InDel primer pair for identifying parthenocarpy QTL (Quantitative Trait Loci) located on No.9 chromosome of eggplant and application of InDel primer pair
By developing the InDel primer pair InDel15 on eggplant chromosome 9, PCR amplification was used to identify parthenocarpic materials, solving the problem of difficult identification in existing technologies, achieving rapid and accurate breeding identification, and saving costs and time.
Patent Information
- Application Number
- CN202511214060.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-22
- Publication Date
- 2025-11-25
AI Technical Summary
Existing technologies make it difficult to quickly and accurately identify parthenocarpic materials in eggplant breeding, resulting in high consumption of human and material resources and long breeding cycles.
The InDel primer pair InDel15, located on eggplant chromosome 9, was developed. Parthenocarpic materials, including non-parthenocarpic, homozygous parthenocarpic, and heterozygous parthenocarpic materials, were identified by detecting the size of PCR product fragments through PCR amplification.
It enables rapid and accurate identification of parthenocarpic materials, saving breeding costs and time and improving breeding efficiency.
Smart Images
Figure CN121006415A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to an Indel primer pair for identifying parthenocarpic QTLs located on chromosome 9 of eggplant and its application, belonging to the field of eggplant molecular breeding technology. Background Technology
[0002] Eggplant is a warm-season vegetable crop. Under greenhouse cultivation conditions and in early spring open-field cultivation, low temperatures and weak light during the flowering period often lead to poor pollination and fertilization, resulting in flower and fruit drop and reduced yield. In production, plant growth regulators such as 2,4-dichlorophenoxyacetic acid (2,4-D) are commonly used to treat the plants. However, improper use of this technique can cause phytotoxicity and induce deformed fruit, thus reducing the marketability of the fruit; it also increases labor input and production costs. Parthenocarpy refers to the phenomenon where the ovary develops directly into fruit after flowering without pollination or other stimulation. Parthenocarpy can overcome the flower and fruit drop obstacles caused by low temperatures, enhance fruit setting ability, significantly increase yield, and also improve fruit quality and reduce cultivation costs. Therefore, the selection and utilization of eggplant materials with parthenocarpy capability has important practical significance in eggplant production.
[0003] Currently, the selection of eggplant materials with parthenocarpy mainly relies on the stigma removal method during the bud stage. This involves manually removing the stigma 1-2 days before flowering to prevent pollination, and then observing the fruit development during the fruit-setting period to determine the parthenocarpy ability. This method is easily affected by the external environment and is time-consuming and labor-intensive. Molecular marker-assisted selection technology plays an irreplaceable role in modern crop breeding. The core of this technology lies in using DNA molecular markers closely linked to the target trait to perform early selection and evaluation of breeding materials at the genomic level. Using molecular markers associated with parthenocarpy, seedlings and even seeds can be identified quickly and accurately, greatly saving manpower and resources, shortening the breeding cycle, and accelerating the breeding process. Therefore, it is urgent to develop molecular markers with high specificity and reproducibility, and to use primer pairs that can identify molecular markers to develop the identification of parthenocarpy in eggplant from phenotypic identification to genotypic identification, which is of great significance for breeding new eggplant varieties with parthenocarpy ability. Summary of the Invention
[0004] One objective of this invention is to provide an InDel primer for identifying parthenocarpic QTLs located on chromosome 9 of eggplant. The primer is InDell5, with the upstream primer sequence shown in SEQ ID NO.1 and the downstream primer sequence shown in SEQ ID NO.2.
[0005] The second objective of this invention is to provide the application of the above primer pairs in the genetic improvement of parthenocarpic eggplant.
[0006] The application involves using InDel15 as a template, with eggplant DNA as a template, to perform PCR amplification and obtain PCR products. If the size of the amplified PCR product fragment is 124 bp, it indicates that the detected eggplant material is a non-parthenocarpic material; if the size of the amplified PCR product fragment is 104 bp, it indicates that the detected eggplant material is a homozygous parthenocarpic material; if the size of the amplified PCR product fragment is both 124 bp and 104 bp, it indicates that the detected eggplant material is a heterozygous parthenocarpic material.
[0007] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0008] This invention provides an Indel primer pair for identifying parthenocarpic QTLs located on chromosome 9 of eggplant. The upstream and downstream sequences of the primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. PCR amplification using these primers yields a PCR product fragment of 124 bp, indicating that the tested eggplant material is non-parthenocarpic; a PCR product fragment of 104 bp indicates that the tested eggplant material is homozygous parthenocarpic; and PCR product fragments of both 124 bp and 104 bp indicate that the tested eggplant material is heterozygous parthenocarpic. Therefore, in breeding, this primer pair can be used to identify the parthenocarpic ability of eggplant materials, allowing for the early elimination of non-parthenocarpic plants, which can significantly save land and subsequent management costs, and improve breeding efficiency. Attached Figure Description
[0009] Figure 1 Distribution of ΔINDEL-index across the genome and individual chromosomes in the two offspring.
[0010] Figure 2 The amplification diagram of the primer pairs in 24 randomly selected F2 single plants in this invention.
[0011] Figure 3 The amplification results of the primer pairs in 24 randomly selected EP74-derived materials in this invention are shown in the figure. Detailed Implementation
[0012] The present invention will be further described below with reference to specific embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.
[0013] Example 1: Screening for InDel molecular markers linked to parthenocarpy.
[0014] The test materials were the eggplant parthenocarpic inbred line EP74 and the non-parthenocarpic inbred line EP26, independently bred by the Eggplant Innovation Team of the Vegetable Research Institute of Jiangsu Academy of Agricultural Sciences, and the F2 segregating population constructed using them as parents. EP74 was a green-calyx, purple-long eggplant, and EP26 was a purple-calyx, purple-long eggplant. Flower buds were collected 1-2 days before flowering, and the stigma was manually removed with tweezers before attaching a date tag. During fruit development, the ovary was observed to swell three times. If the ovary swelled and the fruit length was greater than 20 cm, the plant was considered parthenocarpic.
[0015] Thirty strong parthenocarpy and 30 non-parthenocarpy plants were selected from the F2 population to construct strong parthenocarpy pools and non-parthenocarpy pools, respectively. High-throughput sequencing was performed on both parents, the strong parthenocarpy pools, and the non-parthenocarpy pools using the Illumina HiSeq sequencing platform. Using the eggplant HQ-1315 genome as a reference genome, 108,590 InDel loci were obtained through variant detection. Based on the genotyping results, homozygous InDel loci in the parents and markers with polymorphism between parents were selected. Using one parent as a reference, the InDels in the two mixed pools were analyzed, and the InDel-index (i.e., the frequency of InDels) of the marker loci in the two offspring was calculated, and Δ(InDel-index) was analyzed. Based on the association threshold, candidate regions closely associated with parthenocarpy were obtained on chromosome 9, with physical locations on the chromosome ranging from 87.36 to 89.64 Mb. Figure 1 ).
[0016] InDel variant sites were extracted from the candidate regions mentioned above, and InDel markers were developed for the 87.36-89.64 Mb range and surrounding areas of chromosome 9. The InDel marker design principle was that the base difference between the parents should be greater than or equal to 20 bp, and the amplified fragment size should be 100-200 bp to facilitate detection using high-concentration agarose gel electrophoresis. Polymorphic primers were screened using the parents and their F1 strains. The markers were categorized based on the banding patterns observed in the parents. Banding patterns from the non-parthenocarpy parent EP26 were denoted as 'a', banding patterns from the parthenocarpy parent EP74 as 'b', heterozygous banding as 'h', and unclear or missing data as '-'. The results showed that 19 pairs exhibited good polymorphism between the parents and F1 strains.
[0017] DNA was extracted from individual plants of the F2 population using magnetic beads, and PCR amplification was performed using the 19 pairs of polymorphic primers obtained above. PCR amplification conditions were: 95℃ pre-denaturation for 3 min, 30 cycles of 95℃ denaturation for 15 s, 60℃ annealing for 15 s, and 72℃ extension for 20 s, followed by a final extension at 72℃ for 5 min. The reaction mixture consisted of: 10 μl of 2×Taq mix, 1 μl each of F and R, 1 μl of DNA template, and 7 μl of ddH2O. PCR products were separated using a 1.8% agarose gel electrophoresis, and banding was observed to obtain the genotypes of individual plants in the F2 population. Based on phenotypic data and the genotypes of the exchanged plants, the parthenocarpy gene was located within the region 88973510-89112943 on eggplant chromosome 9. Combining the one-to-one correspondence between phenotypic data and genotypes, a molecular marker linked to parthenocarpy in eggplant was further obtained, and the primer pair for detecting this marker was InDel15.
[0018] The nucleotide sequences of the InDel15 primers are as follows: InDel15-F: AAGTTTGGCTGCTTCTTTC, InDel 15-R: AAAAGAAAGTCTCCACGAG.
[0019] Example 2: Application of the InDel15 primer pair provided by the present invention in identifying parthenocarpy in eggplant
[0020] Twenty-four F2 single plants and twenty-four EP74-derived materials were randomly selected, and DNA was extracted from these 48 materials using the magnetic bead method. PCR amplification was performed using the InDel15 primer pair and the PCR method described in Example 1.
[0021] like Figure 2 As shown, if the size of the PCR product fragment obtained by amplification is 124bp, it indicates that the eggplant material being tested is a non-parthenocarpy material; if the size of the PCR product fragment obtained by amplification is 104bp, it indicates that the eggplant material being tested is a homozygous parthenocarpy material; if the size of the PCR product fragment obtained by amplification is both 124bp and 104bp, it indicates that the eggplant material being tested is a heterozygous parthenocarpy material.
[0022] The 48 materials were subjected to parthenocarpic identification using the artificial stigma removal method described in Example 1. Fruit set was investigated, and it was found that materials yielding a 124 bp band using the primer pair were indeed non-parthenocarpic, while materials yielding a 104 bp band using the primer pair were indeed parthenocarpic. Therefore, the primer pair provided by this invention for identifying parthenocarpic QTLs located on eggplant chromosome 9 can accurately detect parthenocarpic characteristics in eggplant, enabling marker-assisted selection for improving parthenocarpic progeny.
[0023] The above description is only a specific implementation example of the present invention, but any changes or modifications made with reference to the present invention are covered within the scope of the present invention.
Claims
1. An InDel primer pair for identifying parthenocarpic QTLs located on chromosome 9 of eggplant, characterized in that, The primer pair is the InDel15 primer pair, the upstream primer of the InDel15 primer pair is the nucleotide sequence shown in SEQ ID NO.1, and the downstream primer of the InDel primer is the nucleotide sequence shown in SEQ ID NO.
2.
2. The application of the primer pair according to claim 1 in detecting parthenocarpy in eggplant.
3. The application according to claim 2, characterized in that, Using InDel15, with eggplant DNA as a template, PCR amplification was performed to obtain PCR products. If the PCR product fragment size obtained by amplification is 124bp, it indicates that the eggplant material being tested is a non-parthenocarpy material. If the PCR product fragment size obtained by amplification is 104bp, it indicates that the eggplant material being tested is a homozygous parthenocarpic material. If the PCR product fragments obtained by amplification are 124bp and 104bp in size, it indicates that the eggplant material being tested is a heterozygous parthenocarpic material.