An InDel marker for identifying the presence or absence of tubercles on the surface of Momordica charantia fruits, its detection primers and applications

Abstract

The present invention discloses an InDel marker for identifying the presence of granulomas on the surface of bitter melon fruit, as well as its detection primers and applications. The InDel marker of the present invention is a deletion of a base T between the 82nd bp and the 83rd bp of the nucleotide sequence shown in SEQ ID NO.1; the upstream primer nucleotide sequence of the detection primer is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4. The present invention has developed an InDel molecular marker that is completely co-segregated with the presence of granulomas in bitter melon. This can be used to determine the presence of granulomas in bitter melon based on the results of molecular marker gene typing at the bitter melon seed or seedling stage, assist in the selection of bitter melon tumor types, and improve the breeding efficiency of bitter melon varieties with ideal tumor types.

Description

Technical Field

[0001] The invention belongs to the technical field of vegetable molecular breeding, and particularly relates to an InDel marker for identifying the presence or absence of granulomas on the surface of bitter melon fruit, and a detection primer and application thereof. Background Art

[0002] Bitter melon (Momordica charantia L.) is an annual climbing herb in the genus Momordica of the Cucurbitaceae family. It is native to Africa and is now widely distributed in tropical and subtropical regions of Asia and Africa. The most characteristic trait of bitter melon is its tubercles, the warty bumps on the surface of the fruit. Based on the morphological characteristics of the tubercles, bitter melon can be broadly divided into two types: those with numerous, small tubercles and those without tubercles. Consumers in different regions of my country have varying preferences for bitter melons with different tuber types. Therefore, tubercles are an important appearance quality trait that influences consumer choices and are a key target for breeders in genetic improvement of new bitter melon varieties.

[0003] Currently, breeders rely primarily on traditional field observation and identification methods to select bitter melon varieties for their nodule characteristics. This method, however, suffers from low identification efficiency and long lead times, significantly limiting the efficiency of breeding new bitter melon varieties with desired nodule characteristics. Therefore, the development of molecular markers that co-segregate for bitter melon nodule characteristics, allowing for the use of molecular markers to assist in the selection and breeding of nodule types, could enable rapid early identification of individual nodule types at the seed or seedling stage, significantly enhancing the efficiency and level of genetic improvement of new bitter melon varieties. Summary of the Invention

[0004] The present invention aims to provide an InDel marker for identifying the presence or absence of granulomas on the surface of bitter melon fruit and its detection primers and applications, which can be used for molecular marker-assisted breeding of bitter melon fruit tumor traits.

[0005] The present invention selects the bitter melon material 'K44' with granules and the bitter melon material 'K8-201' without granules as the maternal and paternal parents, respectively, obtains an F2 population with segregated fruit nodule traits through artificial hybridization and self-pollination, selects 50 individual plants with granules and 50 individual plants without granules from the F2 population to construct a gene pool with granules and a gene pool without granules, respectively, and performs whole-genome resequencing together with the parents. By statistically analyzing the SNP-index difference (ΔSNP-index) of the two gene pools, candidate regions significantly associated with the bitter melon fruit nodule trait are identified; then, polymorphic InDel (insertion and deletion) molecular markers that exist between the parents are further developed in the candidate regions. Finally, the polymorphic InDel molecular markers are used to genotype the F2 population and the inbred line population with segregated fruit nodule traits, aiming to obtain InDel molecular markers that co-segregate with the fruit nodule trait, ultimately achieving early and rapid identification of bitter melon fruit nodule traits at the seed or seedling stage, and improving the efficiency of bitter melon fruit nodule selection breeding. The details are as follows:

[0006] Identification of candidate genomic regions significantly associated with bitter melon granuloma

[0007] The nodule-bearing bitter melon material 'K44' and no-nodule oil bitter melon material '8-201' were selected as the maternal and paternal parents, respectively, and an F2 population containing 230 individuals with segregated nodule traits was obtained through artificial hybridization and self-pollination. The nodule types of the 230 F2 individuals were identified, and 50 individuals with and without nodule were selected from the F2 population to construct gene pools with and without nodule, respectively. High-depth (average depth 40.76×) whole-genome resequencing was performed together with the parents. Statistical analysis of the SNP-index difference (ΔSNP-index) between the two gene pools revealed a candidate region on chromosome 4 that was significantly (confidence level = 0.99) associated with the nodule trait in bitter melon. The physical coordinates of the candidate region range from 19431924 to 24253081, with a physical interval length of 4.82Mb.

[0008] 2. Development of InDel Molecular Markers for Co-segregation with Granuloma

[0009] Eight InDel markers were uniformly developed within the 4.82 Mb candidate region. These eight InDel markers were used to genotype 230 F2 plants. One marker (named fw4) was found to completely co-segregate with the presence or absence of the bitter melon seed tumor phenotype in these 230 F2 plants. Further genotyping of the 230 F2 plants was performed using molecular markers fw3 and fw5 on either side of fw4. Plants with recombinant exchange between fw3 and fw5 were screened, resulting in the identification of 14 recombinant plants. Seven new InDel and SNP markers were uniformly developed between fw3 and fw5, and 14 recombinant plants were genotyped using fw3, fw4, fw5, and the seven newly developed markers. One InDel marker (fw3.6) was found to completely co-segregate with the presence or absence of the fruit seed tumor phenotype in all 14 recombinant plants. Finally, 74 bitter melon inbred lines with different nodule types were genotyped using the fw3.6 marker and its primers. The results showed that the fw3.6 genotyping completely corresponded to the presence or absence of nodule phenotype.

[0010] Therefore, the first object of the present invention is to provide an InDel marker for identifying the presence of granulomas on the surface of bitter melon fruit, wherein the InDel marker is a deletion of a base T between the 82nd bp and the 83rd bp of the nucleotide sequence shown in SEQ ID NO.1.

[0011] Preferably, the upstream primer nucleotide sequence of the InDel-labeled detection primer is shown as SEQ ID NO.3, and the downstream primer nucleotide sequence is shown as SEQ ID NO.4.

[0012] The present invention also provides a detection primer for identifying the presence of granulomas on the surface of bitter melon fruit. The upstream primer nucleotide sequence of the detection primer is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4.

[0013] The invention also provides a kit for identifying whether granulomas are present on the surface of bitter melon fruit, which contains the detection primers.

[0014] The present invention also provides the use of the InDel marker and the detection primer in identifying whether granulomas are present on the surface of bitter melon fruit.

[0015] The present invention also provides a method for identifying the presence or absence of granulomas on the surface of bitter melon fruit, comprising the following steps:

[0016] a. Extracting the bitter melon DNA to be identified;

[0017] b using the nucleotide sequence as shown in SEQ ID NO.3 upstream primer, the nucleotide sequence as shown in SEQ ID NO.4 downstream primer, to be identified bitter melon DNA as a template for PCR amplification;

[0018] c. The PCR amplification product was digested with endonuclease Dde I, and the digested product was detected by electrophoresis;

[0019] d. Result determination:

[0020] When the enzyme digestion product showed only a 117 bp characteristic band, the bitter melon was determined to be a granuloma bitter melon type;

[0021] When the enzyme digestion product has only 82bp and 34bp characteristic bands, the bitter melon is determined to be a type of bitter melon without granuloma;

[0022] When the enzyme digestion product contains characteristic bands of 117 bp, 82 bp and 34 bp at the same time (the genotype is heterozygous), the bitter melon is determined to be a granuloma bitter melon type.

[0023] Preferably, the PCR amplification reaction system is: 1 μL of 50 ng / μL DNA template, 5 μL of Taq MasterMix, 3 μL of ddH2O, 0.5 μL of 10 μM upstream primer, and 0.5 μL of 10 μM downstream primer.

[0024] Preferably, the PCR amplification reaction program is 95°C for 3 min; 95°C for 30 s, 54°C for 30 s, 72°C for 1 min, for a total of 34 cycles; 72°C for 5 min.

[0025] Preferably, the Dde I enzyme digestion reaction system is: 2 μL PCR amplification product, 1 μL 10×CutSmart buffer, 5 μL U Dde I, 6.5 μL ddH 2 O; the reaction procedure is 37° C. for 1 hour, 65° C. for 20 minutes, and storage at 4° C.

[0026] This study established a system for developing and applying InDel markers to identify the presence of granulomas in bitter melon. First, a hybrid group analysis method was used to identify a region significantly associated with the presence of granulomas in bitter melon. InDel markers were then developed within this region that completely cosegregated with the presence or absence of granulomas in bitter melon. These markers were then validated in families and natural populations with segregating granuloma presence or absence phenotypes.

[0027] The present invention has the following beneficial effects:

[0028] A mixed group analysis method using widely distributed, numerous, and highly accurate SNP markers was used to rapidly locate regions significantly associated with bitter melon seed nodules. The team developed an InDel molecular marker that completely co-segregates with the presence of bitter melon seed nodules. This allows for the determination of bitter melon seed nodules based on molecular marker genotyping results at the seed or seedling stage. This aids in the selection of bitter melon nodules and improves the efficiency of breeding bitter melon varieties with ideal nodules. This addresses the shortcomings of conventional field observation and identification methods used in the selection of bitter melon fruit nodules, which are cumbersome, time-consuming, and labor-intensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a comparison of the appearance of the fruit of bitter melon 'K44', a female bitter melon with nodules, and 'K8-201', a male bitter melon without nodules. The scale is 5 cm.

[0030] Figure 2 It is a fine positioning map of the presence or absence of characteristics of bitter melon granuloma.

[0031] Figure 3 The results are shown in the genotyping of 74 bitter melon samples using the fw3.6 marker and its primers.

[0032] Figure 4 Schematic diagram of introducing a mismatched base (G) 4 bp downstream of the InDel site (T) to create a restriction enzyme cutting site (Dde I). DETAILED DESCRIPTION

[0033] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0034] Example 1

[0035] (1) Experimental materials and phenotypic identification

[0036] The bitter melon inbred line 'K44' with granules was used as the female parent, and the bitter melon inbred line 'K8-201' without granules was used as the male parent. Figure 1 ) were hybridized to obtain an F1 population, which was then selfed to obtain an F2 population (n = 230) segregating for the nodule trait. Seedlings from the parental, F1, and F2 populations were simultaneously soaked and germinated, then planted in the field with standard fertilizer and water management. Eighteen days after pollination, the nodule phenotype of each bitter melon fruit from each individual plant in the parental, F1, and F2 populations was visually inspected.

[0037] (2) DNA extraction

[0038] (1) Weigh 100 mg of fresh young true leaves, grind them into powder quickly in liquid nitrogen, and transfer them into a 2 mL centrifuge tube.

[0039] (2) Add 700 μL of CTAB extraction buffer preheated at 65°C, mix well, and incubate in a 65°C water bath for 45 min.

[0040] (3) After cooling, add equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1), gently invert several times to mix, and centrifuge at 12000 rpm for 5 minutes.

[0041] (4) Transfer the supernatant to a new 2 mL centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), mix by inversion, and centrifuge at 12000 rpm for 5 min.

[0042] (5) Transfer the supernatant to a new 2 mL centrifuge tube, add 2 / 3 volume of pre-cooled isopropanol, gently invert to mix, and place at 4°C for 30 min.

[0043] (6) Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, wash the precipitate twice with 70% ethanol, and air-dry.

[0044] (7) Dissolve the precipitate in 50 μL of 1×TE, add RNase, and digest in a 37°C incubator for 3 hours to remove RNA. Finally, store the DNA solution in a -20°C refrigerator.

[0045] (III) BSA-seq localization and linkage verification of the presence or absence of bitter melon fruit granule tumor traits

[0046] Fifty individuals with and without granulomas were selected from the 'K44'×'K8-201'F2 population to construct gene pools for granulomas and granulomas, respectively. High-depth (average depth 40.76×) whole-genome resequencing was performed along with the parents. Statistical analysis of the SNP-index difference (ΔSNP-index) between the two gene pools revealed a candidate region on chromosome 4 that was significantly (confidence level = 0.99) associated with the fruit tumor trait in bitter melon. The candidate region had physical coordinates from 19431924 to 24253081, with a physical interval length of 4.82 Mb ( Figure 2 a).

[0047] Based on the genetic variant loci obtained by resequencing the parents, eight primer pairs were developed that were polymorphic between 'K44' and 'K8-201' within the initial BSA-seq mapping interval. These eight polymorphic primer pairs were then used to genotype 230 'K44' × 'K8-201' F2 individuals. The PCR reaction system consisted of 1 μL of DNA template (50 ng / μL), 5 μL of Taq Master Mix, 3 μL of ddH2O, 0.5 μL of the upstream primer (10 μM / L), and 0.5 μL of the downstream primer (10 μM / L). The PCR reaction procedure was denaturation at 95°C for 3 min, followed by 34 cycles of denaturation at 95°C for 30 s, annealing at 54-58°C for 30 s, and extension at 72°C for 1 min, followed by extension at 72°C for 5 min. PCR products were separated by 6% polyacrylamide gel electrophoresis, silver stained, and band patterns were counted to determine sample genotypes. Based on the genotyping results of 8 polymorphic primers, local linkage mapping was performed for the presence or absence of fruit granulation. The results showed that 8 markers were linked to the presence or absence of fruit granulation to varying degrees, among which fw4 was completely co-segregated with the presence or absence of fruit granulation. Based on this, the gene controlling fruit granulation was located between markers fw3 and fw5 ( Figure 2 b).

[0048] (IV) Fine mapping of genes controlling bitter melon fruit granule tumors

[0049] Using markers fw3 and fw5, which are located on both sides of the co-segregating marker fw4 at the initial population level, 230 'K44'×'K8-201'F2 plants were screened for recombinant strains. A total of 14 recombinant plants were identified and transplanted into the field for fruit tumor phenotype identification. At the same time, seven new molecular markers were developed between fw3 and fw5, and genotyping was performed on the 14 recombinant plants. The results showed that the fw3.6 marker completely co-segregated with the fruit tumor phenotype in all 14 recombinants ( Figure 2 c).

[0050] The fw3.6 marker reflects a 1-base InDel variation between the parental lines. The maternal line, 'K44', has a T insertion at physical coordinate 21927645 on chromosome 4 compared to the paternal line, 'K8-201'. The upstream primer sequence for the fw3.6 marker is 5'-TGCATCAAGACTTCTGCCAA-3' (SEQ ID NO. 3), and the downstream primer is 5'-TCTTCCAGGCTTCTGCATTATCAAAAAACACTGA-3' (SEQ ID NO. 4). The nucleotide sequence of the genomic DNA segment amplified by PCR from the paternal line, 'K8-201', is shown in SEQ ID NO. 1, and the nucleotide sequence of the genomic DNA segment amplified by PCR from the maternal line, 'K44', is shown in SEQ ID NO. 2. The InDel marker is a T deletion between bp 82 and bp 83 of the nucleotide sequence shown in SEQ ID NO. 1. In the primer design of the present invention, a mismatch base (G) is introduced 4 bp downstream of the target InDel mutation site (T) to artificially create a Dde I recognition site ( Figure 4 PCR amplification of the parental DNA was performed using the above primers, and the PCR products were then digested with the endonuclease Dde I. The product amplified using the granuloma-bearing maternal 'K44' DNA as a template was not recognized by the endonuclease Dde I due to the insertion of a T in the Dde I recognition site, and therefore could not be digested by Dde I. Electrophoresis revealed only a single 117 bp band. The product amplified using the granuloma-free paternal 'K8-201' DNA as a template was recognized by Dde I and digested by Dde I to yield two bands of 82 bp and 34 bp. The PCR amplification reaction system was as follows: 1 μL DNA (50 ng / μL) template, 5 μL Taq Master Mix, 3 μL ddH2O, 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM). The reaction procedure was 95°C for 3 min, followed by 34 cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 1 min, followed by 72°C for 5 min and storage at 12°C. The Dde I digestion reaction system was as follows: 2 μL PCR amplification product, 1 μL 10× CutSmart buffer, 5 μL U Dde I, and 6.5 μL ddH2O. The reaction procedure was 37°C for 1 h, 65°C for 20 min, and storage at 4°C.

[0051] Example 2

[0052] Tender leaves of 33 bitter melon inbred line materials with granules and 41 bitter melon inbred line materials without granules from different sources were collected at the seedling stage to extract DNA. Genotyping was performed using the fw3.6 molecular marker and fw3.6 marker upstream and downstream primers (the same primer sequences as in Example 1). The specific PCR amplification reaction and enzyme digestion reaction system are shown in Example 1. After the enzyme digestion reaction, polyacrylamide gel electrophoresis was performed, and band statistical analysis was performed:

[0053] (1) If the electrophoresis band of a material is the same size as the parent 'K44' electrophoresis band (117 bp characteristic band), the material is judged to be a granulomatous bitter melon type;

[0054] (2) If the electrophoresis bands of a material are the same size as those of the parent 'K8-201' (characteristic bands of 82 bp and 34 bp), the material is judged to be a type of bitter melon without granulomas;

[0055] (3) If the electrophoresis bands of a material show both the maternal 'K44' (117bp characteristic band) and the paternal 'K8-201' (82bp and 34bp characteristic bands), that is, the genotype is heterozygous, then the material is determined to be a granular tumor bitter melon type. Based on the above judgment rules, by comparing the marker typing results of fw3.6 of 74 bitter melon inbred lines and the fruit tumor phenotype results ( Figure 3 , Table 1), and the accuracy was 100%. Therefore, the developed molecular marker fw3.6 and its primers can be used for early identification of the presence or absence of granulomas in bitter melon fruit at the seedling stage.

[0056] Table 1 Phenotypes of fruit nodules and typing statistics of fw3.6 marker in 74 bitter melon inbred lines

[0057]

[0058] Sequence Listing <110> South China Agricultural University <120> An InDel marker for identifying the presence of granulomas on the surface of bitter melon fruit, its detection primers and applications <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 116 <212> DNA <213> Bitter Melon K8-201 (Momordica charantia L. K8-201) <400> 1 tgcatcaaga cttctgccaa tcattagaat gatgaagaaa accacaaaaa taaaagacag 60 gtgagctacc agagaacatt tctcagtgtt ttttgataat gcagaagcct ggaaga 116 <210> 2 <211> 117 <212> DNA <213> Momordica charantia L. K44 <400> 2 tgcatcaaga cttctgccaa tcattagaat gatgaagaaa accacaaaaa taaaagacag 60 gtgagctacc agagaacatt tcttcagtgt tttttgataa tgcagaagcc tggaaga 117 <2;10> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 tgcatcaaga cttctgccaa 20 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <400> 4 tcttccaggc ttctgcatta tcaaaaaaca ctga 34

Claims

1. A detection primer for identifying the presence or absence of granulomas on the surface of bitter melon fruit, characterized in that: The upstream primer nucleotide sequence of the detection primer is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.

4.

2. A kit for identifying the presence or absence of granulomas on the surface of bitter melon fruit, characterized in that: Contains the detection primer according to claim 1.

3. A method for identifying the presence or absence of granulomas on the surface of bitter melon fruit, characterized in that: The following steps are involved: a. Extraction of bitter melon DNA to be identified; the bitter melon to be identified is a separation generation obtained by hybridization of bitter melon 'K44' and bitter melon 'K8-201' without granuloma as parents; b using the nucleotide sequence as shown in SEQ ID NO.3 upstream primer, the nucleotide sequence as shown in SEQ ID NO.4 downstream primer, to be identified bitter melon DNA as a template for PCR amplification; c. The PCR amplification product was digested with endonuclease Dde I, and the digested product was detected by electrophoresis; d. Result determination: When the enzyme digestion product showed only a 117 bp characteristic band, the bitter melon was determined to be a granuloma bitter melon type; When the enzyme digestion product has only 82bp and 34bp characteristic bands, the bitter melon is determined to be a type of bitter melon without granuloma; When the enzyme digestion product contains characteristic bands of 117 bp, 82 bp and 34 bp at the same time, the bitter melon is determined to be of the granuloma bitter melon type.

4. The method according to claim 3, characterized in that The PCR amplification reaction system is as follows: 1 μL of 50 ng / μL DNA template, 5 μL of Taq Master Mix, 3 μL of ddH2O, 0.5 μL of 10 μM upstream primer, and 0.5 μL of 10 μM downstream primer.

5. The method according to claim 3, characterized in that The PCR amplification reaction program is 95°C for 3 min; 95°C for 30 s, 54°C for 30 s, 72°C for 1 min, for a total of 34 cycles; 72°C for 5 min.

6. The method according to claim 3, characterized in that The Dde I enzyme digestion reaction system is as follows: 2 μL PCR amplification product, 1 μL 10×CutSmart buffer, 5 U Dde I, 6.5 μL ddH2O; the reaction procedure is 37°C for 1 hour, 65°C for 20 minutes, and storage at 4°C.

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