WO2023208078A1 - Variation de structure du génome pour réguler la teneur en solides solubles dans les fruits de tomate, produit associé, et application - Google Patents

Variation de structure du génome pour réguler la teneur en solides solubles dans les fruits de tomate, produit associé, et application Download PDF

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WO2023208078A1
WO2023208078A1 PCT/CN2023/091012 CN2023091012W WO2023208078A1 WO 2023208078 A1 WO2023208078 A1 WO 2023208078A1 CN 2023091012 W CN2023091012 W CN 2023091012W WO 2023208078 A1 WO2023208078 A1 WO 2023208078A1
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sequence
tomato
plant
ssc
seq
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Chinese (zh)
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黄三文
周姚
吴坤
张金喆
张智洋
鲍志贵
赵婀姿
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中国农业科学院农业基因组研究所
中国农业科学院深圳农业基因组研究所
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    • A23L21/20—Products from apiculture, e.g. royal jelly or pollen; Substitutes therefor
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Definitions

  • the present invention relates to the technical field of plant molecular breeding, and in particular to the genome structure variation that regulates the soluble solid content of tomato fruits and related products and applications.
  • Tomato as the world's largest vegetable crop, has important economic and nutritional value.
  • Tomato fruit quality as its core and key commodity trait, has an increasing impact on industrial development.
  • Soluble solids content (SSC) is an important indicator for measuring the quality and yield of tomato fruits. It is mainly composed of nutrients such as soluble sugars and organic acids.
  • the level of SSC directly affects the flavor quality and yield of tomatoes (especially processed tomatoes) , and SSC plays an important role in tomato stress resistance.
  • SSC-related QTL have been reported or cloned in tomatoes through classical genetic methods or whole-genome resequencing association analysis, which are located on tomato chromosomes 9 and 11 respectively.
  • Genome structural variations are a type of large-length sequence changes and positional relationship changes on the genome, including long fragment insertions, deletions, duplications of more than 50 bp, chromosomal inversions, translocations, and copy number variations.
  • SSR microsatellite repeats
  • InDel insertion and deletion
  • SNP single nucleotide polymorphism
  • the SVs in the tomato genome discovered in this application can quickly and effectively aggregate or screen beneficial allelic variations in SSC content, and can be used for commercial breeding applications such as tomato quality improvement and Genetic research has very important practical and theoretical significance.
  • the SV-2 is at least one of the following sequences:
  • nucleosides of this application are all derived from the nucleosides of this application. acid sequence and is equivalent to the sequence of the present application.
  • the plant parts are roots, stems, leaves, flowers, fruits, plant cells, pollen or seeds.
  • the present application also provides a molecular marker, comprising a molecular marker for identifying the above-mentioned genome structural variation.
  • the molecular marker used to identify the above-mentioned genomic structural variation is the KASP molecular marker.
  • the types of molecular markers are not limited thereto. All types of molecular markers recognized by those skilled in the art are within the scope of the present invention.
  • the molecular marker used to identify SV-1 is KASP1;
  • the KASP1 amplification primer pair includes the primers shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
  • the molecular marker used to identify SV-2 is KASP2;
  • the amplification primer pair of KASP2 includes the primers shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
  • the two SVs of this application can be further combined with other related SNPs, InDel and other different genetic variation sites to form a complete molecular marker detection panel related to controlling tomato fruit SSC, and the genotype of the molecular marker Panel can be identified.
  • the analysis enables more accurate prediction and effective screening of SSC content in tomato fruits.
  • the target segment genes can also be realized by customizing targeted capture sequencing probes, gene chip probes, or designing multiplex PCR primers. Type of accurate identification and testing, optimization, adjustment or replacement will be implemented according to different project requirements and purposes.
  • This application also provides a hybridization capture probe for capturing the above-mentioned genomic structural variation.
  • This application also provides a primer for amplifying the above-mentioned genomic structural variation.
  • This application also provides the above-mentioned genome structural variation SV, molecular markers, hybridization capture probes or primers. Any application in genotype identification of target segments of tomatoes, prediction of soluble solids content in tomato fruits, or genomic selection breeding.
  • the primer sequences are detailed in the Examples and Sequence Listing.
  • the sequence information and materials of the above KASP marker combinations are useful in assisting the identification of SSC content in tomato fruits, preparing related identification or auxiliary identification products, and tomato molecular breeding.
  • the innovative application of species and germplasm resources should also be within the scope of protection of the present invention.
  • Pan-genome refers to the full name of all genes of a species, genes that are different from individual genomes, including core genes (Core genes, which are genes common to all samples of the species) , non-core genes (Dispensable genes, i.e., genes present in some samples of the species) and individual-specific genes (Strain-specific genes, i.e., genes unique to an individual); Graph genome is relative to the linear reference genome.
  • Structural variation It is a type of chromosomal variation. It is the result of the joint action of internal and external factors. External factors include various rays, chemicals, drastic changes in temperature, etc., and internal factors include disorders of metabolic processes in organisms and aging. wait. The main types are deletions, duplications, inversions, and translocations;
  • KASP is the abbreviation of competitive allele-specific PCR (Kompetitive Allele Specific PCR), which can perform accurate biallelic determination of SNPs and InDels at specific sites in a wide range of genomic DNA samples;
  • HiFi or ONT third-generation genome resequencing of 132 tomato core germplasm resources was combined with second-generation resequencing data of 706 germplasm resources, of which 32 materials were obtained by constructing a HiFi SMRTbell library and sequencing on the PacBio Sequel II platform.
  • 39Gb of high-quality, long-fragment third-generation sequencing data was combined with the ONT data of 46 materials.
  • the genome assembly and annotation were completed according to the process developed by the project team.
  • the tomato pan-graph genome TGG1.1 was constructed and the linear genome was updated.
  • KASP marker PCR amplification reaction system (10 ⁇ L) includes 5 ⁇ L of genomic DNA (50-100 ng), and 0.14 ⁇ L of primer mixed working solution (the preferred primer mixture ratio is 12 ⁇ L of each specific typing primer, 30 ⁇ L of common primer, and 46 ⁇ L of ddH 2 O Mix to prepare a primer working solution, in which the concentration of the specific typing primer and the common primer stock solution is 100 pmol/L.
  • the same detection purpose can also be achieved by using other reasonable primer mixing ratios), LGC company KASP 2 ⁇ Master Mix (Low Rox) 5.0 ⁇ L.
  • KASP 2 ⁇ Master Mix (Low Rox) mainly contains fluorescent probe A, fluorescent probe B, quenching probe A, quenching probe B, high-fidelity Taq enzyme, dNTP, Mg 2+ and low concentration Rox ( As internal reference for fluorescence signal) and other components.
  • KASP1 and KASP2 identified two The fruit SSC values of mainstream commercial varieties whose genotypes at all SV loci are substitution type A or heterozygous genotype H are significantly higher than those of commercial varieties with only a single locus of A or H, and the SSC values of the latter are significantly higher than those of both.
  • Each locus is a commercial product of the reference type R, see Table 3 for details.
  • the DNA extraction method, KASP marker amplification method, conditions and requirements are the same as in Example 2.
  • the test samples included 336 F2 single plants in the above population, three replicates each of two parents and hybrid F1, plus 3 ddH 2 O as NTC blank control, a total of 348 samples.
  • the genetic model chi-square test analysis was performed on the two target SVs sites in the F2 population, and it was confirmed that both sites conformed to the obvious completely independent single-gene genetic segregation model (1:2:1 segregation, ⁇ 2 ⁇ 3.84), the two-locus genotype combination also conforms to the typical genetic segregation model of two completely independent genes ( ⁇ 2 ⁇ 5.99).
  • Table 4 This also proves from the perspective of classical genetics the real existence of the two genomic structural variation sites significantly associated with SSC, and that the two sets of KASP markers successfully transformed and developed above can accurately and efficiently identify the genotype of the target SV site.
  • this application has discovered two tomato fruit SSC-related genome structural variation SVs through pan-graph genome combined with multi-omics methods, enriching the existing tomato fruit quality and yield-related genetic variation types and source sites; and these two The favorable allelic variant genotypes of each SV site contribute a strong cumulative additive effect to tomato fruit SSC in tomato core germplasm resources, mainstream commercial varieties and parental genetic populations. This can be achieved by analyzing the above two genomic structural variation sites. Artificial selection or transformation to achieve different levels of regulation of tomato fruit SSC. Both target SV locus genotypes can be effectively identified by means such as resequencing or molecular markers.
  • This application preferably provides two sets of high-throughput, low-cost KASP markers that can efficiently and accurately identify the genotypes of two target SV sites in different genetic backgrounds, thereby achieving auxiliary selection of tomato fruit SSCs. Therefore, the two tomato fruit SSC-related genome structural variants and the correspondingly developed two sets of high-efficiency KASP markers provided in this application can be directly used for genetic improvement of tomato fruit quality and commercial applications of molecular breeding.

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Abstract

L'invention concerne un gène de variation de structure génomique pour réguler la teneur en solides solubles dans les fruits de tomate, et une application de celui-ci. Le gène de variation de structure génomique est tel que représenté dans SEQ ID NO : 1 et/ou SEQ ID NO : 2. L'invention concerne également un matériau biologique pour l'inactivation artificielle du gène tel que représenté dans SEQ ID NO : 1 et/ou l'insertion du gène tel que représenté dans SEQ ID NO : 2, et un marqueur moléculaire pour identifier la variation de structure génomique.
PCT/CN2023/091012 2022-04-27 2023-04-27 Variation de structure du génome pour réguler la teneur en solides solubles dans les fruits de tomate, produit associé, et application WO2023208078A1 (fr)

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CN202210453401 2022-04-27
CN202210453401.7 2022-04-27
CN202310437293.9A CN116814673A (zh) 2022-04-27 2023-04-20 调控番茄果实可溶性固形物含量的基因组结构变异及相关产品和应用
CN202310437293.9 2023-04-20

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117683938A (zh) * 2024-02-02 2024-03-12 山东永盛农业发展有限公司 与番茄果实宽度紧密连锁的kasp分子标记及其应用
CN117802262A (zh) * 2023-11-27 2024-04-02 广东省农业科学院蔬菜研究所 一种与南瓜可溶性糖性状紧密连锁的snp分子标记及其应用

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US6124528A (en) * 1995-01-15 2000-09-26 Calgene Llc Modification of soluble solids in fruit using sucrose phosphate synthase encoding sequence
US20090205063A1 (en) * 2004-07-14 2009-08-13 Mendel Biotechnology Plant polynucleotides for improved yield and quality
CN104611345A (zh) * 2015-02-05 2015-05-13 中国热带农业科学院热带生物技术研究所 一种改良植物果实品质的基因及其编码产物与应用
CN110129478A (zh) * 2019-05-21 2019-08-16 上海市农业科学院 一种快速鉴定或辅助鉴定番茄可溶性固形物的dCAPS分子标记及应用
CN112522282A (zh) * 2020-12-15 2021-03-19 华中农业大学 一种调控番茄可溶性固形物含量的基因及应用
CN114107322A (zh) * 2021-12-10 2022-03-01 浙江省农业科学院 调控番茄果实可溶性固形物含量的基因的用途

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6124528A (en) * 1995-01-15 2000-09-26 Calgene Llc Modification of soluble solids in fruit using sucrose phosphate synthase encoding sequence
US20090205063A1 (en) * 2004-07-14 2009-08-13 Mendel Biotechnology Plant polynucleotides for improved yield and quality
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