Methods and related molecular markers for predicting melon fruit length based on QTL loci interaction

Abstract

The invention discloses a method for predicting melon fruit length based on QTL site interaction and related molecular markers, belongs to the field of molecular genetic breeding technology, specifically relates to melon fruit length main effect QTL sites qFL2.1 and qFL8.1, further provides the interaction mode of different genotypes between two main effect sites to coordinately regulate fruit length, and uses the interaction mode to predict melon fruit length. And it relates to InDel molecular markers closely linked to two main effect sites and their applications, molecular marker A02-InDel is closely linked to QTL site qFL2.1, and can be amplified by primer pairs as shown in SEQ ID NO.1-2; molecular marker A08-InDel is closely linked to QTL site qFL8.1, and can be amplified by primer pairs as shown in SEQ ID NO.3-4. Using the molecular marker combination of the present invention, the interaction mode between different genotypes between sites can be predicted, so that melon materials with different fruit lengths can be accurately selected, with the outstanding characteristics of high selection accuracy, and can effectively improve the breeding efficiency of melon fruit length.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding, and specifically relates to a mode in which two QTL sites coordinately regulate melon fruit length, a fruit length prediction method and related molecular markers. Background Art

[0002] The traits that affect crop quality and yield are usually complex quantitative traits, and the selection of quantitative traits is one of the key and difficult links in breeding. In the traditional breeding process, a large number of phenotypic identifications are required to select individuals that meet the requirements from the population. This selection method is very likely to be affected by inaccurate phenotypic identification, deviations between individual phenotypes and genotypes, environmental factors, and other factors. In addition, the field planting and phenotypic identification process of crops takes a long time and a lot of cost. Many factors lead to the low efficiency and insufficient accuracy of traditional breeding for quantitative traits. For quantitative traits, QTL mapping technology can identify multiple major effect QTL sites related to phenotypes in the population, develop molecular markers that are closely linked or co-segregated with the major effect sites, and use molecular markers to quickly detect sites that are closely linked to the target traits during the breeding process. Ma Jian et al. used the long-fruited melon variety "B8" and the oblate-fruited melon variety "HP22" as materials to genetically locate the melon fruit shape. They found a QTL controlling the length of melon on chromosome 8 and developed a molecular marker FS based on the location of the QTL interval and the sequence differences between the parents. Generally speaking, there are multiple QTL loci with different effects that affect the phenotype of quantitative traits, and there are generally interactions between different QTL loci. This interaction relationship manifests as an enhancement or reduction effect. They jointly affect the phenotype of quantitative traits, resulting in complex variations in the expression of traits, which greatly reduces the effectiveness of molecular markers of single QTLs in breeding, thereby interfering with the accuracy of molecular marker-assisted selection. Therefore, on the basis of QTL positioning and the development of linked molecular markers, the interaction relationship between different major effect QTL loci is revealed, and molecular markers closely linked to the major effect loci are used in combination according to the interaction effects between loci, so that complex quantitative traits can be predicted more accurately.

[0003] High-throughput sequencing technology can quickly develop a large number of molecular markers in the whole genome. It is an important means to construct a high-density genetic map, and can efficiently perform genotyping on segregated populations, providing accurate and rapid preliminary preparation for subsequent QTL analysis. QTL analysis is a gene positioning method that performs linkage analysis based on the phenotypic data and molecular marker genotypic data of segregated population samples. Multiple gene loci that control quantitative traits can be located on the genetic map. To analyze the interaction relationship between QTLs, it is usually necessary to construct the corresponding chromosome segment substitution system. The population construction process is very complicated and requires continuous backcrossing and molecular marker selection, which is time-consuming and costly. With the update and development of QTL analysis software, based on high-throughput genotyping, the analysis and verification of QTL interaction relationships in early populations can be realized. The effectplot function in the r / qtl package can analyze the interaction relationship between QTL loci based on the genotypic data and phenotypic data of multiple QTL loci in the segregated population, which can effectively improve the efficiency of interaction verification and improve the accuracy of quantitative trait selection.

[0004] InDel (Insertion-deletion marker) is developed based on the insertion or deletion of a certain number of bases in the whole genome between parents. Forward primers and reverse primers are designed on both sides of the insertion or deletion site, and PCR amplification is performed using this pair of primers. The obtained amplified product is detected by gel electrophoresis, and the polymorphism of the site in different materials is judged based on the length of the product fragment. InDel markers can be used to distinguish between heterozygous and homozygous genotypes. After PCR amplification, no enzyme digestion is required, and gel electrophoresis detection can be performed directly. It is low-cost, easy and quick to operate, and the results are stable.

[0005] Muskmelon (Cucumis melo L.) is an important cash crop in the Cucurbitaceae family. China is the largest producer of melons. The annual output of melons in China is about the sum of the annual output of the rest of the world. It plays an important role in agricultural product exports and agricultural industrialization adjustment. The appearance of melon fruit is rich and diverse, mainly including: round, oval, pear-shaped, elliptical, cylindrical and long fruit. Currently, the popular varieties are mainly round fruit and oval varieties, and there are also long fruit varieties, such as Yangjiaocui, which are increasingly favored by consumers. The cultivated area in North China and Central my country is gradually increasing. Therefore, the cultivation of long fruit varieties has become a new trend in melon breeding. More long fruit varieties with different flavors can enrich market diversity, better meet consumer needs, and have outstanding economic value. The appearance of melon fruit is mainly affected by fruit length, which is a typical quantitative trait. At present, several QTL loci have been identified in the study of melon fruit length, but the effect of the interaction between loci on the phenotype has not been reported. Summary of the invention

[0006] One of the objectives of the present invention is to provide two stable main effect QTL loci of melon fruit length and the effect of their interaction on melon fruit length.

[0007] The second object of the present invention is to provide a molecular marker that is closely linked to the above two QTL loci.

[0008] The third object of the present invention is to provide the application of the above molecular marker combination in molecular breeding of melon fruit length.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The invention provides a main effect QTL locus for melon fruit length, including qFL2.1 and qFL8.1, wherein qFL2.1 is located in the bin130-bin142 interval of the A02 chromosome, and qFL8.1 is located in the bin744-bin760 interval of the A08 chromosome.

[0011] The present invention provides the regulatory effects of the above two major QTL loci on the phenotype in the F2 segregation population and the interaction mode between the loci. The "a" allele comes from the long fruit parent flex09, and the "b" allele comes from the round fruit parent cata05. The specific interaction mode affects the length of melon fruit as follows:

[0012] (1) When the qFL2.1 locus was the "aa" homozygous long-fruit genotype, the fruit length increased by an average of 40% to 70%.

[0013] (2) When the qFL2.1 locus was the "ab" heterozygous genotype, the fruit length increased by an average of 20% to 40%.

[0014] (3) When the qFL8.1 locus was the "aa" homozygous long-fruit genotype, the fruit length increased by an average of 20% to 45%.

[0015] (4) When the qFL8.1 locus was the "ab" heterozygous genotype, the fruit length increased by an average of 5% to 15%.

[0016] (5) When both qFL2.1 and qFL8.1 loci are homozygous long-fruit genotypes of "aa", or when one of the qFL2.1 and qFL8.1 loci is homozygous long-fruit genotypes of "aa" and the other is heterozygous "ab", the two loci produce a significant cumulative effect, and the fruit length is extremely long, increasing by an average of 70% to 100%.

[0017] (6) When both qFL2.1 and qFL8.1 loci were heterozygous for the "ab" genotype, or when one of the qFL2.1 and qFL8.1 loci was homozygous for the long-fruit genotype for the "aa" genotype and the other was homozygous for the round-fruit genotype for the "bb" genotype, there was no obvious additive effect between the two loci, and the fruit length was longer, increasing by an average of 20% to 35%.

[0018] (7) When one of the qFL2.1 and qFL8.1 loci is a "bb" pure and round fruit genotype and the other is a "ab" heterozygous genotype, the pure and round fruit genotype locus is epistatic, the increase in fruit length is inhibited, and the fruit length is shorter, with an average increase of 5% to 20%.

[0019] Preferably:

[0020] (1) When the qFL2.1 locus was the "aa" homozygous long-fruit genotype, the fruit length increased by an average of 13.33±1.20 cm, with an increase percentage of 41% to 65%.

[0021] (2) When the qFL2.1 locus was the "ab" heterozygous genotype, the fruit length increased by an average of 7.16±1.60 cm, with an increase of 23% to 36%.

[0022] (3) When the qFL8.1 locus was the "aa" homozygous long-fruit genotype, the fruit length increased by an average of 9.21±1.71 cm, with an increase percentage of 22% to 44%.

[0023] (4) When the qFL8.1 locus was the "ab" heterozygous genotype, the fruit length increased by an average of 3.98±1.45 cm, with an increase percentage of 9% to 12%.

[0024] (5) When both FL2.1 and FL8.1 were homozygous for long fruit genotypes, or one of qFL2.1 and qFL8.1 was homozygous for long fruit genotypes and the other was heterozygous for ab, the fruit length was extremely long, increasing by an average of 18.50±1.77 cm, with an increase of 78% to 97%. There was an obvious additive effect between the two loci.

[0025] (6) When both qFL2.1 and qFL8.1 loci were heterozygous genotypes of "ab", or one of the qFL2.1 and qFL8.1 loci was homozygous for the long-fruit genotype of "aa" and the other was homozygous for the round-fruit genotype of "bb", the fruit length was longer, with an average increase of 7.05±0.65 cm and an increase of 27% to 31%. There was no obvious additive effect between the two loci.

[0026] (7) When one of the qFL2.1 and qFL8.1 loci was a "bb" pure and round fruit genotype and the other was a "ab" heterozygous genotype, the fruit length only increased by 2±1.1 cm, and the round fruit phenotype was advantageous, indicating that the pure and round fruit genotype loci were epistatic, and the increase in fruit length was inhibited, resulting in a shorter fruit length, with an average increase of 9% to 17%.

[0027] The above average increase in fruit length is relative to the "bb" round fruit genotype.

[0028] Therefore, the interaction between the two major QTL loci mentioned above can be used to predict melon fruit length.

[0029] The present invention also discloses that the interaction relationship of the main effect QTL of melon fruit length not only exists in the F2 segregation population constructed with parents flex09 and cata05, but also commonly exists in general melon resources. Therefore, it can also be used to predict the fruit length of melons other than the F2 segregation population constructed with parents flex09 and cata05.

[0030] The present invention also provides a molecular marker tightly linked to the two major effect QTL loci, wherein the molecular marker is A02-Indel or A08-Indel.

[0031] Among them, the molecular marker A02-Indel is linked to the QTL site qFL2.1, and the upstream and downstream primers corresponding to the molecular marker A02-Indel are:

[0032] Upstream primer A02-Indel-F: 5′-AAATGAAAACGTTCGAAAAACC-3′ (SEQ ID NO. 1);

[0033] Downstream primer A02-Indel-R: 5′-TCCATGAGCCTAAATGTTCAAA-3′ (SEQ ID NO. 2);

[0034] The molecular marker A08-Indel is linked to the QTL locus qFL8.1, and the upstream and downstream primers of the A08-Indel are:

[0035] Upstream primer A08-Indel-F: 5'-ATTACCTATTTTCACATTTGTATGGA-3' (SEQ ID NO. 3); downstream primer A08-Indel-R: 5'-TCAATTGAGGCCAAACTTTTC-3' (SEQ ID NO. 4).

[0036] The present invention also provides a molecular marker combination linked to the main effect QTL locus of melon fruit length, which is used to identify melon materials with different fruit lengths. The molecular marker combination includes A02-Indel and A08-Indel. A reagent or a kit including the molecular marker A02-Indel and A08-Indel primer pair is also provided. In specific applications, a reagent containing the molecular marker primer pair can be selected to make a kit. The present invention also includes the use of a reagent for detecting the presence of the molecular marker in locating genes related to melon fruit length. The molecular marker of the present invention can be used to locate genes related to melon fruit length. All of the above applications can be carried out according to conventional methods.

[0037] The present invention also discloses the use of a molecular marker or a combination of molecular markers tightly linked to a major effect QTL of melon fruit length in melon molecular breeding:

[0038] (1) Application in assisting identification of melon fruit length phenotype at the molecular level;

[0039] (2) Application in early prediction of melon fruit length;

[0040] (3) Application in molecular marker-assisted breeding to create melons with different fruit lengths;

[0041] (4) Application in screening or identifying melon germplasm resources;

[0042] (5) Application in melon fruit length genotyping.

[0043] The present invention also discloses a method for determining the plant / variety of melon fruit length, the method comprising the following steps:

[0044] (1) extracting DNA from a melon sample to be tested;

[0045] (2) using the DNA extracted in step (1) as a template, and performing PCR amplification on the template using the two pairs of primers described above;

[0046] (3) Detection by gel electrophoresis;

[0047] (4) Determine according to the electrophoresis band results of step (3), the specific criteria are:

[0048] A. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 159 bp, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 249 bp, then the sample to be tested is a very long fruit melon plant / variety;

[0049] B. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 159 bp, and the PCR amplification product of the molecular marker A08-Indel is two characteristic bands of 239 bp and 249 bp respectively, then the sample to be tested is a very long fruit melon plant / variety;

[0050] C. If the PCR amplification product of the molecular marker A02-Indel is two characteristic bands of 159 bp and 169 bp in length, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 249 bp, then the sample to be tested is a very long fruit melon plant / variety;

[0051] D. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 159 bp or the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 239 bp, then the sample to be tested is a long-fruited melon plant / variety;

[0052] E. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 169p, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 249bp, then the sample to be tested is a long-fruited melon plant / variety;

[0053] F. If the PCR amplification product of the molecular marker A02-Indel is two characteristic bands with lengths of 159 bp and 169 bp respectively, and the PCR amplification product of the molecular marker A08-Indel is two characteristic bands with lengths of 239 bp and 249 bp respectively, then the sample to be tested is a long-fruited melon plant / variety;

[0054] G. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 169p, and the PCR amplification product of the molecular marker A08-Indel is two characteristic bands of 239bp and 249bp in length, respectively, then the sample to be tested is a round-fruited melon plant / variety;

[0055] H. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 169p, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 239bp, then the sample to be tested is a round-fruited melon plant / variety;

[0056] I. If the PCR amplification product of the molecular marker A02-Indel is two characteristic bands with lengths of 159 bp and 169 bp respectively, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 239 bp, then the sample to be tested is a round-fruited melon plant / variety.

[0057] Advantages of the present invention:

[0058] 1. The method of the present invention can be used to identify the fruit lengths of various melons. The main effect QTL loci qFL2.1 and qFL8.1 linkage markers of melon fruit length obtained by the present invention, combined with the interaction relationship between the main effect loci, can be applied to molecular marker-assisted breeding of melon varieties with different fruit lengths. Compared with molecular markers of single loci, it has higher accuracy and a wider range of applications.

[0059] 2. The molecular markers of the present invention can be used to predict fruit length during the seedling stage of plant development, greatly shortening the breeding time from plant planting to fruit ripening, reducing the manpower and material resources required in the breeding process, and effectively improving the breeding efficiency of melon varieties with different fruit lengths.

[0060] 3. The molecular markers of the present invention can be used to accurately and quickly identify the fruit length of mature fruits in the early stages of melon plant development, eliminating the interference of environmental factors on the phenotype, and effectively improving the breeding efficiency of ideal melon fruit length in the process of breeding high-quality melon fruit appearance. It has the advantages of simple detection process and stable amplification results. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The results of QTL analysis of melon fruit length among multiple populations over multiple years;

[0062] In the figure, site A represents the location of the main QTL site qFL2.1 of melon fruit length in the present invention on chromosome A02 and the confidence interval of the QTL detected in multiple populations over many years; site B represents the location of the main QTL site qFL8.1 of melon fruit length in the present invention on chromosome A08 and the confidence interval of the QTL detected in multiple populations over many years.

[0063] Figure 2 The interaction relationship between the main QTL loci qFL2.1 and qFL8.1 of melon fruit length in the segregating population;

[0064] In the figure, the vertical axis on the left represents the melon fruit length (cm), the horizontal axis represents the three genotypes aa, ab and bb at the qFL8.1 locus, and the three broken lines represent the three genotypes aa, ab and bb at the qFL2.1 locus from top to bottom. The average fruit length corresponding to the nine different genotype combinations is represented by circles, and the range of plus signs above and below the circles represents the range of phenotypic changes.

[0065] Figure 3 The statistical results of fruit length of 66 melon germplasm resources are shown below;

[0066] The figure shows the statistical results of fruit length of different genotype combination materials. ** represents extremely significant difference at the 0.01 level (t test), and * represents significant difference at the 0.05 level (t test). DETAILED DESCRIPTION

[0067] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0068] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

[0069] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0070] Example 1 Obtaining the main QTL loci for melon fruit length and analyzing their interaction relationships

[0071] Material Description

[0072] Male parent: flex09. After long-term self-pollination and phenotypic observation, this material exhibits extremely long fruit length.

[0073] Female parent: cata05, this material is a common cultivated variety. After many years of field observation, it shows short fruit length. The above materials can be provided through commercial channels or the melon crop genetic breeding research group of Henan Agricultural University.

[0074] 1. Construction of the isolation group

[0075] flex09 is the male parent, which is a snake melon variety among melons. The fruit length is extremely long, and the longest can reach more than 1m. The round-fruit variety cata05 is the female parent, which is a rough-skinned variety among melons. The fruit length is 10 to 15cm.

[0076] In the spring of 2017, the parents were crossed to obtain F1 seeds, and in the autumn of 2017, the F1 plants were strictly selfed to obtain seeds of the F2 segregating population; in the spring of 2018, the F2 segregating population was planted, and 140 F2 plants with normal growth, sufficient seeds, and full grains were selected. 2：3 Retained; repeated planting in spring 2019 and fall 2019 with 140 F 2：3 Each family is planted with 5-9 plants, and each plant retains 1-2 melons.2：3 Each family randomly selected a single melon with normal growth and full seeds for seed preservation, and planted 140 F 2：4 Each family was planted with 5-9 plants, and 1-2 fruits were left on each plant. All materials were treated with normal seedling raising measures and water and fertilizer management, single vine pruning, and female flowers on the 14-18th node side branches were selected for artificial pollination. After pollination, 1-2 normal swollen fruits were selected, and the fruit length was phenotypically observed and counted after the fruit matured for 40-45 days.

[0077] 2. Gene map construction

[0078] 140 F2 plants and sufficient fresh young leaf tissues of the parents were collected and stored in a -80℃ refrigerator for future use. In this experiment, the parents (flex09, cata05) were resequenced and the 140 F2 plants were subjected to simplified genome sequencing. The whole genome DNA was double-digested with restriction endonucleases (HindIII_BfaI), and the size of the digested fragments was between 220 and 600 bp. The digested fragments were then recovered and the library was constructed according to the ddRAD-seq (double digest restriction-site associated DNA sequencing) method. The paired-end (2×150 bp) of the library was sequenced using the next-generation sequencing (NGS) technology based on the Illumina Hiseq sequencing platform.

[0079] The obtained SNP sites were quality controlled and screened, and sites that were homozygous in the two parents and inconsistent between the parents were retained, sites with a sequencing depth of more than 10 in the offspring were retained, sites with an offspring deletion rate of less than 0.5 were retained, and sites that significantly deviated from Mendel's law of inheritance (P-value < 0.05) were deleted.

[0080] The polymorphic SNPs were converted into bin markers, and the 140 F2 individual plant bin markers were imported into the software Jionmap V4.0, and LOD = 6 was selected as the threshold. The Kosambi method was used to sort the markers within the linkage group, and the genetic distance was obtained. The map was constructed in MapInspect. The genetic map contained a total of 4654 genetic markers, covering a total length of 1462.30Mb of the melon genome, and the average distance between markers was 1.39Mb.

[0081] 3. QTL Analysis

[0082] Combining the genotypic data with the phenotypic data from the four environments, the composite interval mapping method using R / qtl software was used to conduct linkage analysis on melon fruit length. The major QTL loci qFL2.1 and qFL8.1 were located on chromosomes A02 and A08, respectively.

[0083] For the main QTL locus qFL2.1, the LOD values ​​in the four environments were 14.50, 14.56, 14.70, and 13.63, respectively, and the phenotypic variation explanation rates were 32.46%, 27.46%, 36.03%, and 30.71%, respectively. The physical interval corresponding to this QTL was 1.6Mb to 1.8Mb in A02 ( Figure 1 ).

[0084] For the main QTL locus qFL8.1, the LOD values ​​in the four environments were 7.31, 6.59, 7.69, and 9.30, respectively, and the phenotypic variation explanation rates were 13.11%, 11.59%, 15.86%, and 14.93%, respectively. The physical interval corresponding to this QTL was 26.5Mb to 27.8Mb in A08 ( Figure 1 ).

[0085] 4. Analysis of interaction relationships between major QTL loci

[0086] According to the positioning results of the main effect QTL locus of melon fruit length, the molecular markers in the region where the LOD value peak was located were selected as the characteristic markers of the QTL loci. The characteristic marker of qFL2.1 was bin136, and the characteristic marker of qFL8.1 was bin752.

[0087] Combining the genotypes of the characteristic markers of the two main effect QTL loci and the phenotypic data of the four environments, the effectplot function of the R / qtl software was used to analyze the interaction relationship between the two main effect QTL loci ( Figure 2 ).

[0088] When the qFL2.1 locus was the "aa" homozygous long-fruit genotype, the fruit length increased by an average of 13.33±1.20 cm, with an increase percentage of 41% to 65%.

[0089] When the qFL2.1 locus was the "ab" heterozygous genotype, the fruit length increased by an average of 7.16±1.60cm, with an increase percentage of 23% to 36%.

[0090] When the qFL8.1 locus was the "aa" homozygous long-fruit genotype, the fruit length increased by an average of 9.21±1.71 cm, with an increase percentage of 22% to 44%.

[0091] When the qFL8.1 locus was the "ab" heterozygous genotype, the fruit length increased by an average of 3.98±1.45cm, with an increase percentage of 9% to 12%.

[0092] When both FL2.1 and FL8.1 were homozygous for long fruit genotypes, or one of qFL2.1 and qFL8.1 was homozygous for long fruit genotypes and the other was heterozygous for ab, the fruit length was extremely long, increasing by an average of 18.50±1.77cm, with an increase of 78% to 97%. There was an obvious cumulative effect between the two loci.

[0093] When both qFL2.1 and qFL8.1 loci were "ab" heterozygous genotypes, or one of qFL2.1 and qFL8.1 loci was "aa" homozygous long-fruit genotype and the other was "bb" homozygous round-fruit genotype, the fruit length was longer, with an average increase of 7.05±0.65cm and an increase percentage of 27% to 31%. There was no obvious additive effect between the two loci.

[0094] When one of the qFL2.1 and qFL8.1 loci was the "bb" pure and round fruit genotype and the other was the "ab" heterozygous genotype, the fruit length only increased by 2±1.1 cm, and the round fruit phenotype had an advantage, indicating that the pure and round fruit genotype loci were epistatic, the increase in fruit length was inhibited, and the fruit length was shorter, with an average increase of 9% to 17%.

[0095] 5. Development of linkage markers for major QTL loci

[0096] Based on the whole genome resequencing data of the parents flex09 and cata05, the variation data of SNPs and InDels between the parents were obtained by comparison. According to the physical intervals corresponding to the two major QTL loci, tightly linked InDel molecular markers were designed, including:

[0097] InDel marker A02-InDel tightly linked to qFL2.1:

[0098] Upstream primer: 5'-AAATGAAAACGTTCGAAAAACC-3' (as shown in SEQ ID NO.1);

[0099] Downstream primer: 5'-TCCATGAGCCTAAATGTTCAAA-3' (as shown in SEQ ID NO.2);

[0100] The primers were used to perform PCR amplification on the melon samples to be tested (parents and F2 populations). The band pattern of the paternal parent was 159bp, the band pattern of the maternal parent was 169bp, the band pattern of the round fruit material in the segregating population was 169bp, and the band pattern of the elongated and extremely elongated fruit material was 159bp or 159bp / 169bp.

[0101] InDel marker A08-InDel tightly linked to qFL8.1:

[0102] Upstream primer: 5'-ATTACCTATTTTCACATTTGTATGGA-3' (as shown in SEQ ID NO. 3);

[0103] Downstream primer: 5'-TCAATTGAGGCCAAACTTTTC-3' (as shown in SEQ ID NO.4);

[0104] The primers were used to perform PCR amplification on the melon samples to be tested (parents and F2 populations). The band pattern of the paternal parent was 249bp, the band pattern of the maternal parent was 239bp, the band pattern of the round fruit material in the segregating population was 239bp or 249bp / 239bp, and the band pattern of the elongated and extremely elongated fruit material was 249bp.

[0105] 5. Polyacrylamide gel electrophoresis detection

[0106] The DNA of 66 melon germplasm resources were amplified by PCR using the two primer pairs of molecular markers A02-InDel and A08-InDel that were closely linked to the main effect loci of melon fruit length.

[0107] Specific material names and results are shown in Table 2. The reaction system for PCR amplification is shown in Table 1.

[0108] Table 1 PCR amplification reaction system

[0109]

[0110]

[0111] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 30 cycles, and finally extension at 72°C for 10 min and insulation at 4°C.

[0112] The amplification product of the marker A02-InDel primer showed the same band pattern as the male parent flex09, that is, when the amplification product fragment was 159 bp (as shown in SEQ ID NO.5), it was recorded as qFL2.1aa (pure and long fruit genotype); it showed the same band pattern as the female parent cata05, that is, when the amplification product fragment was 169 bp (as shown in SEQ ID NO.6), it was recorded as qFL2.1bb (pure and round fruit genotype); when it showed two band patterns, that is, when the amplification product contained fragments of 159 bp and 169 bp, it was recorded as qFL2.1ab (heterozygous genotype).

[0113] The amplification product of the marker A08-InDel primer showed the same band pattern as the male parent flex09, that is, when the amplification product fragment was 249 bp (as shown in SEQ ID NO.7), it was recorded as qFL8.1aa (pure and long fruit genotype); it showed the same band pattern as the female parent cata05, that is, when the amplification product fragment was 239 bp (as shown in SEQ ID NO.8), it was recorded as qFL8.1bb (pure and round fruit genotype); when it showed two band patterns, that is, when the amplification product contained fragments of 239 bp and 249 bp, it was recorded as qFL8.1ab (heterozygous genotype).

[0114] Based on the test results of 66 melon material genotype combinations, combined with the phenotypic data of these materials in four different environments in spring 2015 (2015S), autumn 2015 (2015A), spring 2016 (2016S), and autumn 2016 (2016A), the analysis results are as follows:

[0115] The genotype combination test results were "qFL2.1aa+qFL8.1aa" for 2 materials, "qFL2.1aa+qFL8.1ab" for 1 material, and "qFL2.1ab+qFL8.1aa" for 1 material. The analysis of the fruit length phenotype data of these materials showed that the fruit length of these materials was extremely long, with an average fruit length of 61.28±11.26cm ( Figure 3 ).

[0116] The genotype combination test results were "qFL2.1aa+qFL8.1bb" and "qFL2.1bb+qFL8.1aa" respectively, and the results showed that the fruit length of these materials was relatively long, with an average fruit length of 21.06±5.78cm ( Figure 3 ).

[0117] The genotype combination test results were "qFL2.1ab+qFL8.1bb" materials, including 2 samples, "qFL2.1bb+qFL8.1ab" materials, including 1 sample, and "qFL2.1bb+qFL8.1bb" materials, including 54 samples. The fruit length of these materials was shorter, with an average fruit length of 15.00±3.96cm ( Figure 3 ).

[0118] In summary, the combined use of the molecular markers A02-InDel and A08-InDel tightly linked to the main effect QTL loci qFL2.1 and qFL8.1 of the melon fruit length of the present invention can be used for large-scale identification of melon materials with different fruit lengths, and the early prediction of melon fruit length has high accuracy. The correspondence between genotype and phenotype conforms to the interaction relationship between different genotypes of the two main effect loci, which effectively shortens the breeding cycle, provides accurate genetic laws for melon fruit length breeding, and can be used on a large scale in production.

[0119] Table 2 Fruit length phenotype and genotype of melon materials

[0120]

[0121]

[0122]

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a molecular marker closely linked to a QTL locus in identifying or assisting in identifying the trait of melon fruit length and in genotyping melon fruit length, characterized in that: The QTL loci include qFL2.1 and qFL8.1, qFL2.1 is located in the bin130-bin142 interval of chromosome A02, and qFL8.1 is located in the bin744-bin760 interval of chromosome A08; the molecular markers are A02-Indel and A08-Indel; the upstream and downstream primers of A02-Indel are: Upstream primer A02-Indel-F: 5′-AAATGAAAACGTTCGAAAAACC-3′; Downstream primer A02-Indel-R: 5′-TCCATGAGCCTAAATGTTCAAA-3′; The upstream and downstream primers of the A08-Indel are: Upstream primer A08-Indel-F: 5′-ATTACCTATTTTCACATTTGTATGGA-3′; Downstream primer A08-Indel-R: 5′-TCAATTGAGGCCAAACTTTTC-3′; The identification criteria are: A. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 159 bp, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 249 bp, then the sample to be tested is a very long fruit melon plant / variety; B. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 159 bp, and the PCR amplification product of the molecular marker A08-Indel is two characteristic bands of 239 bp and 249 bp respectively, then the sample to be tested is a very long fruit melon plant / variety; C. If the PCR amplification product of the molecular marker A02-Indel is two characteristic bands of 159 bp and 169 bp in length, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 249 bp, then the sample to be tested is a very long fruit melon plant / variety; D. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 159 bp or the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 239 bp, then the sample to be tested is a long-fruited melon plant / variety; E. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 169p, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 249bp, then the sample to be tested is a long-fruited melon plant / variety; F. If the PCR amplification product of the molecular marker A02-Indel is two characteristic bands with lengths of 159 bp and 169 bp respectively, and the PCR amplification product of the molecular marker A08-Indel is two characteristic bands with lengths of 239 bp and 249 bp respectively, then the sample to be tested is a long-fruited melon plant / variety; G. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 169p, and the PCR amplification product of the molecular marker A08-Indel is two characteristic bands of 239bp and 249bp in length, respectively, then the sample to be tested is a round-fruited melon plant / variety; H. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 169p, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 239bp, then the sample to be tested is a round-fruited melon plant / variety; I. If the PCR amplification product of the molecular marker A02-Indel is two characteristic bands with lengths of 159 bp and 169 bp respectively, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 239 bp, then the sample to be tested is a round-fruited melon plant / variety.

2. A method for determining the plant / variety of melon fruit length, characterized in that: The method comprises the following steps: (1) extracting DNA from a melon sample to be tested; (2) using the DNA extracted in step (1) as a template, and performing PCR amplification on the template using the two pairs of primers described in claim 1; (3) Detection by gel electrophoresis; (4) Determine according to the electrophoresis band results of step (3), the specific criteria are: A. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 159 bp, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 249 bp, then the sample to be tested is a very long fruit melon plant / variety; B. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 159 bp, and the PCR amplification product of the molecular marker A08-Indel is two characteristic bands of 239 bp and 249 bp respectively, then the sample to be tested is a very long fruit melon plant / variety; C. If the PCR amplification product of the molecular marker A02-Indel is two characteristic bands of 159 bp and 169 bp in length, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 249 bp, then the sample to be tested is a very long fruit melon plant / variety; D. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 159 bp or the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 239 bp, then the sample to be tested is a long-fruited melon plant / variety; E. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 169p, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 249bp, then the sample to be tested is a long-fruited melon plant / variety; F. If the PCR amplification product of the molecular marker A02-Indel is two characteristic bands with lengths of 159 bp and 169 bp respectively, and the PCR amplification product of the molecular marker A08-Indel is two characteristic bands with lengths of 239 bp and 249 bp respectively, then the sample to be tested is a long-fruited melon plant / variety; G. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 169p, and the PCR amplification product of the molecular marker A08-Indel is two characteristic bands of 239bp and 249bp in length, respectively, then the sample to be tested is a round-fruited melon plant / variety; H. If the PCR amplification product of the molecular marker A02-Indel is a characteristic band of 169p, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 239bp, then the sample to be tested is a round-fruited melon plant / variety; I. If the PCR amplification product of the molecular marker A02-Indel is two characteristic bands with lengths of 159 bp and 169 bp respectively, and the PCR amplification product of the molecular marker A08-Indel is a characteristic band of 239 bp, then the sample to be tested is a round-fruited melon plant / variety.

3. A kit for identifying the length trait of melon fruit, characterized in that: Comprising the primer pair described in claim 1.

4. Use of a primer pair for detecting the presence of the molecular markers A02-Indel and A08-Indel described in claim 1 in locating genes related to melon fruit length, wherein the upstream and downstream primers of the molecular marker A02-Indel are: Upstream primer A02-Indel-F: 5′-AAATGAAAACGTTCGAAAAACC-3′; Downstream primer A02-Indel-R: 5′-TCCATGAGCCTAAATGTTCAAA-3′; The upstream and downstream primers of the molecular marker A08-Indel are: Upstream primer A08-Indel-F: 5′-ATTACCTATTTTCACATTTGTATGGA-3′; Downstream primer A08-Indel-R: 5′-TCAATTGAGGCCAAACTTTTC-3′.

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