水稻基因组育种芯片及其应用

水稻基因组育种芯片是将大规模基因组测序、水稻功能基因组研究的最新成果与国际最先进的SNP(single nucleotide polymorphism)芯片技术相结合,构建的服务于育种的基因组技术工具。实践表明,水稻基因组育种芯片在种质资源多样性分析、基因鉴定、基因定位、育种材料基因型选择、品种基因指纹检测等方面可发挥重要作用。水稻基因组育种芯片的应用将推动水稻育种的技术革命。     

EMS诱变技术在植物育种中的研究进展

甲基磺酸乙酯（Ethyl methane sulfonate,EMS）是一种常用的化学诱变剂,能诱发产生高密度的系列等位基因点突变。在当前种质资源极为匮乏,基因资源日益枯竭的状况下,采用EMS诱发突变技术创造有用基因资源具有极其重要的意义。本文通过对EMS的诱变原理和技术要领、应用实例、以及该技术在现代分子生物学中的应用前景加以阐述,对EMS诱变技术在农业生产中的应用具有重要作用。     

SSR和SNP标记在荔枝遗传育种中的应用

荔枝育种长期以来主要依赖实生选种和表型选择,遗传改良进展缓慢,主要源于其种质资源遗传背景不清与品种名称混乱、杂种早期鉴定与功能基因发掘等现代高效育种技术欠缺等原因,因此亟待完善荔枝分子标记辅助选择育种技术,克服传统育种技术障碍,为荔枝遗传改良提供新的技术支撑。对近年来荔枝SSR和SNP两类特异性强的分子标记应用于种质亲缘关系研究、核心种质构建、种质精准鉴定与分子条码构建、真假杂种鉴别以及遗传图谱构建方面的进展进行了综述,以期为荔枝特异性分子标记在育种中的应用提供理论及实践参考。     

农业发展对作物功能基因组研究需求

作物生命科学研究对我国农业的可持续发展提供了强大的科技支撑,功能基因组研究是作物生命科学研究的核心领域之一。现通过提出我国农业发展中面临的一些主要问题,基于作物功能基因组学特别是水稻功能基因组学的最新研究成果,分析农业发展对作物功能基因组研究的多种需求,包括种质资源创新、重大应用价值基因的克隆和功能解析、基因编辑技术和全基因组分子育种。为了进一步满足农业发展对基因组学研究的需求,我国需要推进功能基因组研究,促进作物育种行业的转型、发掘更多的优异种质资源、培育重大突破的新品种,提升我国种业创新能力,不断增强我国功能基因组学对农业生物技术的贡献力度。     

分子标记技术在苹果育种中的应用

DNA分子标记是现代分子生物学发展出来的一类重要的遗传标记,已广泛应用于遗传图谱的构建、种质资源的管理和鉴定、基因的定位与克隆等。综述了分子标记在苹果育种中的应用。     

泛基因组及其在植物功能基因组学研究中的应用

参考基因组是现代功能基因组学的核心框架,以此为基础的现代基因组学技术在过去20年对植物遗传变异发掘、功能基因克隆等研究起了巨大的推动作用。然而,越来越多的研究发现,单一或少数参考基因组不能完整代表和呈现物种或特定群体内的所有基因组变异,因此其在功能基因组学研究中应用存在很大的局限性,甚至会导致错误的结果。泛基因组是指物种或特定群体内全部基因或基因组序列的总和。泛基因组通过完整捕获和呈现群体内全部的基因或基因组序列,代替单一参考基因组应用于功能基因组学研究,可以突破单一参考基因组的局限性。泛基因组在植物功能基因组学研究中有广泛的应用,以泛基因组为基础,结合最新的基因组学技术可以高效、精准鉴定种质资源中的遗传变异。泛基因组研究是目前植物基因组学研究的前沿和热点。本文综述了泛基因组概念的起源和发展,泛基因组组装的技术和策略,以及泛基因组在植物基因组学研究和分子育种方面的应用和最新进展,最后对植物泛基因组研究目前存在的问题和今后研究方向进行了展望。本综述可为植物泛基因组研究和应用提供参考。     

基于引物扩增受阻突变技术开发水稻耐冷基因CTB4a特异性分子标记

水稻(Oryza sativa)作为热带与亚热带起源的作物对低温敏感.对水稻种质进行耐冷性鉴定,能筛选出耐冷性强的种质,发展耐冷基因分子标记,能够有效鉴别种质中耐冷基因的基因型.本研究使用芽期4℃低温处理10d对41份水稻材料进行芽期耐冷鉴定,对品种的芽期耐冷能力进行评价,获得了参试材料中除了'昆明小白谷'之外的芽期耐冷性最强的品种'南特号'.对已克隆的耐冷基因CTB4a开发分子标记,能够辅助选择水稻的耐冷育种.水稻孕穗期耐冷基因CTB4a来源于'昆明小白谷',能够影响水稻抵抗低温的能力.参照公布的CTB4a序列信息,从中挑选出序列中的作用位点SNP(单核苷酸多态性,single nucleotide polymorphism),结合引物扩增受阻突变技术(Penta-primer amplification refractory mutation system,PARMS),用Primer 6.0设计引物,建立CTB4a基因荧光分子标记GM-CTB4a,使用荧光分子标记GM-CTB4a对41份水稻品种进行鉴定,使用酶标仪在'昆明小白谷'中检测到利用标记扩增产物中包含'昆明小白谷'特异SNP、T碱基引物携带的FAM荧光信号,在另外40份品种的扩增产物中检测到包含作用位点的C碱基引物携带的HEX荧光信号.本研究利用设计的分子标记,鉴定了 41份水稻品种的耐冷性和基因型.比对分析耐冷性和基因型鉴定结果,说明我们开发的分子标记GM-CTB4a特异性较强,具有实际应用价值.研究结果为利用水稻孕穗期耐冷基因CTB4a培育强耐冷水稻品种奠定坚实基础.     

作物诱变育种研究进展

作物新品种的培育能保持粮食稳产增收,利用自然和诱变获得遗传变异是作物育种的基础。随着栽培作物基因库的日益贫乏,诱变育种技术已成为目前种质资源创新和挖掘新基因的一个重要途径。本文综述了植物诱变育种的发展简史,物理诱变、化学诱变的主要类型、特点、异同及其在农业育种上的应用。由于诱变育种存在的主要问题是有益突变频率仍然较低,变异的方向和性质尚难控制,因此提高诱变效率,迅速鉴定和筛选突变体以及探索定向诱变的途径,是今后研究的重要课题。     

水稻苗期抗稻瘟病基因全基因组关联分析

含抗性基因的水稻品种易被病原菌克服,因此为进一步发掘新的稻瘟病抗性基因,利用水稻多样性群体Ⅱ(RDP-Ⅱ)中的470份种质资源,在湖南省桃江县稻瘟病高发区的自然病圃中进行水稻苗期的抗病性鉴定,并通过全基因组关联分析(GWAS)鉴定稻瘟病的抗性相关位点,最终鉴定出25份苗期抗性较好的品种,可作为抗性育种材料。采用混合线性模型(MLM)对700 000个单核苷酸多态性(SNP)基因型和苗期的稻瘟病表型数据进行GWAS研究,在水稻基因组中鉴定了24个抗性关联位点,除4号和7号染色体外,在其余10条染色体上均有分布。在这些关联位点中,5个位点包含已克隆或定位的6个稻瘟病基因,其余19个位点是新的抗性位点。此外,通过对水稻亚群抗性规律分析发现,热带粳稻亚群的平均抗性水平最高,温带粳稻亚群的平均抗性水平最低。这些研究结果为分子辅助抗稻瘟病育种提供了分子标记,也为后续抗稻瘟病基因的克隆提供了基因组定位信息。     

水稻抗虫功能基因组研究进展

水稻是重要的粮食作物,也是功能基因组研究的模式植物,而虫害是影响水稻产量的主要因素之一。现从水稻抗虫种质资源、水稻抗虫基因的定位与克隆、水稻抗虫的分子和生理机制,以及抗虫水稻的培育等方面,介绍水稻抗虫功能基因组研究的进展和重要研究成果。期望水稻与昆虫相互作用的功能基因组研究能促进水稻抗虫新品种的培育,为持续控制水稻害虫提供更有效的手段。     

TILLING - a shortcut in functional genomics

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING.     

TILLING,high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding

Induced mutations have been used effectively for plant improvement. Physical and chemical mutagens induce a high frequency of genome variation. Recently, developed screening methods have allowed the detection of single nucleotide polymorphisms (SNPs) and the identification of traits that are difficult to identify at the molecular level by conventional breeding. With the assistance of reverse genetic techniques, sequence variation information can be linked to traits to investigate gene function. Targeting induced local lesions in genomes (TILLING) is a high-throughput technique to identify single nucleotide mutations in a specific region of a gene of interest with a powerful detection method resulted from chemical-induced mutagenesis. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of genome size and ploidy level. However, TILLING requires laborious and time-consuming steps, and a lack of complete genome sequence information for many crop species has slowed the development of suitable TILLING targets. Another method, high-resolution melting (HRM), which has assisted TILLING in mutation detection, is faster, simpler and less expensive with non-enzymatic screening system. Currently, the sequencing of crop genomes has completely changed our vision and interpretation of genome organization and evolution. Impressive progress in next-generation sequencing (NGS) technologies has paved the way for the detection and exploitation of genetic variation in a given DNA or RNA molecule. This review discusses the applications of TILLING in combination with HRM and NGS technologies for screening of induced mutations and discovering SNPs in mutation breeding programs.     

Discovery of chemically induced mutations in rice by TILLING

Background  

Rice is both a food source for a majority of the world's population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non-transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING (Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method.     

How Can We Use Genomics to Improve Cereals with Rice as a Reference Genome?

Rice serves as a model crop for cereal genomics. The availability of complete genome sequences, together with various genomic resources available for both rice and Arabidopsis, have revolutionized our understanding of the genetic make-up of crop plants. Both macrocolinearity revealed by comparative mapping and microcolinearity revealed by sequence comparisons among the grasses indicate that sequencing and functional analysis of the rice genome will have a significant impact on other cereals in terms of both genomic studies and crop improvement. The availability of mutants, introgression libraries, and advanced transformation techniques make functional genomics in rice and other cereals more manageable than ever before. A wide array of genetic markers, including anchor markers for comparative mapping, SSRs and SNPs are widely used in genetic mapping, germplasm evaluation and marker assisted selection. An integrated database that combines genome information for rice and other cereals is key to the effective utilization of all genomics resources for cereal improvement. To maximize the potential of genomics for plant breeding, experiments must be further miniaturized and costs must be reduced. Many techniques, including targeted gene disruption or allele substitution, insertional mutagenesis, RNA interference and homologous recombination, need to be refined before they can be widely used in functional genomic analysis and plant breeding.     

Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population

Background  

Sorghum [Sorghum bicolor (L.) Moench] is ranked as the fifth most important grain crop and serves as a major food staple and fodder resource for much of the world, especially in arid and semi-arid regions. The recent surge in sorghum research is driven by its tolerance to drought/heat stresses and its strong potential as a bioenergy feedstock. Completion of the sorghum genome sequence has opened new avenues for sorghum functional genomics. However, the availability of genetic resources, specifically mutant lines, is limited. Chemical mutagenesis of sorghum germplasm, followed by screening for mutants altered in important agronomic traits, represents a rapid and effective means of addressing this limitation. Induced mutations in novel genes of interest can be efficiently assessed using the technique known as Targeting Induced Local Lesion IN Genomes (TILLING).     

Progress in TILLING as a tool for functional genomics and improvement of crops

Food security is a global concern and substantial yield increases in crops are required to feed the growing world population. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. Targeting Induced Local Lesions in Genomes（TILLING）, which combines traditional chemical mutagenesis with high‐throughput genome‐wide screening for point mutations in desired genes, offers a powerful way to create novel mutant alleles for both functional genomics and improvement of crops. TILLING is generally applicable to genomes whether small or large, diploid or evenallohexaploid, and shows great potential to address the major challenge of linking sequence information to the function of genes and to modulate key traits for plant breeding. TILLING has been successfully applied in many crop species and recent progress in TILLING is summarized below, especially on the developments in mutation detection technology, application of TILLING in gene functional studies and crop breeding. The potential of TILLING/EcoTILLING for functional genetics and crop improvement is also discussed. Furthermore, a small‐scale forward strategy including backcross and selfing was conducted to release the potential mutant phenotypes masked in M2（or M3） plants.     

A large-scale whole-exome sequencing mutant resource for functional genomics in wheat

Hexaploid wheat (Triticum aestivum), a major staple crop, has a remarkably large genome of ~14.4 Gb (containing 106 913 high-confidence [HC] and 159 840 low-confidence [LC] genes in the Chinese Spring v2.1 reference genome), which poses a major challenge for functional genomics studies. To overcome this hurdle, we performed whole-exome sequencing to generate a nearly saturated wheat mutant database containing 18 025 209 mutations induced by ethyl methanesulfonate (EMS), carbon (C)-ion beams, or γ-ray mutagenesis. This database contains an average of 47.1 mutations per kb in each gene-coding sequence: the potential functional mutations were predicted to cover 96.7% of HC genes and 70.5% of LC genes. Comparative analysis of mutations induced by EMS, γ-rays, or C-ion beam irradiation revealed that γ-ray and C-ion beam mutagenesis induced a more diverse array of variations than EMS, including large-fragment deletions, small insertions/deletions, and various non-synonymous single nucleotide polymorphisms. As a test case, we combined mutation analysis with phenotypic screening and rapidly mapped the candidate gene responsible for the phenotype of a yellow-green leaf mutant to a 2.8-Mb chromosomal region. Furthermore, a proof-of-concept reverse genetics study revealed that mutations in gibberellic acid biosynthesis and signalling genes could be associated with negative impacts on plant height. Finally, we built a publically available database of these mutations with the corresponding germplasm (seed stock) repository to facilitate advanced functional genomics studies in wheat for the broad plant research community.     

A functional genomics resource for Brassica napus: development of an EMS mutagenized population and discovery of FAE1 point mutations by TILLING

Two ethylmethanesulfonate (EMS) mutant populations of the semi-winter rapeseed cv. Ningyou7 were constructed with high mutant load, to provide a TILLING platform for functional genomics in Brassica napus, and for introduction of novel allelic variation in rapeseed breeding. Forward genetic screening of mutants from the M2 populations resulted in identification of a large number of novel phenotypes. Reverse genetic screening focused on the potentially multi-paralogous gene FAE1 (fatty acid elongase1), which controls seed erucic acid synthesis in rapeseed. A B. napus BAC library was screened, and loci in a reference mapping population (TNDH) were mapped to conclude that there are two paralogous copies of FAE1, one on each of the B. napus A and C genomes. A new procedure is demonstrated to identify novel mutations in situations where two or more very similar paralogous gene copies exist in a genome. The procedure involves TILLING of single plants, using existing SNPs as a positive control, and is able to distinguish novel mutations based on primer pairs designed to amplify both FAE1 paralogues simultaneously. The procedure was applied to 1344 M2 plants, with 19 mutations identified, of which three were functionally compromised with reduced seed erucic acid content.     

A whole‐genome SNP array (RICE6K) for genomic breeding in rice

The advances in genotyping technology provide an opportunity to use genomic tools in crop breeding. As compared to field selections performed in conventional breeding programmes, genomics‐based genotype screen can potentially reduce number of breeding cycles and more precisely integrate target genes for particular traits into an ideal genetic background. We developed a whole‐genome single nucleotide polymorphism (SNP) array, RICE6K, based on Infinium technology, using representative SNPs selected from more than four million SNPs identified from resequencing data of more than 500 rice landraces. RICE6K contains 5102 SNP and insertion–deletion (InDel) markers, about 4500 of which were of high quality in the tested rice lines producing highly repeatable results. Forty‐five functional markers that are located inside 28 characterized genes of important traits can be detected using RICE6K. The SNP markers are evenly distributed on the 12 chromosomes of rice with the average density of 12 SNPs per 1 Mb and can provide information for polymorphisms between indica and japonica subspecies as well as varieties within indica and japonica groups. Application tests of RICE6K showed that the array is suitable for rice germplasm fingerprinting, genotyping bulked segregating pools, seed authenticity check and genetic background selection. These results suggest that RICE6K provides an efficient and reliable genotyping tool for rice genomic breeding.     

一种能表现水稻基因组DNA分化特征的重复DNA顺序

用ＤＮＡ 复性动力学方法克隆到一个水稻中度重复顺序。Ｓｏｕｔｈｅｒｎ 杂交、限制性内切酶分析及序列分析资料表明,该重复顺序在水稻基因组中具有串联重复和散布状态两种存在方式。以该ＤＮＡ 片段作探针,用Ｓｏｕｔｈｅｒｎ 杂交方法分析了多种野生稻种和栽培稻品种的基因组分化特征。某些限制性内切酶消化过的水稻ＤＮＡ,其图谱呈现出多达４０ 条以上的杂交带,包括强杂交带和弱杂交带两种类型。重复实验结果证明,强杂交带表现为ＢＢＣＣ染色体组型特异而弱带则在栽培稻各品种间显示出丰富的多态性,表明该重复顺序片段在水稻理论研究和育种实践中可能具有重要意义