STS molecular marker closely linked with mature character of apple fruit and application of STS molecular marker

Abstract

The invention discloses an STS molecular marker closely linked with mature characters of apple fruits and application of the STS molecular marker, and relates to the technical field of fruit tree molecular marker-assisted selective breeding. In early-maturing apples, the InDel variation site and the flanking nucleotide sequence are shown as SEQ ID NO.1, and in late-maturing apples, the InDel variation site and the flanking nucleotide sequence are shown as SEQ ID NO.2. A primer of the STS molecular marker is designed according to an InDel variation site flanking sequence and is used for marker-assisted selection in breeding, and the morning and evening of the mature period of apples can be predicted in the early stage of seedlings; the primer sequences of the molecular marker are as shown in SEQ ID NO. 3 and SEQ ID NO. 4. (1) InDel sequence variation identified in the invention and used for developing the STS marker is a key reason for causing difference in the mature period of the apple fruits; (2) the STS molecular marker can be used for predicting the morning and evening of the ripening period of the apple fruits; (3) the STS molecular marker found by the invention can guide crossbreeding of apples, screening can be carried out in advance, the breeding time is saved, and the breeding efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular marker-assisted selection breeding of fruit trees, and particularly relates to an STS molecular marker closely linked to the apple fruit maturity trait and its application. Background Art

[0002] Apple is one of the important economic fruit trees in the world and is widely planted around the world. Rich in various nutrients such as soluble sugars, organic acids, vitamins, minerals, polyphenols and flavonoids, apples are deeply loved by consumers. As one of the five major apple gene centers in the world, China has a long cultivation history. As early as more than 2,000 years ago, "Ting, Nai, Magnolia officinalis" were recorded in Sima Xiangru's "Ode to the Upper Forest", where "Nai" refers to Mian apple. Thanks to the superior geographical conditions and climate environment, the apple planting area and output in China have been expanding year by year. The rapid development of the apple industry plays an important role in increasing farmers' income and promoting the development of the agricultural economy. However, at the same time, the apple industry also faces many problems: firstly, the variety structure is single and unreasonable, mainly dominated by late-maturing varieties of Fuji series (accounting for nearly 70%), while the proportions of early-maturing and mid-maturing varieties are small; secondly, the fruit quality of different maturity periods is uneven and the competitiveness is weak. These problems have greatly restricted the healthy and sustainable development of the apple industry. Therefore, studying the regulatory mechanism related to apple fruit maturity has important theoretical value and practical significance.

[0003] Fruit maturity is an important agronomic trait that determines the market supply cycle and commercial value of fruits. It covers a series of complex biological processes from fruit setting to swelling and直至成熟等一系列复杂生物学过程,是一个受遗传因素和环境因素共同影响的复杂数量性状。果树中关于成熟期的遗传定位也开展了广泛的研究,许多具有显著效应的数量性状位点(QTL)也被成功鉴定。然而,果实成熟期的具体遗传调控机制仍未完全明晰。相比之下,果实成熟的调控机制已经得到了较为深入的研究。成熟过程中,果肉的颜色、风味、香气和质地发生显著变化,这些变化主要由植物激素调控,包括乙烯、脱落酸(ABA)和生长素。按照成熟过程中是否出现呼吸跃变现象可将果实分为呼吸跃变型果实和非呼吸跃变型果实两种类型,前者在成熟期间表现出乙烯释放和呼吸速率的峰值,而后者则无此特征。脱落酸是非呼吸跃变型果实成熟的关键调节因子,同时它与乙烯的互作也参与了两类果实的成熟过程。此外,生长素通过与乙烯或ABA的相互作用,分别参与调控呼吸跃变型和非呼吸跃变型果实的成熟。

[0004] Transcription factors play an important role in the fruit ripening process. In tomatoes, RIPENING INHIBITOR (RIN) and NON-RIPENING (NOR) are two key transcription factors related to ripening. RIN belongs to the SEPALLATA MADS-box gene family and regulates multiple aspects of fruit development and ripening. It can directly activate the transcription of ripening-related genes and work synergistically with other regulatory factors such as TAGL1 and FUL1 / 2. NOR regulates fruit ripening through ethylene and ABA signaling pathways. Studies have shown that homologous genes of NOR also have similar regulatory effects in a variety of fruit trees (such as strawberries, bananas and melons). Not only that, in peach, two adjacent NAC transcription factors PpNAC1 and PpNAC5 have been shown to regulate fruit ripening, a finding consistent with the results of studies that ethylene is involved in vegetative and reproductive growth in tomatoes. In addition, the importance of epigenetic regulation in fruit ripening has also been verified.

[0005] Apple (Malus×domestica Borkh.) is a typical respiratory climacteric fleshy fruit, and its maturity period has attracted much attention because it is positively correlated with fruit hardness. Fruit hardness plays a decisive role in the storage and shelf life of apples. In a linkage mapping study based on a biparental population, researchers found multiple QTLs related to maturity period. At the same time, genome-wide association analysis (GWAS) based on unrelated individuals revealed a significant effect QTL on chromosome 3, in which one NAC transcription factor MdNAC18.1 was considered to be a key candidate gene for maturity period. Although the MdNAC18.1 locus contains many DNA polymorphisms, its specific causal variation still needs to be further analyzed.

[0006] In recent years, with the increasing popularity of high-throughput sequencing technology, high-density genetic variation maps have greatly made up for the defects of genetic linkage maps constructed earlier. Various important agronomic traits have been identified in many species using GWAS. Therefore, we used natural apple populations to construct a phenotypic database of apple maturity, used high-throughput sequencing technology to detect whole-genome variations, and conducted whole-genome association studies on apples. We found an InDel variation that was significantly associated with the apple maturity phenotype. We used the STS markers developed from the polymorphic sequence of this site to screen seedlings of apple resources and predict the early or late maturity of the fruit, which will help achieve the goal of efficient molecular-assisted breeding. Summary of the invention

[0007] The purpose of the present invention is to provide an STS molecular marker closely linked to apple fruit maturity traits and its application. Specifically:

[0008] 1. Combining genetics and molecular biology techniques, we identified an InDel mutation site that is closely linked to the apple fruit maturity trait;

[0009] 2. Based on the DNA variation type of the InDel variation site closely linked to the apple fruit maturity trait in apple resource varieties, an STS molecular marker was developed for use in the seedling stage to predict the early or late fruit maturity.

[0010] The object of the present invention is achieved in that:

[0011] By using the genetic variation map of the apple resource population and the phenotypic database of fruit maturity traits, we conducted genome-wide association analysis, located the genetic loci of apple fruit maturity traits, identified InDel variations significantly associated with maturity traits within the candidate interval, and finally developed molecular markers that can be used to identify the early maturity of apple seedlings, forming the technical solution of this invention. The identification of the early maturity of apple plants can greatly reduce the waste of land and labor costs in the early stage of breeding, and has important theoretical significance and high economic value.

[0012] STS molecular markers tightly linked to apple fruit ripening traits

[0013] The InDel mutation site and flanking nucleotide sequence in early-maturing apples are shown in SEQ ID NO.1, and the InDel mutation site and flanking nucleotide sequence in late-maturing apples are shown in SEQ ID NO.2.

[0014] Application of STS molecular markers tightly linked to apple fruit maturity traits

[0015] The primers of STS molecular markers are designed based on the flanking sequences of InDel mutation sites and used for marker-assisted selection in breeding. The early or late maturity of apples can be predicted at the early seedling stage.

[0016] The primer sequences of the molecular markers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0017] Specifically:

[0018] 1. Identification of InDel mutation sites closely linked to apple fruit ripening period

[0019] Through the identification of fruit maturity traits of more than 400 apple resource populations, a phenotypic database of maturity traits of apple resource populations was constructed. The whole genome variation map of more than 400 apple resource populations was constructed using the second-generation sequencing technology of the Illumina platform. A QTL interval on chromosome 3 significantly associated with maturity traits was located using genome-wide association analysis (GWAS). Figure 1), and further genotyping of different significantly associated variant sites was performed. An InDel variant site was found in the promoter region of the MdNAC18.1 gene within the candidate interval. The genotyping results showed that when there was a 58bp deletion fragment at this site, the corresponding samples were mainly early-maturing varieties, and when there was a 58bp insertion fragment at this site, the corresponding samples were mainly late-maturing varieties. This indicates that the variant site is co-segregated with the maturity stage of apple fruit.

[0020] 2. Development of STS molecular markers closely linked to apple fruit maturity traits

[0021] Based on the flanking sequences of InDels that are closely linked to apple fruit maturity traits, STS molecular markers for identifying maturity traits of apple hybrid populations were developed. The nucleotide sequence of the specific fragment amplified in early-maturing apples is shown in SEQ ID NO.1, and the nucleotide sequence of the specific fragment amplified in late-maturing apples is shown in SEQ ID NO.2; the primer sequences of the STS molecular markers are shown in SEQ.ID No3 and SEQ.ID No4.

[0022] The genomic DNA of the tested individuals was amplified by PCR using the primers of the above-mentioned STS molecular markers, and then tested by agarose gel electrophoresis. It was found that the tested individuals could be divided into the following three types:

[0023] 1) The PCR amplification product was a single band of 225 bp, and the fruit maturity was early;

[0024] 2) Individuals whose PCR amplification product was a single band of 283 bp had late fruit maturity;

[0025] 3) For individuals whose PCR amplification products contained two bands of 225 bp and 283 bp, the fruit maturity period was between early and late.

[0026] Therefore, the molecular marker composed of this pair of primers can be used for molecular marker-assisted selection of fruit maturity traits of apple hybrid offspring.

[0027] Compared with the existing methods, the present invention has the following advantages and positive effects:

[0028] ① The InDel sequence variation identified for developing STS markers is the key reason for the difference in apple fruit ripening period;

[0029] ②STS molecular markers can be used to predict the early or late maturity of apple fruit;

[0030] ③The STS molecular markers found can guide apple hybrid breeding, enable early screening, save breeding time and improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a genome-wide association analysis map of apple fruit ripening;

[0032] The results showed that genome-wide association analysis revealed that a candidate interval significantly associated with apple fruit maturity traits was located at the end of chromosome 3.

[0033] Figure 2 It is the InDel variation site and its flanking DNA sequence that are significantly associated with the ripening period of apple fruit;

[0034] Sequence comparison revealed that, in the apple resource population, early-maturing apple resources all had a 58bp fragment deletion at this InDel mutation site that was significantly associated with the ripening period of apple fruit, while late-maturing apple resources all contained this 58bp DNA fragment insertion.

[0035] Figure 3 It is the DNA structure diagram of the flanking sequence of the STS molecular marker site and the agarose gel electrophoresis diagram of the PCR product of the apple resource population amplified by the primers based on the STS molecular marker;

[0036] A: A schematic diagram showing the DNA structure of the flanking sequence of the STS molecular marker site closely linked to the ripening period of apple fruit. The dotted line represents the InDel variable site significantly associated with the ripening period of apple fruit. F1 and R1 represent the specific primers SEQ.ID No3 and SEQ.ID No4 of the developed STS molecular marker, respectively.

[0037] B: Agarose gel electrophoresis of the products of PCR amplification using primers SEQ.ID No3 and SEQ.ID No4 in the apple resource population based on STS molecular markers. The single 225bp band corresponds to early-maturing apples, the single 283bp band corresponds to late-maturing apples, and the sample containing both bands corresponds to mid-maturing apples.

[0038] Figure 4 It is the phenotypic trait that indicates the maturity period of apple fruit corresponding to individuals with different genotypes;

[0039] The results showed that among the three types of genotype individuals in apple resources, the '++' genotype represents that the InDel variation site significantly associated with the apple fruit maturity period contains a 58bp insertion fragment on the alleles of both chromosomes. This type of apple resource is late-ripening apples. The '+-' genotype represents that the InDel variation site significantly associated with the apple fruit maturity period contains a 58bp insertion fragment on the alleles of both chromosomes, and the other chromosome does not have this insertion fragment. This type of apple resource is medium-ripening apples. The '--' genotype represents that the InDel variation site significantly associated with the apple fruit maturity period does not contain a 58bp insertion fragment on the alleles of both chromosomes. This type of apple resource is early-ripening apples. DETAILED DESCRIPTION

[0040] The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments. The following is described in detail with reference to the accompanying drawings and embodiments:

[0041] Example 1: Acquisition of STS molecular markers

[0042] 1) Construction of apple fruit maturity phenotype database

[0043] When the size of apple fruit no longer changes, the background color of the peel disappears, and the seed color changes from light green to brown, combined with the previously recorded maturity date, we identified the maturity traits of 500 apple resource populations and constructed a fruit maturity phenotype database of different resources. The results showed that there were significant differences in the maturity phenotypic traits among individuals in the resource population. Combined with previous studies, we divided apples into early-maturing varieties according to their maturity period, that is, the maturity period is within ≤100 days after flowering; mid-maturing varieties, that is, the maturity period is within 100-150 days after flowering; late-maturing varieties, that is, the maturity period is ≥150 days after flowering.

[0044] 2) Construction of genotype database of apple resource population

[0045] Based on the genome data construction and resequencing analysis of 500 natural apple resource populations, GATK software was used to detect population SNP variation, and preliminary screening was performed using the minimum allele frequency (MAF) of 0.01. In the cultivated apple and wild apple populations, 17,720,553 and 23,856,685 high-quality SNP variation sites were identified, respectively, with an average of one SNP variation per 28bp and 38bp in the genome. This high-density apple genome-wide variation map provides an important basis for the construction of a genotype database.

[0046] 3) Identification of genetic variation loci significantly associated with apple fruit ripening

[0047] Based on the previously constructed apple resource population maturity phenotype database and genotype database, a genome-wide association analysis was performed using a mixed linear model (MLM), and a candidate interval significantly associated with the maturity of apple fruit was detected in the 27.37-30.73Mb interval of chromosome 3. Further combined with the candidate gene association analysis, a 58bp InDel was identified in the promoter region of the MdNAC18.1 gene, which co-segregated with the early and late maturity of apple fruit.

[0048] Example 2: Application of STS molecular markers

[0049] 100 mg of young apple leaves were used for apple genomic DNA extraction. The detailed DNA extraction steps were carried out according to the plant genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. The TaKaRa Ex The kit uses the diluted genomic DNA as a template and a pair of PCR amplification primers labeled with STS molecules to perform PCR amplification using apple DNA as a template. The PCR primer sequences are shown in Appendix 1. The 10 μL reaction system for PCR amplification is as follows:

[0050]

[0051] The reaction program was: 95°C, 5 min; 35 cycles of [95°C, 30 s; 60°C, 30 s; 72°C, 3 min]; and finally extension at 72°C for 10 min.

[0052] After the PCR reaction was completed, the products were directly subjected to polymorphism detection using 2.0% agarose gel electrophoresis.

[0053] Table 1 List of primers used for STS molecular marker genotyping

[0054]

Claims

1. An STS molecular marker tightly linked to apple fruit maturity traits, characterized by: The InDel mutation site and flanking nucleotide sequence in early-maturing apples are shown in SEQ ID NO.1, and the InDel mutation site and flanking nucleotide sequence in late-maturing apples are shown in SEQ ID NO.

2.

2. The use of the STS molecular marker tightly linked to the apple fruit maturity trait according to claim 1, characterized in that: The primers of STS molecular markers are designed based on the flanking sequences of InDel mutation sites and used for marker-assisted selection in breeding. The early or late maturity of apples can be predicted at the early seedling stage. The primer sequences of the molecular markers are shown in SEQ ID NO.3 and SEQ ID NO.4.

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