A major quantitative trait locus stably associated with high oil content in peanuts and its application

Abstract

The present invention discloses a major quantitative trait locus stably associated with high oil content in peanuts. The locus is located on peanut chromosome 7, with a marker interval of Chr7_426363 to Chr7_606659. The present invention also includes CAPS molecular markers developed based on the major quantitative trait locus. The present invention provides an effective tool for molecular-assisted breeding of peanut oil content.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular breeding, in particular to a major quantitative trait locus associated with stable high oil content in peanuts and an application thereof. Background Art

[0002] my country is the world's largest peanut producer and consumer. With over 70 million mu (approximately 1.6 million hectares) of peanut planting area and a total output exceeding 18 million tons, China ranks first among domestic oil crops. Approximately 55% of the country's total peanut production is used for oil extraction, resulting in an annual output of 3.2 million tons of peanut oil, making it the second-largest source of domestic vegetable oil. Developing peanut production is one of the most important ways to ensure edible oil safety in my country. The oil content of typical peanut germplasm ranges from 40% to 55%, with some high-oil peanut germplasm reaching over 60%. However, in production, the oil content of peanuts is generally around 50%. Studies have shown that every 1 percentage point increase in peanut oil content can increase economic benefits by 7%, making the development of high-oil peanut varieties a pressing need for the modern peanut industry.

[0003] However, yield and quality traits in almost all crops are complex, with quantitative traits significantly influenced by both genetic and environmental factors (Baring et al. 2013; Wilson et al. 2013; Cui et al. 2020; Chen et al. 2019). For example, Chen et al. (2019) estimated the heritability of several yield-related traits under multiple environments. The heritability of the tested yield-related traits ranged from 0.34 to 0.93, and analysis of variance revealed significant differences between RIL populations, between environments, and in RIL × environment interactions. In some cases, marginal variation in these continuous quantitative traits and between alternating lines can make it difficult to distinguish the best-performing lines in field trials. Therefore, understanding heritability and mode of inheritance can facilitate breeding strategies. Currently, determining oil content requires a difficult process that can only be performed after harvest. In these circumstances, traditional breeding strategies to increase oil content are considered costly, inefficient, and time-consuming. In modern breeding, marker-assisted selection (MAS) allows for rapid selection of traits of interest from germplasm resources at an early stage, without being affected by complex environments. MAS has achieved many successes in breeding elite crops with complex quantitative traits, such as cotton, rice, and soybean (Tian et al. 2021; Hulsekemp et al. 2015; Shi et al. 2021), as well as in animal breeding (Chen et al. 2022). However, the establishment of MAS must be carried out under the conditions where specific, useful genes or QTLs have been successfully mapped.

[0004] To achieve the goals of MAS breeding, breeders have been diligently searching for valuable QTLs. In many contexts around the world, identifying QTLs for peanut oil content and conducting targeted breeding efforts will be crucial components of programs aimed at ensuring the supply of edible oil for human consumption. Unfortunately, despite the recognition of the importance of MAS breeding, research into exploiting genetic resources to increase peanut oil content, particularly the cloning of functional genes, lags significantly behind other oilseed crops such as soybeans. Breeding high-oil peanut varieties using MAS remains challenging due to the lack of relevant, favorable genetic resources. Because artificial selection is regionally specific, after generations of continuous selection by breeders, genotypes within varieties gradually become homogenized, resulting in a narrow genetic base. Utilizing these homogenized genetic resources makes it difficult to create superior varieties. Therefore, enriching peanut genetic diversity, particularly exploiting wild peanut genetic resources, is crucial for future peanut breeding efforts. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a major quantitative trait locus stably associated with high oil content in peanuts and an application thereof.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0007] The major quantitative trait locus stably associated with high oil content in peanut is located on chromosome 7 of peanut, with the marker interval being Chr7_426363-Chr7_606659.

[0008] The CAPS molecular markers developed based on the above-mentioned major quantitative trait loci specifically include 5 pairs of SelectionMarkers, namely SM 1-SM 5.

[0009] The SM 1 includes a primer pair consisting of SEQ ID NO.1 and SEQ ID NO.2 in the nucleotide sequence table.

[0010] The SM 2 includes a primer pair consisting of SEQ ID NO. 3 and SEQ ID NO. 4 in the nucleotide sequence table.

[0011] The SM 3 includes a primer pair consisting of SEQ ID NO. 5 and SEQ ID NO. 6 in the nucleotide sequence table.

[0012] The SM4 comprises a primer pair consisting of SEQ ID NO. 7 and SEQ ID NO. 8 in the nucleotide sequence table.

[0013] The SM 5 comprises a primer pair consisting of SEQ ID NO. 9 and SEQ ID NO. 10 in the nucleotide sequence table.

[0014] A kit for detecting peanut oil content, comprising at least peanut genomic template DNA, dNTPs, and a buffer, is characterized in that the kit particularly comprises primer sequences and / or restriction endonucleases for identifying peanut oil content-related QTL sites.

[0015] The primer sequence is any one of the five pairs of Selection Markers, SM 1 to SM 5.

[0016] The SNP sites are located on the peanut high oil stability main effect quantitative trait locus and are closely linked to it, specifically including 5 SNP sites. Each SNP site can distinguish the genotype of the peanut high oil stability main effect quantitative trait locus alone or in combination; the dCAPS primers corresponding to the 5 SNP sites are SM 1 to SM 5 mentioned above. The beneficial effects of the above technical solution are as follows: the research of the present invention has clarified that the major quantitative trait locus associated with the stable high oil content of peanuts is located on peanut chromosome 7, with the marker interval being Chr7_426363-Chr7_606659, which provides a key target region for genetic research and improvement of the high oil content trait of peanuts. CAPS molecular markers SM1 and SM2 were further developed based on the major quantitative trait locus, providing an effective tool for molecular-assisted breeding of peanut oil content. On this basis, a corresponding detection kit was further constructed, namely a kit for detecting the oil content potential of peanuts, which contains peanut genomic template DNA, dNTPs, a buffer, etc., and in particular, contains primer sequences and / or restriction endonucleases for identifying QTL sites related to peanut oil content. The primer sequences in the kit can be selected to include the front primer shown in SEQ ID NO.1 and / or the back primer shown in SEQ ID NO.2, or the front primer shown in SEQ ID NO.3 and / or the back primer shown in SEQ ID NO.4.

[0017] As shown in the examples below, the genotypes of different peanut plants were distinguished based on the above-mentioned molecular markers. The average oil content of individual plants with the genotype consistent with SW9721 was 51.46, 51.83 and 50.96, respectively, while the average oil content of individual plants with the genotype consistent with ST320, Jihua 201138 and Jihua No. 6 was 49.89, 49.54 and 48.98, respectively, and there was a high degree of statistical difference; that is, after selection with SM primer markers, the average oil content of the offspring can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the phenotypes of SW9721-3 and its parents in Example 1.

[0019] Figure 2This is the genetic linkage map of the RIL population in Example 1.

[0020] Figure 3 Schematic diagram of CAPS marker selection efficiency evaluation in Example 3; in the figure, C1, C2 and C3 represent the cultivated parent ST320, Jihua 201138 and Jihua 6 genotypes, respectively, and SW represents the SW9721 type. DETAILED DESCRIPTION

[0021] The following examples illustrate the present invention in detail. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly purchased from the market.

[0022] In the description of the following embodiments, when used in this specification and the appended claims, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their collections. The term "and / or" used in this specification and the appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations. As used in this specification and the appended claims, the term "if" can be interpreted as "when..." or "once" or "in response to determining" or "in response to detecting" depending on the context. Similarly, the phrase "if it is determined" or "if [described condition or event] is detected" can be interpreted as meaning "once it is determined" or "in response to determining" or "once [described condition or event] is detected" or "in response to detecting [described condition or event]" depending on the context.

[0023] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the sentences "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways. Example 1 Construction of high-density genetic map

[0024] In this example, based on the peanut RIL populations pre-constructed in our previous study under multiple environments (multiple offspring materials were obtained by distant hybridization of conventional peanut cultivated materials and wild peanut germplasm PI210553, among which the obtained SW9721 series, especially SW9721-3 materials, showed high oil content characteristics; see Figure 1 ), based on the analysis of its main quality traits, in preparation for the subsequent exploration of gene loci affecting quality-related traits, the RIL population was genotyped and identified using liquid microarrays. After rigorous screening, a total of 3,290 polymorphic SNP markers were identified. Based on the physical location of the SNP loci on the liquid microarray, these polymorphic markers were anchored to the corresponding 20 peanut chromosomes. The markers were further grouped according to chromosomes, and a high-density genetic linkage map was constructed. The results showed that ( Figure 2 ), the map covers a total length of 4,421.92 cM, with an average length of 221.10 cM per linkage group. Among the 20 linkage groups on chromosomes, chromosome 13 has the longest genetic length, at 350.86 cM, and chromosome 4 has the shortest, at 57.90 cM. Each linkage group has an average of 164.5 polymorphic SNP markers, with chromosome 15 having the highest number of 309 and chromosome 16 having the lowest number of 33. The average distance between adjacent polymorphic markers is 1.34 cM, and the average genetic distance between markers on different chromosomes ranges from 0.90 cM to 4.00 cM.

[0025] Example 2 QTL analysis of quality traits

[0026] In this example, to explore the excellent wild gene resources carried by SW9721-3, QTL detection was performed using ICIM-ADD and confirmed using the R language package "qtl". The test results show (Table 3) that a total of 12 QTL loci were detected for four traits in three environments. The LOD values of these loci ranged from 3.26 to 17.82, and the explained genetic variation ranged from 1.97% to 24.56%. Among them, 7 QTL loci were co-localized using the protein-oil trait, and 6 QTL loci were located using the oleic acid-linoleic acid trait. Among them, qSQ_7 could be stably detected in protein and oil content in three environments, with LOD values ranging from 6.01 to 17.82 and an explained genetic variation of 4.59% to 24.56%. The oil content-enhancing gene originated from SW9721-3, indicating that this QTL locus has important breeding value. The QTL locus qSQ_19 was simultaneously mapped to three traits, oil content, oleic acid, and linoleic acid, under two different environments, indicating that qSQ_19 coordinately regulates multiple quality traits. With the exception of the two loci mentioned above, the remaining 10 QTL loci were detected in only one environment, indicating that their effects are environmentally dependent. The results also revealed that among all these loci, only qSQ_19 was shared between the QTL controlling the protein-oil ratio and the QTL controlling the oleic acid-linoleic acid ratio, indicating that the primary genetic basis of the protein-oil ratio and the oleic acid-linoleic acid ratio are different, consistent with the results of the correlation analysis.

[0027] Table 3. QTL analysis of quality-related traits

[0028]

[0029] Example 3 Development and Utilization of Auxiliary Markers for High Oil Locus qSQ_7

[0030] In this example, considering that qSQ_7 is an environmentally stable major effect QTL, SNP-based CAPS markers were developed based on the results of parental resequencing and named SM 1 (Selection Marker 1) to SM5. In order to verify the availability of the developed CAPS markers, SW9721 was used to prepare hybrid combinations with three cultivated peanut resource materials ST320, Jihua 201138 and Jihua No. 6, and three F2 populations were formed after self-pollination, namely pop1, pop2 and pop3. SM1 and SM2 were used for screening in F2, and 25 individual plants with completely consistent genotypes with ST320, Jihua 201138 and Jihua No. 6 were screened out from the three F2 populations. 25 individual plants with the same genotype as SW9721 were screened out from the remaining three populations. After harvesting these individual plants, the oil content was measured for statistical analysis. The results showed that ( Figure 3), after marker selection, the average oil content of individual plants with the same genotype as SW9721 in pop1, pop2 and pop3 were 51.46, 51.83 and 50.96, respectively, while the average oil content of individual plants with the same genotype as ST320, Jihua 201138 and Jihua 6 were 49.89, 49.54 and 48.98, respectively. That is, after selection with SM primer pairs, the average oil content of the offspring can be significantly increased by 1.57, 2.29 and 1.97 percentage points.

[0031] Example 4 Genotyping method based on the developed tightly linked molecular markers

[0032] The table below lists the sequence information of molecular markers SM 1 to SM5; these molecular markers were developed based on five groups of SNP sites that are closely linked to the genome; the single nucleotide polymorphism variations of the five groups of SNPs are underlined in the table.

[0033]

[0034]

[0035]

[0036] Based on the SNP location and polymorphic variation, 30 bp of sequence upstream and downstream of the genome were extracted. CAPS primers (Neff et al., 2002) were designed using the "dCAPS Finder" program (http: / / helix.wustl.edu / dcaps / dcaps.html) based on the sequence. Using enzyme-recognized sequences generated or eliminated before and after the mutation, a primer was designed above and below the mutation site to amplify the sequence containing the mutation site. Because the amplified products differed between parental materials, enzyme digestion allowed identification of the nucleotide type at the mutation site, thereby determining the gene's origin.

[0037] The loci, primer sequences, endonucleases, and product information used in this experiment are listed in the primer table above. To test whether the designed CAPS marker is polymorphic between the parents, PCR amplification was first performed using each parent as a template. The PCR reaction system and procedure are as follows: PCR reaction system:

[0038]

[0039]

[0040] PCR reaction program: 95°C for 2 min; 95°C for 30 s; 53°C for 30 s; 72°C for 30 s; 72°C for 10 min; 16°C∞; a total of 30 cycles.

[0041] The amplified PCR products were detected by agarose gel electrophoresis, and products with single bands were further subjected to enzyme digestion detection; the endonuclease system used was as follows: (enzyme digestion for 30 minutes at the enzyme's working temperature, followed by conventional band detection on 1% agarose gel electrophoresis)

[0042] 10×NEBBuffer 2.5 μL Restriction enzymes 1.0μL PCR products 10 μL <![CDATA[ddH2O]]> 11.5μL Total 25 μL

[0043] The technical details not elaborated in the above operation steps are routine operations in the field of biomolecular technology.

[0044] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0045] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention, and should all be included in the scope of protection of the present invention.

Claims

1. CAPS molecular marker, characterized by: The molecular markers include CAPS-1 and CAPS-2. The primer pair for identifying CAPS-1 is shown as SEQ ID NO.1 and SEQ ID NO.2, the amplified sequence of this primer pair is shown as SEQ ID NO.11, and the endonuclease is MboII; the primer pair for identifying CAPS-2 is shown as SEQ ID NO.3 and SEQ ID NO.3, the amplified sequence of this primer pair is shown as SEQ ID NO.12, and the endonuclease is MboII.

2. A kit for detecting peanut oil content, comprising at least peanut genomic template DNA, dNTPs, and a buffer, characterized in that: The kit also contains primer sequences and restriction endonucleases for identifying peanut oil content-related QTL sites; the primer sequences are SM 1 and SM 2; SM 1 is a primer pair consisting of SEQ ID NO.1 and SEQ ID NO.2, the sequence of its amplified product is shown in SEQ ID NO.11, and the restriction endonuclease is MboII; SM 2 is a primer pair consisting of SEQ ID NO.3 and SEQ ID NO.4, the sequence of its amplified product is shown in SEQ ID NO.12, and the restriction endonuclease is MboII.

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