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灌木柳耐盐SNP位点的快速鉴定与标记开发
教忠意, 田雪瑶, 郑纪伟, 王保松, 何开跃, 何旭东
南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (5) : 107-113.
PDF(1889 KB)
PDF(1889 KB)
灌木柳耐盐SNP位点的快速鉴定与标记开发
Rapid identification and marker development of SNP loci for salt tolerance in shrub willow
【目的】 对灌木柳(Salix spp.)耐盐相关位点进行快速鉴定并开发单核苷酸多态性(SNP)标记,为耐盐灌木柳早期鉴定与品种选育提供参考。【方法】 以耐盐和敏盐灌木柳无性系为亲本材料建立杂交组合,对子代进行盐胁迫并分别构建耐盐和敏盐混池,利用特定位点参数扩增测定(SLAF-seq)技术对亲本及混池进行简化基因组测序,开发多态性SLAF标签并利用SNP-index法进行关联分析。对候选SLAF标签序列进行功能注释,针对SNP位点设计引物并进行重测序验证。【结果】 4个样本(含2个亲本及2个混池)SLAF测序平均Q30值为94.36%,GC含量为39.39%,测序深度为48.57×。测序共获得175 468个SLAF标签,其中多态性标签25 675个,占比14.63%。关联分析共筛选出与耐盐性状显著关联的SLAF标签18个,包含25个SNP位点。功能注释显示有6、4、3和1个标签序列分别在Nt、NOG、KEGG和Swiss-Prot数据库中有比对结果。重测序结果表明,开发的10个SNP标记在耐盐个体和敏盐个体间发生了碱基位点的替换,与原SLAF测序结果一致。【结论】 本研究利用BSA分析方法结合SLAF测序技术可快速、有效地对目标性状进行初步定位与鉴别,可为柳树功能标记的开发及分子标记辅助育种提供理论依据和技术支持。
【Objective】Due to the rapid reduction of non-agriculture and non-grain land, the afforestation area in China has been drastically decreased in the past two decades and efforts were extended to the afforestation in saline-alkali wastelands in the coastal region. Thus, cultivation and identification of salt tolerance forest germplasm appear to be of particular the importance. However, the salt tolerance in plants is a complex physiological and biochemical process involving with numerous genes, proteins and other multiple coordinative mechanisms. In order to provide a certain significant reference for an early identification and variety selection of salt tolerance shrub willow, we designed this study to screen the SNP loci related with salt tolerance and to develop corresponding SNP markers. 【Method】A hybrid cross was made using salt-tolerance and salt-sensitivity parents identified in the previous study. Cuttings from 1 505 selected seedlings were used for the salt tolerance treatment, of which 50 salt-tolerance and 50 salt-sensitivity individuals were screened for the gene mixing-pool construction. Four libraries, including the parents and two gene pools, were established and sequenced using the SLAF strategy on the Illuminna HiSeq 2500 platform. After filtering and mapping, the polymorphic SLAF tags among the two parents were selected and utilized for an association analysis based on the SNP-index method. The sequences of candidate SLAF tags related tightly with salt tolerance were blasted against multiple databases in NCBI for the functional annotation. The primer pairs for the SNP loci in candidate SLAF tags were designed, and the PCR products were re-sequenced for a loci validation. 【Result】The mean sequencing depth, Q30 value, and GC content of the four samples including two parents and two gene mixing-pools, were 48.57×, 94.36% and 39.39%, respectively. Through the digestion of restriction endonuclease, a total of 175 468 SLAF tags were obtained, of which 25 675 SLAF tags were polymorphic, accounting for 14.63% of the total SLAF tags. According to the genotype and sequencing information of the two parents, 1 774 SLAF tags were determined for a further association analysis. Based on the SNP-index method, 18 SLAF tags containing 25 candidate SNP loci that related closely with salt tolerance were identified. Each three SNP loci were discovered in Marker 116156 and Marker 88668 tags, and each two SNP loci were found in Marker 118929, Marker 108616 and Marker 68843 tags, respectively. Six sequences retrieved proteins with the highest sequence similarity in the Nt database, and four or three sequences were assigned with the NOG and KEGG databases, respectively. Only one sequence had a hit in Swiss-Prot database. Base substitutions among ten SNP markers were detected between salt-tolerance and salt-sensitivity individuals, which was consistent with the original sequence of SLAF-seq. 【Conclusion】 The target trait could be rapidly and effectively located and identified using the bulked segregant analysis combined with the SLAF-seq method in this study, which could provide a theoretical basis and technical support for the development of functional markers and marker-assisted selection in willow.
柳树 / 耐盐 / 连锁分析 / 特定位点参数扩增测定 / 单核苷酸多态性(SNP)
willow / salt tolerance / association analysis / specific-locus amplified frangment sequencing (SLAF-seq) / single nucleotide polymorphism(SNP)
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Willow species (Salix) are important as short‐rotation biomass crops for bioenergy, which creates a demand for faster genetic improvement and breeding through deployment of molecular marker‐assisted selection (MAS). To find markers associated with important adaptive traits, such as growth and phenology, for use in MAS, we genetically dissected the trait variation of a Salix viminalis (L.) population of 323 accessions. The accessions were sampled throughout northern Europe and were established at two field sites in Pustnäs, Sweden, and at Woburn, UK, offering the opportunity to assess the impact of genotype‐by‐environment interactions (G × E) on trait–marker associations. Field measurements were recorded for growth and phenology traits. The accessions were genotyped using 1536 SNP markers developed from phenology candidate genes and from genes previously observed to be differentially expressed in contrasting environments. Association mapping between 1233 of these SNPs and the measured traits was performed taking into account population structure and threshold selection bias. At a false discovery rate (FDR) of 0.2, 29 SNPs were associated with bud burst, leaf senescence, number of shoots or shoot diameter. The percentage of accession variation () explained by these associations ranged from 0.3% to 4.4%, suggesting that the studied traits are controlled by many loci of limited individual impact. Despite this, a SNP in the EARLY FLOWERING 3 gene was repeatedly associated (FDR < 0.2) with bud burst. The rare homozygous genotype exhibited 0.4–1.0 lower bud burst scores than the other genotype classes on a five‐grade scale. Consequently, this marker could be promising for use in MAS and the gene deserves further study. Otherwise, associations were less consistent across sites, likely due to their small estimates and to considerable G × E interactions indicated by multivariate association analyses and modest trait accession correlations across sites (0.32–0.61).
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Willows (Salix spp.) are important biomass crops due to their ability to grow rapidly with low fertilizer inputs and ease of cultivation in short‐rotation coppice cycles. They are relatively undomesticated and highly diverse, but functional testing to identify useful allelic variation is time‐consuming in trees and transformation is not yet possible in willow. Arabidopsis is heralded as a model plant from which knowledge can be transferred to advance the improvement of less tractable species. Here, knowledge and methodologies from Arabidopsis were successfully used to identify a gene influencing stem number in coppiced willows, a complex trait of key biological and industrial relevance. The strigolactone‐related More AXillary growth (MAX) genes were considered candidates due to their role in shoot branching. We previously demonstrated that willow and Arabidopsis show similar response to strigolactone and that transformation rescue of Arabidopsis max mutants with willow genes could be used to detect allelic differences. Here, this approach was used to screen 45 SxMAX1, SxMAX2, SxMAX3 and SxMAX4 alleles cloned from 15 parents of 11 mapping populations varying in shoot‐branching traits. Single‐nucleotide polymorphism (SNP) frequencies were locus dependent, ranging from 29.2 to 74.3 polymorphic sites per kb. SxMAX alleles were 98%–99% conserved at the amino acid level, but different protein products varying in their ability to rescue Arabidopsis max mutants were identified. One poor rescuing allele, SxMAX4D, segregated in a willow mapping population where its presence was associated with increased shoot resprouting after coppicing and colocated with a QTL for this trait.
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Biological assay has been based on analysis of all individuals collected from sample populations. Bulked sample analysis (BSA), which works with selected and pooled individuals, has been extensively used in gene mapping through bulked segregant analysis with biparental populations, mapping by sequencing with major gene mutants and pooled genomewide association study using extreme variants. Compared to conventional entire population analysis, BSA significantly reduces the scale and cost by simplifying the procedure. The bulks can be built by selection of extremes or representative samples from any populations and all types of segregants and variants that represent wide ranges of phenotypic variation for the target trait. Methods and procedures for sampling, bulking and multiplexing are described. The samples can be analysed using individual markers, microarrays and high‐throughput sequencing at all levels of DNA, RNA and protein. The power of BSA is affected by population size, selection of extreme individuals, sequencing strategies, genetic architecture of the trait and marker density. BSA will facilitate plant breeding through development of diagnostic and constitutive markers, agronomic genomics, marker‐assisted selection and selective phenotyping. Applications of BSA in genetics, genomics and crop improvement are discussed with their future perspectives.
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为研究甜瓜蔓枯病（GSB）抗病基因在甜瓜染色体上的定位情况，对甜瓜蔓枯病抗病亲本P181、感病亲本P01及其F<sub>2</sub>遗传分离群体进行蔓枯病抗性鉴定，根据F<sub>2</sub>蔓枯病发病情况，选择2个亲本及F<sub>2</sub>中极端抗病和极端感病的子代各30个，提取DNA并分别构建抗病和感病基因混池，利用SLAF-seq和BSA技术进行蔓枯病抗病基因QTL定位分析。测序共开发得到188 022个SLAF标签，分析过滤掉低质量的SNP位点后，获得5 676个高质量的SNP标记。采用SNP-index关联分析方法，在甜瓜染色体上获得2个与GSB抗性紧密关联的候选区域，一个定位于Chr01上的9 837 447~11 028 838 bp区间，区间大小为1.19 Mb；另一个定位于Chr04上的33 135 224~33 993 540 bp区间，区间大小为0.86 Mb。共有218个基因定位在关联区域内，依据基因功能注释分析，推测可能与甜瓜蔓枯病抗病相关的候选基因有15个，包括NBS-LRR基因1个，E3泛素蛋白连接酶合成基因5个，RPM1相关蛋白基因1个，锌指蛋白相关基因1个，NAC结构域蛋白基因1个，钙依赖蛋白激酶2个，凝集素相关受体蛋白基因3个，RING-H2 finger蛋白基因1个。研究结果将为甜瓜蔓枯病的抗性基因研究提供参考。
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Next-generation DNA sequencing (NGS) projects, such as the 1000 Genomes Project, are already revolutionizing our understanding of genetic variation among individuals. However, the massive data sets generated by NGS—the 1000 Genome pilot alone includes nearly five terabases—make writing feature-rich, efficient, and robust analysis tools difficult for even computationally sophisticated individuals. Indeed, many professionals are limited in the scope and the ease with which they can answer scientific questions by the complexity of accessing and manipulating the data produced by these machines. Here, we discuss our Genome Analysis Toolkit (GATK), a structured programming framework designed to ease the development of efficient and robust analysis tools for next-generation DNA sequencers using the functional programming philosophy of MapReduce. The GATK provides a small but rich set of data access patterns that encompass the majority of analysis tool needs. Separating specific analysis calculations from common data management infrastructure enables us to optimize the GATK framework for correctness, stability, and CPU and memory efficiency and to enable distributed and shared memory parallelization. We highlight the capabilities of the GATK by describing the implementation and application of robust, scale-tolerant tools like coverage calculators and single nucleotide polymorphism (SNP) calling. We conclude that the GATK programming framework enables developers and analysts to quickly and easily write efficient and robust NGS tools, many of which have already been incorporated into large-scale sequencing projects like the 1000 Genomes Project and The Cancer Genome Atlas.
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The majority of agronomic traits are controlled by multiple genes that cause minor phenotypic effects, making the identification of these genes difficult. Here we introduce MutMap, a method based on whole-genome resequencing of pooled DNA from a segregating population of plants that show a useful phenotype. In MutMap, a mutant is crossed directly to the original wild-type line and then selfed, allowing unequivocal segregation in second filial generation (F(2)) progeny of subtle phenotypic differences. This approach is particularly amenable to crop species because it minimizes the number of genetic crosses (n = 1 or 0) and mutant F(2) progeny that are required. We applied MutMap to seven mutants of a Japanese elite rice cultivar and identified the unique genomic positions most probable to harbor mutations causing pale green leaves and semidwarfism, an agronomically relevant trait. These results show that MutMap can accelerate the genetic improvement of rice and other crop plants.
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Global climate change imposes increasing impacts on our environments and crop production. To decipher environmental impacts on crop plants, the concept "envirotyping" is proposed, as a third "typing" technology, complementing with genotyping and phenotyping. Environmental factors can be collected through multiple environmental trials, geographic and soil information systems, measurement of soil and canopy properties, and evaluation of companion organisms. Envirotyping contributes to crop modeling and phenotype prediction through its functional components, including genotype-by-environment interaction (GEI), genes responsive to environmental signals, biotic and abiotic stresses, and integrative phenotyping. Envirotyping, driven by information and support systems, has a wide range of applications, including environmental characterization, GEI analysis, phenotype prediction, near-iso-environment construction, agronomic genomics, precision agriculture and breeding, and development of a four-dimensional profile of crop science involving genotype (G), phenotype (P), envirotype (E) and time (T) (developmental stage). In the future, envirotyping needs to zoom into specific experimental plots and individual plants, along with the development of high-throughput and precision envirotyping platforms, to integrate genotypic, phenotypic and envirotypic information for establishing a high-efficient precision breeding and sustainable crop production system based on deciphered environmental impacts.
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Association mapping through linkage disequilibrium (LD) analysis is a powerful tool for the dissection of complex agronomic traits and for the identification of alleles that can contribute to the enhancement of a target trait. With the developments of high throughput genotyping techniques and advanced statistical approaches as well as the assembling and characterization of multiple association mapping panels, maize has become the model crop for association analysis. In this paper, we summarize progress in maize association mapping and the impacts of genetic diversity, rate of LD decay, population size, and population structure. We also review the use of candidate genes and gene‐based markers in maize association mapping studies that has generated particularly promising results. In addition, we examine recent developments in genome‐wide genotyping techniques that promise to improve the power of association mapping and significantly refine our understanding of the genetic architecture of complex quantitative traits. The new challenges and opportunities associated with genome‐wide analysis studies are discussed. In conclusion, we review the current and future impacts of association mapping on maize improvement along with the potential benefits for poor people in developing countries who are dependent on this crop for their food security and livelihoods.
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从‘嘎啦’苹果中克隆了己糖激酶基因MdHXK1（基因序列号：MDP0000309677）全长，测序发现其包含长为1 497 bp完整的开放阅读框，编码499个氨基酸，预测其编码蛋白质分子量为54.05 kD，等电点为5.76。系统进化树分析表明，HXK1在不同物种间具有高度的序列保守性；其中，苹果MdHXK1与中国白梨PbHXK1亲缘关系最近。功能域分析表明，MdHXK1蛋白含有两个保守的激酶域。Southern blotting结果表明，MdHXK1在苹果基因组中有一个拷贝。亚细胞定位预测表明，MdHXK1主要定位于细胞质。分析MdHXK1基因启动子序列发现含有与抗逆、糖信号及激素信号相关的顺式作用元件。荧光定量PCR分析表明，MdHXK1在苹果的茎和花中表达量较高；在苹果果实发育期间，MdHXK1基因的表达、MdHXK1葡萄糖磷酸化相对酶活性和葡萄糖积累水平都呈现先升高后降低的趋势，MdHXK1基因的表达明显受盐、低温和ABA的诱导。原核诱导并纯化了MdHXK1蛋白，为后续MdHXK1蛋白的功能鉴定奠定基础。
A hexokinase gene named MdHXK1（MDP0000309677）was cloned from‘Gala’apple （Malus × domestica Borkh.）. Sequence analysis showed that the length of MdHXK1 gene was 1 497 bp，which encoded 499 amino acids. It was predicted that the molecular mass of this protein was 54.05 kD，and pI was 5.76. A phylogenetic tree indicated that the HXK1 had a higher sequence conservation among different species，while apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that MdHXK1 protein was mainly localized in cytoplasm. There was an indication that MdHXK1 existed as one copy in apple genome by Southernblotting. In silico analysis suggested that the promoter sequence contained several typical cis-acting elements，including defense responsive elements，sugar signaling responsive elements and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower. During the development of apple fruits，the expression of MdHXK1 gene increased at first and then decreased. The changes on Glc phosphorylation activity（relative）and glucose concentration showed the same trend. Besides，the expression of this gene was induced by salt stress，low temperature and ABA. Finally，we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression，which established the foundation for the further study on the functions of MdHXK1 protein.
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