条;将2个样本整合后共捕获17035个细胞,经过滤及降维聚类后得到18个细胞簇(C0~C17),参考已知和潜在的标记基因,被注释为13种细胞类型及其亚型,基本涵盖了鱼类皮肤组织中的大部分细胞类型。基于伪批量(Pseudo-bulk)处理,从2个样本间筛选出20个DEGs(8个在天子蓝盘丽鱼皮肤组织中高表达,12个在阿莲卡盘丽鱼皮肤组织中高表达),主要注释到与气体传递、氧化代谢相关的GO功能条目。在单细胞维度,选择黑色素细胞和虹彩细胞2个细胞集群进行样本间差异分析,结果显示:在黑色素细胞中存在8个DEGs,主要富集于热应答、未折叠蛋白结合、生长因子活性、金属离子结合等信号通路。在虹彩细胞中存在25个DEGs,KEGG信号通路富集分析发现主要富集于凋亡、沙门氏菌感染和MAPK等信号通路,涉及细胞生存、应激响应和信号传导等功能。【结论】TSPAN3A、PTGES、ATF3、JUNK、GADD45GA、HSP70L、FOSAB等基因在调节天子蓝盘丽鱼皮肤体色、色素合成与沉着等方面发挥重要作用,而黑色素合成不活跃可能是导致天子蓝盘丽鱼体色较阿莲卡盘丽色更加鲜艳和绚丽的主要原因之一。

固相萃取-一标多测高效液相色谱法测定化妆品中5种苯并三唑类防晒剂

建立了固相萃取技术结合高效液相色谱测定化妆品中5种苯并三唑类防晒剂的一标多测定量分析方法。该方法待测样品使用HLB固相萃取小柱净化处理,以亚甲基双-苯并三唑基四甲基丁基酚为内参物,通过建立该物质与另外4种物质的相对校正因子,计算出各物质的含量。研究了不同进样量、不同流动相流速及不同柱温对相对校正因子的影响,比较了一标多测法和外标法的计算结果。结果表明,5种物质的线性关系良好(R^(2)≥0.9997),检测限和定量限分别为4.00~26.43μg/L和10.00~60.42μg/L;各物质的相对校正因子重复性较好,其相对标准偏差(RSD)为0.25%~1.16%;两种方法计算结果无明显差异,其相对平均偏差(RAD)为0.12%~2.19%。该方法操作简单,使用标准物质少,检测效率高,可用于化妆品中5种防晒剂的含量测定和质量控制。

基于SNP标记对嘉定白蚕豆遗传多样性与特异性的鉴定

为了在基因组水平上揭示嘉定白蚕豆的遗传特性,基于简化基因组测序技术,研究了嘉定白蚕豆种质的遗传多样性,及其与其他来源蚕豆种质的遗传差异,并利用检测到的SNP进行了主成分分析、聚类分析与群体遗传结构分析。结果表明:142份蚕豆种质资源中共检测到30984个SNP。主成分分析可大致区分嘉定材料与其他材料,聚类分析将这142份材料划分为上海嘉定、上海其他和其他省份材料为主的三簇,而群体遗传结构分析则显著区分了嘉定材料与其他材料。从上述SNP中筛选了18个在嘉定和其他材料间存在高分化的SNP标记,均匀分布在6对染色体上,利用这些SNP可准确鉴定(准确率78%)嘉定白蚕豆核心材料。本研究可为嘉定白蚕豆种质资源的保护、研究和利用提供重要的技术支撑。

一测多评法测定大黄碳酸氢钠片中5种游离蒽醌类成分的含量

目的建立一测多评法(QAMS)测定大黄碳酸氢钠片中5种游离蒽醌类成分的含量。方法采用高效液相色谱法,以大黄酚为内参物,确立芦荟大黄素、大黄酸、大黄素和大黄素甲醚与大黄酚的相对校正因子,并与外标法测定结果进行比较,以验证一测多评法在测定该成分的合理性和可行性。结果8批不同生产企业的样品,一测多评法计算值与外标法实测值无明显差异,相对平均偏差均小于1.0%。结论本方法准确可靠,可用于大黄碳酸氢钠片中5种游离蒽醌类成分的含量测定。

一测多评法同时测定参芪二皮冬茶颗粒中6种成分的含量

目的 建立一测多评法同时测定参芪二皮冬茶颗粒中毛蕊异黄酮葡萄糖苷、阿魏酸、迷迭香酸、橙皮苷、橘皮素、川陈皮素的含量。方法 分析采用Agilent Eclipse Plus-C_(18)色谱柱(4.6 mm×250 mm, 5μm);流动相乙腈-0.1%磷酸,梯度洗脱;体积流量1.0 mL/min;柱温25℃;检测波长260、330 nm。以橙皮苷为内标,计算其他5种成分的相对校正因子,测定其含量。结果 6种成分在各自范围内线性关系良好(r≥0.999 0),平均加样回收率99.82%~101.18%,RSD 1.26%~2.25%。一测多评法所得结果与外标法接近。结论 该方法简便可靠,重复性好,可用于参芪二皮冬茶颗粒的质量控制。