Indel molecular marker for detecting cold tolerance of eggplant and application thereof

Abstract

The present application relates to a kind of Indel molecular markers for detecting eggplant cold tolerance.The present application finds the candidate gene significantly related to eggplant cold tolerance by GWAS analysis, and develops 17bp Indel specific molecular markers by PCR amplification to the sequence of candidate gene.The 17 bases of the mark are GATTATATTTACTATCA at the 125-141 position of amplification product in cold-sensitive material, while the 17 bases are missing at the 125th position in cold-resistant material.The marker can be used for molecular assisted breeding selection of eggplant cold tolerance.

Description

Technical Field

[0001] The invention relates to an Indel molecular marker for detecting cold tolerance of eggplant and application thereof, belonging to the technical field of eggplant molecular breeding. Background Art

[0002] Eggplant (Solanum melongena L.) is an important vegetable crop in the Solanaceae family, native to tropical Southeast Asia and a thermophilic crop. The optimum temperature for eggplant seed germination is 25-30°C. Temperatures below 15°C slow growth and cause flower drop, while temperatures below 10°C disrupt the plant's metabolism. Consequently, chilling injury can negatively impact all stages of eggplant growth, including seed germination, seedling growth, and flowering and fruiting. This can severely threaten eggplant yield and quality, hindering the sustainable development of winter and spring eggplant production. However, current research on eggplant cold tolerance is limited to identifying cold tolerance in germplasm resources and the effects of cold stress on eggplant physiological parameters, providing limited guidance for genetic improvement of cold tolerance in eggplant.

[0003] With the rapid development of modern molecular biology technology, a variety of new molecular breeding technologies have emerged, including molecular marker-assisted selection. Molecular marker-assisted breeding can provide new ideas for the creation and selection of new varieties. Molecular marker-assisted selection has the following advantages: (1) It can overcome the difficulty of phenotypic identification of traits such as fertility restoration and photothermosensitive sterility; (2) It can overcome the difficulty of genotypic identification of traits when there are few breeding materials and repeated identification is not allowed or there is a certain risk; (3) For important traits such as yield and resistance to diseases that only occur in the late growth period, molecular markers can be used to detect seedlings and even seeds, greatly saving the manpower and material resources wasted in plant cultivation; (4) Molecular marker-assisted selection is a non-destructive trait evaluation and selection; (5) Molecular marker-assisted selection allows more groups to be included in the selection objects, thereby applying a stronger selection pressure on them, and multiple traits can be selected at the same time. The use of molecular marker-assisted selection combined with conventional breeding has become an inevitable choice for crop breeding. Moreover, these advantages are particularly important in the identification of eggplant cold tolerance. Therefore, it is urgent to develop molecular markers with strong specificity and good reproducibility to develop the identification of eggplant cold tolerance from phenotypic identification to genotypic identification, which is of great significance for the breeding of new cold-tolerant eggplant varieties. Summary of the Invention

[0004] One of the purposes of the present invention is to provide an Indel molecular marker for detecting cold tolerance of eggplant, wherein the Indel site is obtained by PCR amplification using eggplant genomic DNA as a template using a primer pair. The sequences of the primer pair are shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0005] The Indel molecular marker is characterized in that the primer pair amplifies the 17 bases GATTATATTTACTATCA at positions 125-141 in the cold-sensitive material, while the amplified product in the cold-resistant material lacks these 17 bases starting from position 125.

[0006] A second object of the present invention is to provide an application of an Indel molecular marker for detecting cold tolerance of eggplant in cold tolerance breeding of eggplant.

[0007] The application uses a primer pair to amplify the indel site obtained by PCR using eggplant genomic DNA as a template. In cold-sensitive materials, the amplified product contains the 17 bases GATTATATTTACTATCA at positions 125-141, while in cold-tolerant materials, the amplified product lacks these 17 bases starting at position 125. The sequences of the primer pair are shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0008] In summary, the beneficial effects of the present invention are as follows:

[0009] The present invention discovered a candidate gene significantly associated with eggplant cold tolerance through GWAS analysis, and developed a 17bp Indel-specific molecular marker by PCR amplification of the candidate gene sequence. The primer pair sequence of the molecular marker is shown in SEQ ID NO.1 and SEQ ID NO.2. The molecular marker can be used to detect the cold tolerance of eggplant and is used for molecular marker-assisted selection of eggplant cold tolerance breeding, providing a new molecular marker for eggplant cold tolerance breeding. In breeding, the marker can be used to identify the cold tolerance of eggplant materials and eliminate cold-sensitive plants early, which can greatly save land and subsequent management costs and improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Expression of candidate associated genes (FPKM) in cold-tolerant and cold-sensitive materials.

[0011] Figure 2 Sequence information of Indel in cold-resistant and cold-sensitive materials.

[0012] Figure 3 Detection of indel markers in the F2 segregating population from the cross between parents A80 and A153. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0014] Example

[0015] 1. Determination of molecular markers for cold tolerance in eggplant

[0016] In this example, 144 representative eggplant materials from major sources such as USDA, AVRDC, national germplasm resource banks, as well as some local varieties and advanced inbred lines were used to form an association population.

[0017] Phenotypic identification

[0018] When eggplant seedlings reached the four-leaf, one-heart stage, they were transferred to a 4°C growth chamber for cold stress. The chilling injury index (CHI) of the treated plants was calculated according to a previously established grading standard for seedling cold stress. The chilling injury index grading scale is as follows: Level 0, no symptoms; Level 1: less than one-third of one leaf exhibits symptoms; Level 2: less than one-third of two to three leaves exhibit symptoms; Level 3: more than one-half of two to three leaves exhibit symptoms; Level 4: only the growing point is normal; Level 5: the entire plant is dead.

[0019] 1.2. Association analysis of cold tolerance in eggplant

[0020] DNA was extracted from 144 eggplant germplasm samples, and each sample's genomic DNA was digested with restriction endonucleases RsaI and HaeIII. The resulting digested fragments (SLAF tags) were 3'-end amplified, ligated with dual-index sequencing adapters, amplified by PCR, purified, pooled, and gel-cut to select target fragments. After quality control, the libraries were sequenced using Illumina HiSeq. SNPs were developed using GATK and samtools. The intersection of the SNP markers generated by the two methods was used as the final reliable SNP marker dataset, resulting in a total of 1,853,626 population SNPs. After filtering based on completeness > 0.5 and MAF > 0.05, 130,387 highly concordant population SNPs were obtained for subsequent analysis. A genome-wide association study of cold tolerance in eggplant was conducted using a mixed linear model (MLM) based on population structure (Q) and kinship (K). A total of 322 SNPs with potentially significant associations with cold tolerance were detected.

[0021] 1.3. Obtaining candidate markers

[0022] Based on the results of genome-wide association analysis, genes within the 100 kb region upstream and downstream of the SNP site at position 137124654 on chromosome 7 were selected as candidate associated genes. Combined with transcriptome analysis, it was found that the candidate associated genes SMEL_007g292440.1 (VQ10), SMEL_007g292400.1 (NCED1), SMEL_007g292480.1 (GA2OX1) and SMEL_007g292490.1 (GA2OX1) can be induced by cold stress ( Figure 1 ). The results of sequence amplification of the coding region and promoter region showed that the coding region sequences of these four genes in different materials were the same, while the promoter sequence of SMEL_007g292490.1.01 had multiple Indels and SNPs. Analysis of these Indels and SNPs found that the Indel 1000bp upstream of the ATG of the SMEL_007g292490.1.01 gene in the cold-resistant material caused the deletion of two TATA boxes ( Figure 2 Based on the sequence information of this interval, primers F: 5'-AGCGTTAACTTTGCCGACAT-3' and R: 5'-TTCACGTCTGTTTATATTTTATACGAA-3' were designed to develop an InDel molecular marker closely linked to cold tolerance.

[0023] Genomic DNA was extracted from the cold-resistant material 'A80' and the cold-sensitive material 'A153'. PCR amplification was performed using the designed InDel primers using the genomic DNA as a template to obtain PCR amplification products. The PCR reaction conditions were: 95°C pre-denaturation for 3 minutes, 30 cycles of 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, and 72°C extension for 20 seconds, followed by a complete extension at 72°C for 5 minutes. The reaction system was: 2×Taq mix 10μl, 1μl each of F and R, 1μl DNA template, and 7μl of ddH2O. The amplified product lengths of the two materials were significantly different. The cold-resistant material amplified a 282bp fragment, while the cold-sensitive material amplified a 299bp fragment ( Figure 3 ), the accuracy of the target sequence was verified and could be further developed into genetic markers for selection of cold tolerance traits in eggplant.

[0024] An F2 population was constructed using the cold-tolerant eggplant material 'A80' and the cold-sensitive eggplant material 'A153'. F2 individual plants were randomly selected and PCR amplified using the genomic DNA of these individual plants as templates using the designed Indel primers. The banding patterns were then counted. Amplification showed that the marker was polymorphic in the F2 individual plants ( Figure 3 Statistical analysis of the phenotypic coincidence rate revealed that the correlation between the genotype and phenotype detected by this SNP marker reached a significant level, indicating that this SNP marker can be well used in the screening of cold-tolerant germplasm.

[0025] The above description is only a specific implementation example of the patent of the present invention, but any changes or modifications made by referring to the patent application of the present invention are included in the patent scope of the present invention.

Claims

1. An indel molecular marker closely related to chilling tolerance in eggplant. The molecular marker is an indel site obtained by PCR amplification using a primer pair with eggplant genomic DNA as a template. The primer pair sequences are shown in SEQ ID NOs. 1 and 2. When the primer pair amplifies a product in a chilling-sensitive material, the 17 bases GATTATATTTACTATCA are present at positions 125-141. However, when the primer pair amplifies a product in a chilling-tolerant material, the 17 bases are missing from position 125 onwards.

2. Use of the Indel molecular marker closely related to eggplant cold tolerance according to claim 1 in eggplant cold tolerance breeding.

3. The use of the Indel molecular marker closely related to cold tolerance of eggplant in cold tolerance breeding of eggplant according to claim 2, characterized in that: Using a primer pair, the Indel site obtained by PCR amplification with eggplant genomic DNA as a template, the 17 bases GATTATATTTACTATCA at positions 125-141 of the amplified product in the cold-sensitive material are missing these 17 bases from position 125, while the amplified product in the cold-resistant material lacks these 17 bases starting from position 125. The sequences of the primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2.

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