Muskmelon fruit length molecular marker and application thereof

Abstract

The invention discloses a muskmelon fruit length molecular marker and application thereof. According to the method, two methods of BSA-seq and GPS-seq are utilized to perform preliminary positioning on the muskmelon fruit length related QTL, a reference genome Mel (DHL92) gene 3.6. 1 and a re-sequencing result are utilized to perform comparative analysis, two algorithms of SNP-index and InDel-index are comprehensively utilized to perform correlation analysis, the results of the two analysis methods are respectively counted, and the results of the two analysis methods are compared with those of the QTL. The larger segment is used as a candidate region associated with the length of the melon fruit, KASP06-3880 and KASP06-3606 are obtained, and the KASP06-3880 is located at the 34305438th basic group of the No.6 chromosome; the KASP06-3606 is located at the base at the 18936169th site of the chromosome 11; the two KASP markers KASP06-3880 and KASP11-3606 are subjected to genotype detection in extreme materials of F2 and RIL populations, and the two KASP markers determine the length of the muskmelon fruits, so that a new way is provided for breeding of the muskmelon fruits with different lengths.

Description

Technical Field

[0001] The present invention relates to the technical field of biological breeding, in particular to a melon fruit length molecular marker and application thereof. Background Art

[0002] Melon fruit exhibits exceptional genetic diversity. Fruit shape is not only an important appearance quality trait but also a key indicator of fruit diversity. Fruit length is a key determinant of fruit shape and is closely related to yield. Research on the genetic mechanisms of fruit length is fundamental to cultivar improvement. Fruit length varies significantly between different melon accessions, ranging from 5 to 200 cm, a nearly 40-fold difference; fruit weight ranges from 0.1 to 10 kg, with a nearly 100-fold difference. Furthermore, this crop exhibits an extremely diverse range of fruit shapes, including round, pear-shaped, ovate, obovate, elliptical, olive-shaped, bottleneck-shaped, cylindrical, and rod-shaped. Therefore, melon is an ideal model crop for studying fruit trait formation and development. Previous studies have investigated the mapping of QTLs for fruit shape and length in melon, but the results vary widely due to varying genetic backgrounds and fruit shapes. This study focused on fruit length, an important indicator affecting melon fruit shape. A genetic population was constructed using two germplasm materials with obvious differences in fruit length. Gene positioning research was carried out and molecular markers were developed in order to lay the foundation for molecular marker-assisted selection of melon. Summary of the Invention

[0003] In view of the defects of the prior art, the object of the present invention is to provide a molecular marker for melon fruit length, which provides a new approach for the breeding of different melon fruit lengths.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A melon fruit length molecular marker, comprising KASP06-3880 and KASP06-3606, wherein KASP06-3880 is located at base 34305438 on chromosome 6; and KASP06-3606 is located at base 18936169 on chromosome 11;

[0006] The primer sequence of KASP06-3880 includes two forward primers and one reverse primer. The nucleotide sequences of the two forward primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3.

[0007] The primer sequence of KASP06-3606 includes two forward primers and one reverse primer. The nucleotide sequences of the two forward primers are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively; the nucleotide sequence of the one reverse primer is shown in SEQ ID NO.6.

[0008] A kit comprises the above-mentioned primer sequence.

[0009] The use of the above-mentioned melon fruit length molecular marker or kit in assisting the breeding of different melon fruit lengths.

[0010] A method for identifying melon fruit length comprises the following steps: performing genotyping detection on two KASP markers, KASP06-3880 and KASP11-3606, in extreme materials of F2 and RIL populations; for the KASP06-3880 marker in the offspring of the F2 and RIL populations, if its genotype is the same as that of the maternal parent 551, it is marked as AA; if it is the same as that of the paternal parent B22, it is marked as aa; similarly, for the KASP11-3606 marker, if it is the same as that of the maternal parent 551, it is marked as BB; if it is the same as that of the paternal parent B22, it is marked as bb; the genotypes of the two loci are integrated into AABB, AAbb, aaBB and aabb; when the genotype is AABB, the fruit length tends to be longer, and when the genotype is aabb, the fruit length tends to be shorter.

[0011] The present invention has the beneficial effects of preliminarily locating QTLs associated with melon fruit length using BSA-seq and GPS-seq methods, performing comparison analysis with the resequencing results using the reference genome Melon (DHL92) genome 3.6.1, and performing association analysis using a combination of SNP-index and InDel-index algorithms. The results of the two analysis methods are statistically analyzed, and larger fragments are used as candidate regions associated with melon fruit length, thereby obtaining KASP06-3880 and KASP06-3606. KASP06-3880 is located at base positions 34305438 on chromosome 6, and KASP06-3606 is located at base positions 18936169 on chromosome 11. Genotyping of the two KASP markers, KASP06-3880 and KASP11-3606, is performed in extreme materials of F2 and RIL populations, and both KASP markers determine melon fruit length, providing a new approach for breeding different melon fruit lengths. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Figure 2 is the frequency distribution diagram of F2 fruit length, where Figure A is the phenotypic distribution of F2 fruit length in autumn 2022; Figure B is the phenotypic distribution of F2 fruit length in spring 2023.

[0013] Figure 2 Map of QTLs for melon fruit length identified by BSA method.

[0014] Figure 3 Map of QTLs for melon fruit length identified using GPS method.

[0015] Figure 4 This is the fine mapping map of the QTL on chromosome 6 for melon fruit length, where Figure A is the physical map, Figure B is the genetic map, Figure C is the QTL mapping result for fruit weight, Figure D is the QTL mapping result for fruit length, and Figure E is the QTL detection information for FW and FL.

[0016] Figure 5 This is the fine mapping map of the QTL on chromosome 11 for melon fruit length. Figure A is the physical map, Figure B is the genetic map, Figure C is the QTL mapping result for fruit weight, Figure D is the QTL mapping result for fruit shape index, Figure E is the QTL mapping result for fruit length, and Figure F is the QTL detection information for FW, FL, and FS.

[0017] Figure 6 This is the linkage analysis map of the genotypes of the two loci KASP06-3880 and KASP11-3606 and the fruit length of the extreme materials of the F2 and RIL populations.

[0018] Figure 7 The genotypes of the two loci and the fruit length phenotypes of typical materials in the F2 and RIL populations are shown. DETAILED DESCRIPTION

[0019] 1. Test Materials

[0020] This experiment used the long-fruited melon material '551' (FL = 165.0 cm, FSI = 18.54) with extremely significant differences in parental phenotypes as the female parent. '551' was introduced from the National Western Melon Medium-Term Bank (No.: ZTG00906) and was homozygous after multiple generations of self-pollination. It is a flexuous variety of thick-skinned melon; the round-fruited melon material 'B22' (FL = 13.5 cm, FSI = 1.00) was used as the male parent. 'B22' is a homozygous material bred by the research group and is a thick-skinned melon inodorus variety. All melon germplasm materials came from the Melon Genetics and Breeding Research Group of the Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences.

[0021] 2. Test methods

[0022] 1. Construction of genetic population and phenotypic statistics

[0023] Using the long-fruited muskmelon material '551' as the female parent (P1) and the round-fruited muskmelon material 'B22' as the male parent (P2), individual seeds were harvested from hybrids to construct an F1 population. The F1 material was strictly selfed and individual seeds were harvested to construct an F2 population. A first-generation backcross was created using P1 as the male parent to construct a BC1P1 population. A first-generation backcross was created using P2 as the male parent to construct a BC1P2 population. Six generations of single-seed selfing from the F2 population created a RIL population. The muskmelon material was planted in a plastic greenhouse with hanging vines, with a plant spacing of 40 cm and a row spacing of 60 cm.

[0024] Mature fruits were measured by cutting them longitudinally and measuring from top to bottom with a ruler. Curved fruits were measured from head to tail with a tape measure. This length was considered the fruit length. Ripe melons were collected for individual fruit length statistics from each generation. Frequency distribution statistics and normal distribution tests were performed for each trait using Excel and Origin.

[0025] 2. Genetic analysis of melon fruit length

[0026] Melon fruit length data were statistically analyzed using Excel and SPSS software, and fruit length trends and segregating generation frequency distributions were plotted. The major gene + polygene mixed genetic model analysis method proposed by Gai Junyi et al. (Gai Junyi et al., 2000) for studying plant quantitative traits was used. Genetic analysis of fruit length data from multiple generations, which had been statistically measured, was performed using the major gene + polygene mixed genetic model software SEA. The analysis included maximum likelihood estimation, AIC criterion screening, candidate genetic model fitness testing, and calculation of the optimal model's first- and second-order genetic parameters.

[0027] 3. Mixed pool construction

[0028] The fruit lengths of all plants in the F2 population were arranged in descending order to construct three pools. Pool 1 was the largest pool, FL-1, containing 16 progeny, accounting for 10% of the F2 population; pool 2 was the intermediate pool, FL-2, containing 16 progeny, accounting for 10% of the F2 population; and pool 3 was the smallest pool, FL-3, containing 16 progeny, accounting for 10% of the F2 population.

[0029] The pooled DNA and parental DNA were sequenced using the Illumina sequencing platform, followed by Ridit analysis to calculate the p-value for each SNP. After Ridit analysis, the data were denoised using a 0.2 Mb window size and a p-value threshold of 10⁻¹. Finally, the regions significantly associated with melon fruit length traits were identified by counting sites with significant p-values. The maximum and minimum pools were subjected to BSA-seq analysis, and the three pools were subjected to GPS-seq analysis. The results of these two analyses were combined to determine the initial localization interval.

[0030] 4. Development and detection of KASP molecular markers

[0031] Based on the GPS sequencing results, SNPs within the candidate interval were filtered to obtain high-quality SNPs. For these high-quality SNPs, KASP markers were developed on the genome. The KASP marker consists of two forward primers and one reverse primer, with the SNP site as the last base at the 3' end of the forward primer. Primers were designed and tested using the software SnapGene and Primer6. Testing criteria were as follows: primer annealing temperature was 55±5°C; four or more consecutive bases of complementary sequences could not appear within a single primer, and five or more bases of complementary sequences could not appear between the forward and reverse primers. Primer sequences that met these criteria were entered into the Cucumis melon genome database (http: / / cucurbitgenomics.org / organism / 18) for BLAST comparison. Primers with only a single matching sequence in the comparison were used for KASP marker development. After primer alignment, FAM and VIC fluorescent-labeled adapter sequences were added to the 5' ends of the two forward sequences, respectively.

[0032] 5. Genetic linkage analysis

[0033] The genotype of each F2 plant was analyzed based on the marker typing results. Genotypes identical to the maternal genotype were recorded as "0," those identical to the paternal genotype were recorded as "2," heterozygous genotypes were recorded as "1," and missing genotypes were recorded as "-1." KASP genotyping and phenotypic data were then analyzed using QTLIciMapping (v4.2).

[0034] 2. Results and Analysis

[0035] 1. Population phenotypic distribution and genetic model analysis

[0036] The melon fruit length phenotype statistics of the four generation groups (P1, P2, F1, F2) prepared by '551' and 'B22' were conducted in two consecutive different seasons (autumn 2022 and spring 2023). The results showed that the fruit length was significantly different between the parents (Table 1). The average fruit length of '551' was 146 cm, the average fruit length of 'B22' was 13 cm, and the average fruit length of F1 was 52 cm, which was between the parents. The fruit length of the F2 segregating population varied continuously, with the maximum fruit length of 111 cm and the minimum value of 10 cm. The fruit length showed a unimodal continuous distribution with a kurtosis of less than 1 and a skewness of less than 1. Therefore, the distribution of fruit length in the F2 generation segregating population conformed to the normal distribution, which was consistent with the genetic characteristics of quantitative traits (such as Figure 1), and the 2-year repeatability was good (Table 2). Using phenotypic data for genetic analysis, six basic generation populations (P1, P2, F1, F2, BC1P1, and BC1P2) were constructed using the long-fruited melon inbred line '551' as the female parent (P1) and the round-fruited melon inbred line 'B22' as the male parent (P2) (Table 3). The maximum likelihood method and the IECM algorithm were used to estimate the genetic pattern of fruit length in these populations (Table 4). Based on the principle of achieving the least number of significant levels and the smallest AIC value for the genetic model of melon fruit length, the optimal genetic model was two pairs of additive-dominant-epistatic major genes + additive-dominant-epistatic polygenes (MX2-ADI-ADI). The heritabilities of the major genes in the genetic populations BC1P1, BC1P2, and F2 were 86.1188%, 100%, and 60.4821%, respectively; and the polygene heritabilities were 1.5882%, 0%, and 29.2478%, respectively. This indicated that the inheritance efficiency of the major genes for melon fruit length was higher in the BC1P1, BC1P2, and F2 generations, and that selection for fruit length in thick-skinned melon occurred in earlier generations and was less affected by environmental factors.

[0037] Table 1. Phenotypic data of fruit length of parents and their F2 generation populations

[0038]

[0039] Table 2. Pearson correlation coefficient of fruit length between different years

[0040] Sampling year 2022 2023 2022 1 0.996 2023 0.996 1

[0041] Table 3. Variation analysis of melon fruit length

[0042] generations Number of plants Average variance Standard deviation Coefficient of variation / % <![CDATA[P1]]> 10 144.429 40.952 6.399 0.0443 <![CDATA[P2]]> 10 12.444 1.278 1.130 0.0908 <![CDATA[F1]]> 10 52.444 29.278 5.411 0.1032 <![CDATA[F2]]> 756 49.794 276.699 16.634 0.3340 <![CDATA[B1]]> 30 78.897 231.167 15.204 0.1927 <![CDATA[B2]]> 30 26.190 27.454 5.240 0.2001

[0043] Table 4. Maximum log-likelihood and AIC values of the genetic model for melon fruit length

[0044] Model Maximum likelihood value AIC value Model Maximum likelihood value AIC value 1MG-AD -3642.429 7292.858 MX1-AD-ADI -3457.816 6939.631 1MG-A -3680.227 7366.453 MX1-AD-AD -3521.291 7060.581 1MG-EAD -3708.999 7423.998 MX1-A-AD -3534.578 7085.155 1MG-NCD -3656.039 7318.078 MX1-EAD-AD -3534.581 7085.161 2MG-ADI -3497.915 7015.83 MX1-NCD-AD -3500.338 7016.676 2MG-AD -3623.931 7259.862 MX2-ADI-ADI -3447.986 6931.972 2MG-A -3698.482 7404.964 MX2-ADI-AD -3456.896 6943.791 2MG-EA -3644.158 7294.317 MX2-AD-AD -3500.252 7022.504 2MG-CD -3695.652 7399.303 MX2-A-AD -3493.802 7005.604 2MG-EAD -3695.652 7397.303 MX2-EA-AD -3534.578 7085.155 PG-ADI -3486.538 6993.076 MX2-CD-AD -3596.031 7210.061 PG-AD -3537.89 7089.781 MX2-EAD-AD -3534.58 7085.161

[0045] Note: MG is the main gene model; MX is the main gene + multi-gene mixed model; PG is the multi-gene model; A is the additive effect; D is the dominant effect; I is the epistatic effect; E is equal

[0046] 2. Preliminary positioning of QTL for melon fruit length

[0047] This study used BSA-seq( Figure 2 ) and GPS-seq( Figure 3) methods were used to preliminarily locate the QTL associated with melon fruit length. The reference genome Melon (DHL92) genome 3.6.1 was used for comparison analysis with the resequencing results. The SNP-index and InDel-index algorithms were combined for association analysis. The results of the two analysis methods were statistically analyzed, and the larger fragments were used as candidate regions associated with melon fruit length. The preliminary positioning intervals of the melon fruit length QTL were obtained, with a total of two candidate regions, located between 33.2Mb and 34.4Mb on chromosome 6 and between 17.6Mb and 24Mb on chromosome 11, respectively.

[0048] 3. Fine mapping of QTL for melon fruit length and development of molecular markers

[0049] A total of 20 pairs of SNP molecules were designed in this study, and 16 pairs of molecular markers with good polymorphism and high genotyping quality were selected (Table 5), including 9 pairs of chromosome 6 ( Figure 4 -A), 7 pairs of chromosomes 11. After calculating the genetic distance using QTLIciMapping software, a linkage interval of 22.71 cM was obtained on chromosome 6 ( Figure 4 -B), using complete interval mapping (ICIM) to detect QTL for melon fruit length, it was found that fruit length was linked to the marker interval KASP06-7 to KASP06-9 ( Figure 4 -C). The peak LOD of this QTL was 2.63, the contribution rate was 7.55%, the additive effect was -8.25, and the dominant effect was -7.56 ( Figure 4 -E), and the fruit weight is also within this range ( Figure 4 -D), the peak LOD of this QTL was 2.79, the contribution rate was 6.23%, the additive effect was -0.52, and the dominant effect was -0.23. The KASP06-7 and KASP06-9 markers are located on chromosome 6 at physical positions of 34205880 bp and 34374748 bp, respectively, with a distance of approximately 168.8 kb between them. By constructing a genetic map with target intervals, the candidate interval for the melon fruit length gene was successfully narrowed from 1.2 MB to 168.8 kb; a 14.97 cM linkage interval was obtained on chromosome 11 ( Figure 5 -B), and found that fruit length ( Figure 5 -E) is linked to the KASP11-2 to KASP11-4 marker interval. The peak LOD of this QTL is 4.94, the contribution rate is 11.68%, the additive effect is -13.88, and the dominant effect is 0.41 ( Figure 5 -F), fruit weight ( Figure 5 -C) and fruit shape index ( Figure 5-D) is also within this interval, with the peak LOD values of its QTL being 3.55 and 2.85, the contribution rates being 8.55% and 6.90%, the additive effects being -0.6 and -1.33, and the dominant effects being 0.01 and -0.07 ( Figure 5 -F). The KASP11-2 and KASP11-4 markers are located on chromosome 11 at physical positions of 18,246,857 bp and 19,190,291 bp, respectively, with a distance of approximately 943.4 kb. By constructing a genetic map of the target interval, the candidate interval for the melon fruit length gene was successfully narrowed from 6.4 MB to 943.4 kb.

[0050] The two KASP markers KASP06-3880 (34305438) and KASP11-3606 (18936169) (Table 6) were genotyped in extreme materials of F2 and RIL populations. For the KASP06-3880 marker in the offspring of the F2 and RIL populations, if its genotype is the same as that of the maternal parent 551, it is marked as AA; if it is the same as that of the paternal parent B22, it is marked as aa. Similarly, for the KASP11-3606 marker, if it is the same as that of the maternal parent 551, it is marked as BB; if it is the same as that of the paternal parent B22, it is marked as bb. The genotypes of these two loci were integrated into AABB, AAbb, aaBB and aabb. The results showed that when the genotype was AABB, the fruit length tended to be longer; while when the genotype was aabb, the fruit length was shorter ( Figure 6 The fruit lengths of the two genotypes differed significantly ( Figure 7 ), indicating that the genotypes of these two loci were closely related to the fruit length of the progeny of the F2 and RIL populations.

[0051] Table 5. Detailed information of labeled primers

[0052]

[0053]

[0054] Table 5

[0055]

[0056] Table 6. Molecular marker primer information

[0057]

Claims

1. A melon fruit length molecular marker, characterized in that The molecular markers include KASP06-3880 and KASP06-3606, wherein KASP06-3880 is located at base 34305438 of chromosome 6; and KASP06-3606 is located at base 18936169 of chromosome 11; The primer sequence of KASP06-3880 includes two forward primers and one reverse primer. The nucleotide sequences of the two forward primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the nucleotide sequence of the one reverse primer is shown in SEQ ID NO.

3. The primer sequence of KASP06-3606 includes two forward primers and one reverse primer. The nucleotide sequences of the two forward primers are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively; the nucleotide sequence of the one reverse primer is shown in SEQ ID NO.

6.

2. A kit, characterized in that Comprising the primer sequence of claim 1.

3. Use of the melon fruit length molecular marker according to claim 1 or the kit according to claim 2 in assisting breeding of different melon fruit lengths.

4. A method for identifying the length of melon fruit, characterized in that The method comprises the following steps: performing genotype detection on two KASP markers, KASP06-3880 and KASP11-3606, in extreme materials of F2 and RIL populations; for the KASP06-3880 marker in the offspring of the F2 and RIL populations, if its genotype is the same as that of the maternal parent 551, it is marked as AA; if it is the same as that of the paternal parent B22, it is marked as aa; similarly, for the KASP11-3606 marker, if it is the same as that of the maternal parent 551, it is marked as BB; if it is the same as that of the paternal parent B22, it is marked as bb; the genotypes of the two sites are integrated into AABB, AAbb, aaBB and aabb; when the genotype is AABB, the fruit length tends to be longer, and when the genotype is aabb, the fruit length is shorter.

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