Strengthen the Research of Molecular Biology, Promote the Sustainable Development of Apple Industry
CONG PeiHua, ZHANG CaiXia, HAN XiaoLei, ZHANG LiYiResearch Institute of Pomology, Chinese Academy of Agricultural Sciences/Key Laboratory of Fruit Germplasm Resources Utilization, Ministry of Agriculture/National Apple Breeding Center, Xingcheng 125100, Liaoning责任编辑: 赵伶俐
收稿日期:2019-11-22接受日期:2019-11-29网络出版日期:2019-12-01
| 基金资助: |
Received:2019-11-22Accepted:2019-11-29Online:2019-12-01
作者简介 About authors
丛佩华,congph@163.com
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丛佩华, 张彩霞, 韩晓蕾, 张利义. 加强分子生物学研究,促进苹果产业持续发展[J]. 中国农业科学, 2019, 52(23): 4320-4321 doi:10.3864/j.issn.0578-1752.2019.23.012
CONG PeiHua, ZHANG CaiXia, HAN XiaoLei, ZHANG LiYi.
随着高质量苹果多品种基因组全序列及重测序数据不断涌现,其对苹果乃至蔷薇科作物分子育种产生十分重要的影响[1,2,3,4,5],也昭示苹果分子生物学研究步入后基因组时代。如何基于基因组所提供的信息,发展和利用新的技术手段,如最新的基因编辑技术,在全基因组水平上全面分析基因的功能至关重要[6]。同时,多组学联合发展使生物学研究从对单一研究基因表达模式转向多个基因或多个家族基因系统分析,包括研究基因间的相互关系及其网络关系。然而,由于苹果基因组存在高度杂合、遗传背景复杂、自交不亲合与童期长等特点,使苹果重要性状相关的基因功能验证等分子生物学研究较其他农作物仍然落后[7]。在苹果分子生物学研究上,需要从多个层面解决一些特色如柱状性状、氮素等营养元素利用以及生物胁迫和非生物胁迫等一系列重要生物学问题背后的分子机制,以促进苹果产业可持续发展[8,9,10]。
在后基因组时代,转录因子家族的研究仍然是分子生物学热点,这是由于功能基因调控分子机制的解析离不开转录因子。本栏目以“苹果分子生物学研究”专题的形式刊发4篇与转录因子基因克隆和表达分析的相关文章,其中《苹果LIM基因家族生物信息学及表达分析》一文开展苹果LIM转录因子家族成员的生物信息学及表达分析研究。发现MdLIM8和MdLIM11可能与果锈的形成有关,为进一步从分子水平上研究果锈的调控提供了线索。《苹果乙烯响应因子MdERF72对非生物胁迫的响应分析》一文通过转基因技术获得MdERF72过表达苹果愈伤组织,在对MdERF72的表达量与高盐、低温等非生物胁迫之间的关联分析基础上,探究MdERF72对非生物胁迫的响应,这有助于为苹果砧木的遗传改良提供理论参考。《苹果NLP转录因子基因家族全基因组鉴定及表达模式分析》一文首次对苹果NLP转录因子全基因组成员进行鉴定,并从基因和蛋白水平上系统地检测MdNLPs的组织表达、氮响应过程及非生物胁迫变化情况,这可对提高果树氮肥利用效率与提质增效带来新的思考。《柱状苹果Co基因的筛选与候选基因分析》一文在前期基因定位的基础上,通过转录组在柱状和普通型苹果茎尖中筛选到一个候选转录因子基因MdMYB15,其在柱状苹果中显著上调表达,推测该基因可能与柱状苹果树型形成有关。
另外,CRISPR/Cas9基因组编辑系统突破性的问世,使其必然成为生命科学研究领域的助力器。目前,该系统已成功应用于多种植物基因功能研究和新种质创制[11,12,13,14,15],在分子育种中表现出巨大的前景。其中,驱动U6 snRNA转录的U6启动子常作为CRISPR/Cas9基因编辑载体中驱动sgRNA转录的重要元件。但是,目前还未有苹果内源U6启动子介导的CRISPR/Cas9基因编辑体系。《苹果U6启动子的克隆及功能分析》一文从苹果基因组克隆6条U6启动子,并筛选出一条转录活性高且片段长度较短的U6启动子,将促进CRISPR/Cas9基因编辑体系在苹果育种方面发挥工具的作用。
以上5篇论文围绕苹果产业面临的主要科学和实践问题,从分子水平深入研究苹果响应非生物逆境胁迫的调控机制,为苹果抗逆种质的创制、定向遗传改良和基因编辑提供了理论参考。希望上述论文的发表能够给苹果分子生物学研究奠定更多基础,进一步推动苹果分子育种技术的发展。
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被引期刊影响因子
DOI:10.1038/ng.654URLPMID:20802477 [本文引用: 1]
We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.
DOI:10.1186/s13742-016-0139-0URLPMID:27503335 [本文引用: 1]
Domesticated apple (Malus?×?domestica Borkh) is a popular temperate fruit with high nutrient levels and diverse flavors. In 2012, global apple production accounted for at least one tenth of all harvested fruits. A high-quality apple genome assembly is crucial for the selection and breeding of new cultivars. Currently, a single reference genome is available for apple, assembled from 16.9?×?genome coverage short reads via Sanger and 454 sequencing technologies. Although a useful resource, this assembly covers only ~89?% of the non-repetitive portion of the genome, and has a relatively short (16.7?kb) contig N50 length. These downsides make it difficult to apply this reference in transcriptive or whole-genome re-sequencing analyses.
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Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development.
DOI:10.1038/s41467-019-09518-xURLPMID:30940818 [本文引用: 1]
A complete and accurate genome sequence provides a fundamental tool for functional genomics and DNA-informed breeding. Here, we assemble a high-quality genome (contig N50 of 6.99?Mb) of the apple anther-derived homozygous line HFTH1, including 22 telomere sequences, using a combination of PacBio single-molecule real-time (SMRT) sequencing, chromosome conformation capture (Hi-C) sequencing, and optical mapping. In comparison to the Golden Delicious reference genome, we identify 18,047 deletions, 12,101 insertions and 14 large inversions. We reveal that these extensive genomic variations are largely attributable to activity of transposable elements. Interestingly, we find that a long terminal repeat (LTR) retrotransposon insertion upstream of MdMYB1, a core transcriptional activator of anthocyanin biosynthesis, is associated with red-skinned phenotype. This finding provides insights into the molecular mechanisms underlying red fruit coloration, and highlights the utility of this high-quality genome assembly in deciphering agriculturally important trait in apple.
DOI:10.1038/s41467-017-00336-7URLPMID:28811498 [本文引用: 1]
Human selection has reshaped crop genomes. Here we report an apple genome variation map generated through genome sequencing of 117 diverse accessions. A comprehensive model of apple speciation and domestication along the Silk Road is proposed based on evidence from diverse genomic analyses. Cultivated apples likely originate from Malus sieversii in Kazakhstan, followed by intensive introgressions from M. sylvestris. M. sieversii in Xinjiang of China turns out to be an "ancient" isolated ecotype not directly contributing to apple domestication. We have identified selective sweeps underlying quantitative trait loci/genes of important fruit quality traits including fruit texture and flavor, and provide evidences supporting a model of apple fruit size evolution comprising two major events with one occurring prior to domestication and the other during domestication. This study outlines the genetic basis of apple domestication and evolution, and provides valuable information for facilitating marker-assisted breeding and apple improvement.Apple is one of the most important fruit crops. Here, the authors perform deep genome resequencing of 117 diverse accessions and reveal comprehensive models of apple origin, speciation, domestication, and fruit size evolution as well as candidate genes associated with important agronomic traits.
DOI:10.1038/s41438-019-0141-7URLPMID:30962944 [本文引用: 1]
In 2010, a major scientific milestone was achieved for tree fruit crops: publication of the first draft whole genome sequence (WGS) for apple (Malus domestica). This WGS, v1.0, was valuable as the initial reference for sequence information, fine mapping, gene discovery, variant discovery, and tool development. A new, high quality apple WGS, GDDH13 v1.1, was released in 2017 and now serves as the reference genome for apple. Over the past decade, these apple WGSs have had an enormous impact on our understanding of apple biological functioning, trait physiology and inheritance, leading to practical applications for improving this highly valued crop. Causal gene identities for phenotypes of fundamental and practical interest can today be discovered much more rapidly. Genome-wide polymorphisms at high genetic resolution are screened efficiently over hundreds to thousands of individuals with new insights into genetic relationships and pedigrees. High-density genetic maps are constructed efficiently and quantitative trait loci for valuable traits are readily associated with positional candidate genes and/or converted into diagnostic tests for breeders. We understand the species, geographical, and genomic origins of domesticated apple more precisely, as well as its relationship to wild relatives. The WGS has turbo-charged application of these classical research steps to crop improvement and drives innovative methods to achieve more durable, environmentally sound, productive, and consumer-desirable apple production. This review includes examples of basic and practical breakthroughs and challenges in using the apple WGSs. Recommendations for "what's next" focus on necessary upgrades to the genome sequence data pool, as well as for use of the data, to reach new frontiers in genomics-based scientific understanding of apple.
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Columnar apple is a dwarfed mutant with thick, upright main stems and shortened internodes, and generates short fruit spurs instead of long lateral branches. It is a good resource for high dense planting and high yield production in the modern apple industry. Therefore, to understand its unique growth habit was highly interested for all research groups. The current research achievements are summarized as follows: (1) The growth habits of columnar apple is closely related with its endogenous hormones. The free IAA to total IAA ratio was found higher in the axillary buds of columnar apple trees than in the standard type apple trees. The columnar apple producing high number of spurs is because the higher level of zeatin-like growth substances exists in both apical and lateral shoots. The dwarfed growth phenotype is probably correlated with the lower GA level in the columnar apple trees. (2) The columnar phenotype is controlled by a single dominant allele of the columnar gene, which is clustered with the genes controlling main stem growth, branching habit, leaf feature and fruit quality. The Co gene has been fine-mapped to chromosome 10 within the region of 18.51-19.09 Mb. (3) Five Co candidate genes has been reported till today. As the observation that expression of 91071 in apple and tobacco led to shortened internodes in transgenic plants, 91071 was taken as the most promising Co candidate gene, although more studies are needed to clarify whether the 91071 gene also causes the reduced lateral branches and increased spurs in columnar apple. Since co gene is closed related with both plant hormone metabolism and signal transduction, studies on its biological functions by RNAi and transgenic technologies can not only reveal the molecular mechanism of the unique growth characteristics of columnar apple tree, but also provide the theoretical basis for the molecular breeding of columnar apple with improved quality.
DOI:10.3864/j.issn.0578-1752.2017.22.018URL [本文引用: 1]
Columnar apple is a dwarfed mutant with thick, upright main stems and shortened internodes, and generates short fruit spurs instead of long lateral branches. It is a good resource for high dense planting and high yield production in the modern apple industry. Therefore, to understand its unique growth habit was highly interested for all research groups. The current research achievements are summarized as follows: (1) The growth habits of columnar apple is closely related with its endogenous hormones. The free IAA to total IAA ratio was found higher in the axillary buds of columnar apple trees than in the standard type apple trees. The columnar apple producing high number of spurs is because the higher level of zeatin-like growth substances exists in both apical and lateral shoots. The dwarfed growth phenotype is probably correlated with the lower GA level in the columnar apple trees. (2) The columnar phenotype is controlled by a single dominant allele of the columnar gene, which is clustered with the genes controlling main stem growth, branching habit, leaf feature and fruit quality. The Co gene has been fine-mapped to chromosome 10 within the region of 18.51-19.09 Mb. (3) Five Co candidate genes has been reported till today. As the observation that expression of 91071 in apple and tobacco led to shortened internodes in transgenic plants, 91071 was taken as the most promising Co candidate gene, although more studies are needed to clarify whether the 91071 gene also causes the reduced lateral branches and increased spurs in columnar apple. Since co gene is closed related with both plant hormone metabolism and signal transduction, studies on its biological functions by RNAi and transgenic technologies can not only reveal the molecular mechanism of the unique growth characteristics of columnar apple tree, but also provide the theoretical basis for the molecular breeding of columnar apple with improved quality.
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Nitrate reductase is a key enzyme in nitrogen assimilation, and it catalyzes the nitrate-to-nitrite reduction process in plants. A variety of factors, including nitrate, light, metabolites, phytohormones, low temperature, and drought, modulate the expression levels of nitrate reductase genes as well as nitrate reductase activity, which is consistent with its physiological role. Recently, several transcription factors involved in controlling the expression of nitrate reductase genes have been identified in Arabidopsis. NODULE-INCEPTION-like proteins (NLPs) are transcription factors responsible for nitrate-inducible expression of nitrate reductase genes. Since NLPs also control nitrate-inducible expression of genes encoding nitrate transporter, nitrite transporter, and nitrite reductase, the expression levels of nitrate reduction pathway-associated genes are coordinately modulated by NLPs in response to nitrate. LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factors (LBD37-LBD39) are strong candidates for transcription factors mediating negative feedback regulation in response to increases in the contents of nitrogen-containing metabolites, whereas LONG HYPOCOTYL 5 (HY5) that promotes photomorphogenesis in light may be a transcription factor involved in light-induced expression of a nitrate reductase gene. Furthermore, unidentified transcription factors likely mediate other signals and regulate the expression of nitrate reductase genes. This review presents a summary of our current knowledge of such transcription factors. (C) 2014 Elsevier Ireland Ltd.
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DOI:10.1016/j.ymeth.2017.03.009URLPMID:28315486 [本文引用: 1]
The increasing burden of the world's population on agriculture necessitates the development of more robust crops. As the amount of information from sequenced crop genomes increases, technology can be used to investigate the function of genes in detail and to design improved crops at the molecular level. Recently, an RNA-programmed genome-editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9 has provided a powerful platform to achieve these goals. By combining versatile tools to study and modify plants at different molecular levels, the CRISPR/Cas9 system is paving the way towards a new horizon for basic research and crop development. In this review, the accomplishments, problems and improvements of this technology in plants, including target sequence cleavage, knock-in/gene replacement, transcriptional regulation, epigenetic modification, off-target effects, delivery system and potential applications, will be highlighted.
DOI:10.1007/978-1-4939-6859-6_25URLPMID:28220435 [本文引用: 1]
Multiplex CRISPR-Cas9 nuclease mediated genome editing is an efficient method for disrupting gene function in plants. Use of CRISPR-Cas9 has escalated rapidly in recent years and is expected to become routine practice in molecular biology and related fields of research. Due to the relatively novel and widespread adoption of this technology, first-time users may not have regular access to experienced guidance or technical support from peers or mentors. Here, we offer guidance and technical support in the form of a detailed and tested protocol for simultaneous targeting of three separate loci on the TRANSPARENT TESTA 4 (TT4) gene in Arabidopsis thaliana using multiplex CRISPR-Cas9. Although we target multiple loci on a single gene in Arabidopsis, the same approach can be used to target multiple genes or alleles in other plant species as well. We recommend the use of a molecular toolkit to streamline the process and make recommendations for this type of approach. The protocol starts with an overview of the reagents and covers designing of gRNAs and assembly of components into a final T-DNA delivery molecule through Golden Gate cloning and Multisite Gateway LR recombination.
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