CN110106270B - A molecular marker co-separated with the yellow seed coat of melon and its application - Google Patents
A molecular marker co-separated with the yellow seed coat of melon and its application Download PDFInfo
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Abstract
本发明公开了一种与甜瓜黄色种皮共分离的分子标记及其应用。本发明提供了检测待测甜瓜基因组中是否含有序列3所示的第25‑38位的核苷酸的物质如下1)‑4)中至少一种中的应用：1)鉴定或辅助鉴定待测甜瓜的种皮颜色是否为黄色；2)制备鉴定或辅助鉴定待测甜瓜的种皮颜色是否为黄色产品；3)选育携带黄色种皮基因型的甜瓜品种；4)制备选育携带黄色种皮基因型的甜瓜品种产品。利用本发明的分子标记鉴定甜瓜黄色种皮的准确性可达100％，进一步可用于甜瓜分子标记辅助育种、基因聚合育种等，在甜瓜生长的任意阶段进行筛选、鉴定，效率高、特异性好、准确度高，大大节约时间和成本，对加速甜瓜黄色种皮育种具有重要意义。The invention discloses a molecular marker co-separated with the yellow seed coat of melon and its application. The present invention provides the application in at least one of the following 1)-4) for detecting whether the melon genome to be tested contains the nucleotide at position 25-38 shown in sequence 3: 1) identification or auxiliary identification to be tested Whether the seed coat color of the melon is yellow; 2) Preparation and identification or auxiliary identification of whether the seed coat color of the melon to be tested is a yellow product; 3) Breeding the melon variety carrying the yellow seed coat genotype; 4) Preparation and breeding of the yellow seed Peel genotypes of melon varieties. The accuracy of identifying the yellow seed coat of melon by using the molecular marker of the invention can reach 100%, and further can be used for molecular marker-assisted breeding of melon, gene aggregation breeding, etc., screening and identification at any stage of melon growth, high efficiency and good specificity , high accuracy, greatly saves time and cost, and is of great significance for accelerating the breeding of melon yellow seed coats.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a molecular marker coseparated with a yellow seed coat of a melon and application thereof.
Background
The melon (Cucumis melo L.) is an annual herbaceous plant of Cucumis melo of Cucumis of Cucurbitaceae, and has fragrant and sweet fruit and rich nutrition, and is an important economic crop widely planted in the world. In recent years, with the adjustment of rural industrial structures and the increasing improvement of the living standard of people, the open field and protected field cultivation of the melons in China are greatly developed, and the annual supply level of the melons is remarkably improved. Because the muskmelon has stronger heterosis, the production and cultivation of the muskmelon hybrid seeds have great economic value. At present, most melons in production belong to male flower amphoteric flower varieties, the artificial pollination mode is mainly adopted in the production process of first-filial generation seeds, the problems of large workload, high cost, easiness in mixing and the like exist, and field identification or indoor molecular marker detection is mostly adopted for purity identification of the hybrids, so that the time consumption and the cost are high. If the macroscopic morphological marker is used as the indicating character, the cost of purity identification in seed production can be greatly saved.
The color of the seed coat of the melon is divided into white, yellow, tan and the like. In 1936, the melon seed coat color was first analyzed by Hagiwara and Kamimura, and it was thought that the white color of the seed coat was dominant over yellow and tan colors, as controlled by the gene wt (white tasta). Genetic analysis of white seed coat traits of melon resource PI 414723 by Pirin et al (1999) showed that white seed coats are dominant to yellow seed coats, controlled by a single dominant gene pair Wt-2, and localized this gene to IV linkage groups. Zhang Kexin et al (2018) located white gene (WT) controlling melon seed coat color on linkage group 5, with linkage markers HD0520 and HD0519 at both ends, and the genetic distance from the linkage markers is 13.3cM and 7cM, respectively. In the first generation seed reproduction process of the melon hybrid, the color of yellow seed coats is easy to be visually identified, so that the yellow seed coats can be used as the marker characters for hybrid purity identification and applied to mechanical mixing and biological mixing of seeds and visual characters for preliminary identification of hybrid seed purity. However, the yellow seed coat of melon is recessive gene control, and the seed coat is developed from maternal cells, the character of the seed is controlled by maternal genes, and the character controlled by the genotype of the seed is expressed in the next generation, which is typically delayed inheritance, so that the seed must be planted and the genotype of the previous generation can be judged according to the separation condition of the progeny seed. Therefore, the conventional hybridization method is labor-consuming in breeding yellow seed coat varieties, the development of specific molecular markers related to yellow seed coat genes is carried out, the molecular marker-assisted selection of the yellow seed coat genes is carried out, the selection efficiency can be improved in the next generation breeding process, the breeding cost is reduced, the yellow seed coat characters are applied to the purity identification of hybrid seeds as soon as possible, and the method has important significance in promoting the industrialized development of muskmelons.
Disclosure of Invention
The invention aims to solve the technical problem of providing a molecular marker coseparated with a melon yellow seed coat and application thereof, and the invention provides the following technical scheme:
the invention aims to provide application of a substance for detecting whether a 25 th to 38 th nucleotides shown in a sequence 3 are contained in a genome of a melon to be detected.
The invention provides application of a substance for detecting whether a 25 th-38 th nucleotide shown in a sequence 3 is contained in a genome of a melon to be detected in at least one of the following 1) -6):
1) identifying or assisting in identifying the seed coat color genotype of the melon to be detected;
2) preparing and identifying or assisting in identifying the seed coat color genotype of the muskmelon to be detected;
3) breeding a melon variety carrying a yellow seed coat genotype;
4) preparing and breeding a product of a melon variety carrying a yellow seed coat genotype;
5) breeding a melon variety with a non-yellow seed coat;
6) preparing and breeding a product of a melon variety with a non-yellow seed coat;
the seed coat color genotype is yellow seed coat genotype A2A2, non-yellow seed coat genotype A1A1 or non-yellow seed coat genotype A1A 2;
the genotype A2A2 of the yellow seed coat is nucleotide containing the 25 th to 38 th sites of the sequence 4 or no nucleotide containing the 25 th to 38 th sites of the sequence 3 in 2 homologous chromosomes of the melon genome;
the genotype A1A1 of the non-yellow seed coat is that 2 homologous chromosomes of the melon genome all contain sequence 3 or all contain nucleotides at 25 th to 38 th positions of the sequence 3;
the genotype A1A2 of the non-yellow seed coat is that 1 homologous chromosome of the melon genome contains the sequence 3 or contains the 25 th to 38 th nucleotides of the sequence 3, and the other homologous chromosome contains the sequence 4 or does not contain the 25 th to 38 th nucleotides of the sequence 3.
Another purpose of the invention is to provide a use for detecting whether a substance of sequence 3 and/or sequence 4 is contained in the genome of the melon to be detected.
The invention provides an application of detecting whether a substance containing a sequence 3 and/or a sequence 4 in a genome of a melon to be detected is at least one of the following substances 1) to 6):
1) identifying or assisting in identifying the seed coat color genotype of the melon to be detected;
2) preparing and identifying or assisting in identifying the seed coat color genotype of the muskmelon to be detected;
3) breeding a melon variety carrying a yellow seed coat genotype;
4) preparing and breeding a product of a melon variety carrying a yellow seed coat genotype;
5) breeding a melon variety with a non-yellow seed coat;
6) preparing and breeding a product of a melon variety with a non-yellow seed coat;
the seed coat color genotype is yellow seed coat genotype A2A2, non-yellow seed coat genotype A1A1 or non-yellow seed coat genotype A1A 2;
the genotype A2A2 of the yellow seed coat is nucleotide containing the 25 th to 38 th sites of the sequence 4 or no nucleotide containing the 25 th to 38 th sites of the sequence 3 in 2 homologous chromosomes of the melon genome;
the genotype A1A1 of the non-yellow seed coat is that 2 homologous chromosomes of the melon genome all contain sequence 3 or all contain nucleotides at 25 th to 38 th positions of the sequence 3;
the genotype A1A2 of the non-yellow seed coat is that 1 homologous chromosome of the melon genome contains the sequence 3 or contains the 25 th to 38 th nucleotides of the sequence 3, and the other homologous chromosome contains the sequence 4 or does not contain the 25 th to 38 th nucleotides of the sequence 3.
In the above application, the substance for detecting whether the genome of the melon to be detected contains the 25 th-38 th nucleotides shown in the sequence 3 or the substance for detecting whether the genome of the melon to be detected contains the sequence 3 and/or the sequence 4 is 1) or 2):
1) the primer set comprises a single-stranded DNA molecule (YS-F) shown as a sequence 1 in a sequence table or a derivative thereof, and a single-stranded DNA molecule (YS-R) shown as a sequence 2 in the sequence table or a derivative thereof;
2) a PCR reagent or a kit containing the primer set.
The substitution and/or deletion and/or addition of one or more nucleotides is the substitution and/or deletion and/or addition of no more than 10 nucleotides.
The primer pair satisfies the following conditions: the DNA fragment obtained by PCR amplification with the melon genome DNA as the template contains the sequence shown in the sequence 3 or the sequence 4, wherein the difference between the sequence 3 and the sequence 4 is whether the 25 th-38 th nucleotide of the sequence 3 is contained.
In the above complete set of primers, the molar ratio of the primer YS-F to the primer YS-R is 1: 1.
PCR reagents containing the primer pairs are also within the scope of the present invention.
In the PCR reagent, the concentration of the primer YS-F and the primer YS-R in the primer pair in the PCR reagent is 0.5 mu mol/L.
In the application, the derivative of the single-stranded DNA molecule shown in the sequence 1 in the sequence table is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1;
the derivative of the single-stranded DNA molecule shown in the sequence 2 in the sequence table is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2.
It is a further object of the invention to provide a product.
The product provided by the invention comprises a substance for detecting whether the 25 th-38 th nucleotide shown in the sequence 3 is contained in the genome of the melon to be detected, or whether the substance for detecting the sequence 3 and/or the sequence 4 is contained in the genome of the melon to be detected;
the product has at least one function of 1) to 3) as follows:
1) identifying or assisting in identifying the seed coat color genotype of the melon to be detected;
2) breeding a melon variety carrying a yellow seed coat genotype;
3) and breeding the melon variety with non-yellow seed coat.
The invention also provides a biological material which comprises 25 th to 38 th nucleotides shown in a sequence 3 or DNA molecules shown in the sequence 3 and/or DNA molecules shown in the sequence 4;
or, the invention also provides the application of the biological material in at least one of the following 1) to 3):
1) identifying or assisting in identifying the seed coat color genotype of the melon to be detected;
2) breeding a melon variety carrying a yellow seed coat genotype;
3) and breeding the melon variety with non-yellow seed coat.
The invention also provides a method for identifying or assisting in identifying the seed coat color genotype of the muskmelon to be detected, which comprises the following steps: detecting whether the genome of the melon to be detected contains 25 th-38 th nucleotides shown in a sequence 3 or not, and if the genome of the melon to be detected does not contain the 25 th-38 th nucleotides shown in the sequence 3, determining that the seed coat color genotype of the melon to be detected is a yellow seed coat genotype A2A 2; if the genome of the melon to be detected contains 25 th-38 th nucleotides shown in the sequence 3, the seed coat color genotype of the melon to be detected is non-yellow seed coat genotype A1A1 or non-yellow seed coat genotype A1A 2;
the genotype A2A2 of the yellow seed coat is nucleotide containing the 25 th to 38 th sites of the sequence 4 or no nucleotide containing the 25 th to 38 th sites of the sequence 3 in 2 homologous chromosomes of the melon genome;
the genotype A1A1 of the non-yellow seed coat is that 2 homologous chromosomes of the melon genome all contain sequence 3 or all contain nucleotides at 25 th to 38 th positions of the sequence 3;
the genotype A1A2 of the non-yellow seed coat is that 1 homologous chromosome of the melon genome contains the sequence 3 or contains the 25 th to 38 th nucleotides of the sequence 3, and the other homologous chromosome contains the sequence 4 or does not contain the 25 th to 38 th nucleotides of the sequence 3.
The invention also provides a method for identifying or assisting in identifying the seed coat color of the offspring of the muskmelon to be detected, which comprises the following steps:
detecting whether the genome of the melon to be detected contains 25 th-38 th nucleotides shown in a sequence 3 or not, and if the genome of the melon to be detected does not contain the 25 th-38 th nucleotides shown in the sequence 3, determining that the seed coat of the progeny of the melon to be detected is yellow or is selected as a candidate; and if the genome of the melon to be detected contains 25 th-38 th nucleotides shown in the sequence 3, the seed coat color of the melon progeny to be detected is not yellow or the candidate is not yellow.
The invention also provides a method for breeding the melon variety carrying the yellow seed coat genotype or breeding the melon variety with yellow seed coat as a progeny, which is used for breeding the melon to be tested with the yellow seed coat genotype A2A2 as the seed coat color genotype.
The invention also provides a method for breeding a melon variety carrying a non-yellow seed coat genotype or a melon variety with a non-yellow seed coat as a seed substitute, which is used for breeding the melon to be tested, the seed coat color genotype of which is the non-yellow seed coat genotype A1A1 or A1A 2.
In the above method, the method for detecting whether the genome of the melon to be detected contains the 25 th to 38 th nucleotides shown in the sequence 3 is as follows:
1) direct sequencing;
2) amplifying the muskmelon to be detected by using the set of primers, and detecting an amplification product;
if the amplification product only contains a 117bp fragment, the genome of the melon to be detected does not contain the 25 th-38 th nucleotides shown in the sequence 3;
and if the amplification product contains a 131bp fragment, the genome of the melon to be detected contains 25 th-38 th nucleotides shown in a sequence 3.
The primer pair is utilized to carry out PCR amplification, and the following reaction system can be specifically adopted to carry out PCR amplification: 0.8 μ L Buffer, 0.8 μ L dNTPs (concentration of each of four dNTPs is 2mmol/L), the YS-F and the YS-R, 1 μ L of the melon genomic DNA to be tested (50ng/μ L), 0.1 μ L
DNA Polymerase for PAGE，ddH2And O is supplemented to 10 mu L. Buffer and
DNA Polymerase for PAGE was a product of Beijing Quanjin Biotechnology Ltd.
The annealing temperature for PCR amplification using the primer pair may be 59 ℃. The following reaction conditions can be specifically adopted for PCR amplification by using the primer pair: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 5 min.
Experiments prove that the molecular marker (YS) of the yellow seed coat of the melon is related to the yellow seed coat of the melon, and the seed of the A2A2 genotype melon progeny, of which two chromosomes do not contain the 25 th to 38 th nucleotides of the sequence 3 in the sequence table, is the yellow seed coat. The accuracy of identifying whether the melon has the yellow seed coat genotype or not by using the melon yellow seed coat molecular marker provided by the invention can reach 100%, the melon yellow seed coat molecular marker provided by the invention can be further used for melon molecular marker assisted breeding, screening and identifying are carried out at any stage of melon growth, the efficiency is high, the specificity is good, the accuracy is high, the time and the cost are greatly saved, and the melon yellow seed coat molecular marker has an important significance for accelerating melon short vine breeding.
Drawings
FIG. 1 shows the phenotypes of melon varieties "oriental honey I" (white seed coat) and "Huangzi" gold jade (yellow seed coat).
FIG. 2 is the genotype analysis of the parents and the offspring by the marker YS; wherein P1: oriental honey I; p2: gold and jade; f1: hybrid F prepared by using oriental honey I as female parent and using Huangzi Jinyu as male parent1(ii) a 1-36 is yellow seed coat F2Generating single plants; m: 100bp DNA ladder; .
FIG. 3 shows the results of the test of 12 melon varieties with the marker YS; wherein 1-12 are oriental honey I, selected nectar crisp, Hongdao side, extra-large white Sha honey, Meilun No. eleven, Xue Su No. I, Huangzi Jinyu, Huangzi Jinxiangyu, Huangzi Jinyu, Xinchun, Zhaojun No. nine, early spring honey, Xueli crisp and Ruijin respectively; m: 100bp DNA ladder.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The following examples are intended to further illustrate the invention and are not to be construed as limiting the invention. In the examples, unless otherwise specified, all experimental methods used were conventional methods; the reagents used, etc., are commercially available.
The test materials used in the following examples, wherein the white seed coat material eastern honey No. is described in: "Zhang qun, Wangya, Wangming, Oriental Honey No. I high-yield cultivation technique [ J ]. Shanghai vegetables, 2016 (1): 77-78 "article, available to the public from the applicant, for the inventor from Happy seeds of Sinkiang Limited, the biomaterial was used only for repeating the relevant experiments of the invention and was not used for other purposes; yellow seed coat material Huangzi Jinyu is described in 'Dazuyun, Zhaohui, Xia Chengdong, Yangbai, Liu Yongzhong' breeding of early-maturing storable strong yellow-peel melon variety Huangzi Jinyu [ J ].2006 (1): 23-25 "article, which was purchased by the inventor from scientific and technological resources of the gardening industry of Jianghuai, Anhui, and publicly available from the applicant, the biomaterial was used only for repeating the related experiments of the present invention, and was not used for other purposes. All of the remaining materials used are commercially available.
Example 1 screening of molecular markers associated with seed coat color of Cucumis melo
1. Construction and genetic analysis of segregating populations
A white seed coat thick-skin melon variety oriental honey I and a yellow seed coat thick-thin intermediate melon variety yellow golden jade are screened from the collected melon varieties (figure 1). The oriental honey I is used as a female parent and the yellow-seed golden jade is used as a male parent to prepare a hybrid combination, and the obtained F1The seed coat is white, and the oriental honey I is used as male parent and the yellow-seed golden jade is used as female parent to prepare hybrid combination, so as to obtain F1The seed coat is yellow, indicating that the color of the seed coat shows a delayed genetic effect, and F1The seed coat develops from the maternal cells.
Using Oriental honey A as female parent and using Huangzi Jinyu as female parentMale parent hybridization obtained F1F obtained after selfing2The total 470 seeds are planted in the field separately, each plant is selfed separately, and the seeds are harvested in the mature period, namely F3And thirdly, inspecting the phenotype of each single plant seed after the seed is aired, thereby obtaining F2The generation plant genotype. Statistical analysis of the data shows that F2Seed (F) of the institute3Substitute) seed coat color separation ratio is white: yellow 358:112, showing a mendelian genetic segregation ratio (χ) of 3:12＝0.28<3.84, p-value ═ 0.59), these data indicate that the yellow seed coat trait is controlled by a single stealth nuclear gene.
2. Development and screening of molecular markers
Specific primers were designed using the online software Primer-BLAST (http:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST/index. cgilink _ LOC ═ BlastHome) based on the melon reference genomic sequence (https:// www.melonomics.net /).
Respectively extracting parent Oriental honey I, Huangzi Jinyu, F1 (filial generation obtained by hybridization of Huangzi Jinyu as male parent and Oriental honey I as female parent) and yellow seed coat F by CTAB method2Genomic DNA of the individual. Selecting 15 yellow seed coats F2The genomic DNA of the single plant is mixed in equal amount to establish a mixed pool, and three samples of two parents are added to screen the synthesized 120 pairs of melon primers. The reaction system for PCR amplification is as follows: mu.L Buffer, 0.8. mu.L dNTPs (2mmol/L each), 1. mu.L Primers, 1. mu.L genomic DNA (50 ng/. mu.L), 0.1. mu.L
DNA Polymerase for PAGE (from Beijing Quanjin Biotech Co., Ltd.) and 6.3. mu.L ddH2And O. The reaction program of PCR amplification is pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 30s, and running for 35 cycles; extension at 72 ℃ for 5 min. And detecting the PCR amplification product by 8% polyacrylamide gel electrophoresis.
The results show that: the mixed pool DNA, the gold jade and the chromosome 5 marker YS show a linkage relation.
The marker YS consists of a primer F shown in a sequence 1 and a primer R shown in a sequence 2,
and (3) primer F: CTTATCCGCCTCCGACACC (SEQ ID NO: 1);
and (3) primer R: AGTTAACGCACCCGAACCC (SEQ ID NO: 2).
Therefore, the mark YS can be used for identifying the color of the seed coat of the melon to be detected.
Example 2 identification of seed coat color genotype of melon by molecular marker YS
Using the YS-labeled primers obtained in example 1, the parent Oriental Honey I, Huangzi Jinyu, F1(F obtained by hybridization of Oriental honey I as female parent and Huangzi Jinyu as male parent1) And 470 of F as described above2Genotype analysis was performed on the progeny plants (358 white seed coat plant, 112 yellow seed coat plant) as follows:
and (3) performing PCR amplification by using the single-plant genome DNA extracted by the CTAB method as a template and using the primer F and the primer R marked with YS to obtain a PCR amplification product.
The reaction system for PCR amplification is as follows: mu.L Buffer, 0.8. mu.L dNTPs (2mmol/L each), 1. mu.L Primers (10. mu. mol/L, F + R (final reaction concentration of each primer is 0.5. mu. mol/L), 1. mu.L genomic DNA (50 ng/. mu.L), 0.1. mu.L
DNA Polymerase for PAGE (from Beijing Quanjin Biotech Co., Ltd.) and 6.3. mu.L ddH2O。
The reaction procedure of the PCR amplification is pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 30s, and running for 35 cycles; extension at 72 ℃ for 5 min.
Detecting the PCR amplification product by 8% polyacrylamide gel electrophoresis.
Partial results are shown in FIG. 2, with the marker YS versus parent and partial F2Detecting the PCR amplification product of the generation individual plant; p1: oriental honey I; p2: gold and jade; 1-36: yellow seed coat F2Carrying out single plant cultivation; m: 100bp DNA ladder; as can be seen, the PCR amplification products in the two parents are fragments with the range of 100-200bp and different sizes, and the F1 generation contains the PCR amplification products in the two parents;
sequencing from P1The nucleotide sequence of the target fragment amplified in (Oriental Honey I) is sequence 3, the size is 131bp, and the represented genotype is named as A1A1(2 homologous chromosomes contain sequence 3);
from P2The nucleotide sequence of the amplified target fragment in (gold jades) is sequence 4, the size is 117bp, and the represented genotype is named as A2A2(2 homologous chromosomes contain sequence 4);
F1the seed coat is white in color, contains nucleotide sequences shown in sequence 3 and sequence 4, and has a genotype of A1A2 (one of 2 homologous chromosomes contains sequence 3 and the other contains sequence 4);
470F2Among the individual generations, 358 strains of white seed coat included 120 strains of A1A1 genotype (2 homologous chromosomes all contain sequence 3), 238 strains of A1A2 heterozygous genotype (2 homologous chromosomes contain sequence 3 and 4); the 112 yellow seed coat strains have the genotype of A2A2(2 homologous chromosomes contain a sequence 4).
The results in FIG. 2 show that 1-36 yellow seed coats F2Individual and parent P2The (gold and jade) band patterns consistently showed coseparation.
Through sequence alignment, the fragments of the sequence 3 and the sequence 4 are different in that the nucleotide shown in the 25 th to 38 th positions of the sequence 3 is not contained in the sequence 4.
The experimental results show that the marker YS and the amplified product sequence 3 or sequence 4 thereof are related to the seed coat color genotype of the melon, and can be used for detecting the seed coat color genotype of the melon.
Therefore, whether the seed coat color genotype of the melon to be detected or whether the seed coat of the progeny seed of the melon to be detected is yellow is identified or assisted to be identified by detecting whether the 25 th to 38 th nucleotides shown in the sequence 3 are contained in the genome of the melon to be detected, which is specifically as follows:
detecting whether the genome of the melon to be detected contains 25 th-38 th nucleotides shown in a sequence 3, if the genome does not contain the 25 th-38 th nucleotides shown in the sequence 3, the seed coat color of the offspring of the melon to be detected is yellow, and the seed coat color genotype of the melon to be detected is yellow seed coat genotype A2A 2; if the test melon contains 25 th-38 th nucleotides shown in the sequence 3, the seed coat color of the progeny of the test melon is not yellow, and the seed coat color genotype of the test melon is non-yellow seed coat genotype A1A1 or non-yellow seed coat genotype A1A 2;
the yellow seed coat genotype A2A2 is that 2 homologous chromosomes of a melon genome to be detected all contain a sequence 4;
the genotype A1A1 of the non-yellow seed coat is that 2 homologous chromosomes of a melon genome to be detected all contain a sequence 3;
the genotype A1A2 of the non-yellow seed coat is that 1 homologous chromosome of the muskmelon genome to be detected contains a sequence 3, and the other homologous chromosome contains a sequence 4;
or identifying or assisting to identify the seed coat color genotype of the melon to be detected or whether the seed coat of the progeny seed of the melon to be detected is yellow by detecting whether the sequence 3 and/or the sequence 4 are contained in the genome of the melon to be detected, which is concretely as follows:
detecting whether the genome of the melon to be detected contains DNA molecules shown in a sequence 3 and/or a sequence 4,
if the genome of the melon to be detected does not contain the sequence 3 and contains the sequence 4, the seed coat color of the offspring of the melon to be detected is yellow or the candidate is yellow, and the seed coat color genotype of the melon to be detected is yellow seed coat genotype A2A 2;
if the genome of the melon to be detected only contains the sequence 3 and does not contain the sequence 4, the seed coat color of the offspring of the melon to be detected is not yellow or the candidate is not yellow, and the seed coat color genotype of the melon to be detected is non-yellow seed coat genotype A1A 1;
and if the genome of the melon to be detected contains the sequence 3 and the sequence 4, the seed coat color of the progeny of the melon to be detected is not yellow or the candidate is not yellow, and the seed coat color genotype of the melon to be detected is non-yellow seed coat genotype A1A 2.
The method for detecting whether the genome of the melon to be detected contains the 25 th-38 th nucleotides shown in the sequence 3 is as follows:
1) direct sequencing;
2) amplifying the muskmelon to be detected by using a primer pair marked with YS, and detecting an amplification product;
if the amplification product only contains 117bp, the 25 th-38 th nucleotides shown in the sequence 3 are not contained in the genome of the melon to be detected;
if the amplified product contains a 131bp fragment (only 131bp fragment or 117bp and 131bp fragments), the genome of the melon to be detected contains 25 th-38 th nucleotides shown in the sequence 3.
The method for detecting whether the genome of the melon to be detected contains the DNA molecules shown in the sequence 3 and/or the sequence 4 comprises the following steps:
1) direct sequencing;
2) amplifying the muskmelon to be detected by using a primer pair marked with YS, and detecting an amplification product;
if the size of the amplification product is only 117bp, the genome of the melon to be detected contains the sequence 4 and does not contain the sequence 3;
if the size of the amplification product is only 131bp, the genome of the melon to be detected contains the sequence 3 and does not contain the sequence 4;
if the sizes of the amplification products are 131bp and 117bp, the genome of the melon to be detected contains a sequence 3 and a sequence 4.
Example 3 application of marker YS in molecular marker-assisted selection of species of melon yellow seed coat
In order to develop molecular marker-assisted selective breeding of yellow seed coat genes and verify the accuracy of the marker YS in seed coat color, according to the method in the embodiment 2, 12 melon varieties with different genetic backgrounds except for the first oriental honey and the gold jade are actually detected by utilizing the marker YS, and 14 melon varieties in the table 1 are identified in total. The method comprises the following specific steps:
table 1 shows the information of 14 melon varieties
Genomic DNA of each material was extracted by the CTAB method, and 14 melon materials in Table 1 were PCR-amplified using the marker YS according to the method in example 2 to obtain PCR amplification products.
If the amplification product only contains 117bp, the 25 th-38 th nucleotides shown in the sequence 3 are not contained in the genome of the melon to be detected;
if the amplified product contains a 131bp fragment (only 131bp fragment or 117bp and 131bp fragments), the genome of the melon to be detected contains 25 th-38 th nucleotides shown in the sequence 3.
If the genome of the melon to be detected does not contain the sequence 3 and contains the sequence 4, the seed coat color of the offspring of the melon to be detected is yellow or the candidate is yellow, and the seed coat color genotype of the melon to be detected is yellow seed coat genotype A2A 2;
if the genome of the melon to be detected only contains the sequence 3 and does not contain the sequence 4, the seed coat color of the offspring of the melon to be detected is not yellow or the candidate is not yellow, and the seed coat color genotype of the melon to be detected is non-yellow seed coat genotype A1A 1;
and if the genome of the melon to be detected contains the sequence 3 and the sequence 4, the seed coat color of the progeny of the melon to be detected is not yellow or the candidate is not yellow, and the seed coat color genotype of the melon to be detected is non-yellow seed coat genotype A1A 2.
The results are shown in FIG. 3, where 1-6, 7-12, 13 and 14 correspond to 14 varieties in Table 1, respectively; wherein 5 parts of white seed coat varieties (selected nectar crisp, red-channel side, extra-large white sand honey, melen No. eleven and snow crisp No. I) are consistent with eastern honey No. I, and the genotype is A1A 1; the genotypes of two white seed coat varieties (Xuelicui, Ruijin) are heterozygous A1A 2; 5 parts of yellow seed coat varieties (yellow seed gold fragrant jade, Huangzi gold jade, Xinchun, Zhaojun No. and early spring honey) are consistent with the yellow seed gold jade, the genotype is A2A2, the identification accuracy is 100%, the explanation mark YS can quickly identify the color of the seed coat of the melon, and the genotypes of A1A1, A1A2 and A2A2 are distinguished.
Based on the results, the yellow seed coat material and the white seed coat material are utilized to adopt a conventional sexual hybridization and backcross breeding method, and are assisted with the marker YS for molecular marker assisted selection, so that a new melon variety carrying the yellow seed coat genotype can be rapidly cultured, and the breeding efficiency is greatly improved.
The above embodiments are merely illustrative of the present invention, but the embodiments of the present invention are not limited by the variety or materials of the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be regarded as equivalent replacements within the protection scope of the present invention.
Sequence listing
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Claims (6)
1. The application of the following substances 1) to 6) in detecting whether the muskmelon genome to be detected contains the substances of the sequence 3 and/or the sequence 4 is/are:
1) identifying or assisting in identifying the seed coat color genotype of the melon to be detected;
2) preparing and identifying or assisting in identifying the seed coat color genotype of the muskmelon to be detected;
3) breeding a melon variety carrying a yellow seed coat genotype;
4) preparing and breeding a product of a melon variety carrying a yellow seed coat genotype;
5) breeding a melon variety with white seed coat as a progeny;
6) preparing a product for breeding a melon variety with white seed coats as progeny;
the color of the seed coat is yellow or white;
the seed coat color genotype is yellow seed coat genotype A2A2, white seed coat genotype A1A1 or white seed coat genotype A1A 2;
the genotype A2A2 of the yellow seed coat is that 2 homologous chromosomes of the melon genome all contain a sequence 4;
the genotype A1A1 of the white seed coat is that 2 homologous chromosomes of a melon genome all contain a sequence 3;
the genotype A1A2 of the white seed coat is that 1 homologous chromosome of the melon genome contains a sequence 3, and the other homologous chromosome contains a sequence 4;
the method for detecting whether the melon genome to be detected contains the substances of the sequence 3 and/or the sequence 4 is (1) or (2) as follows:
(1) the primer set consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table;
(2) PCR reagents or kits containing sets of primers; the primer set consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.
2. A product comprising the test of claim 1 for the presence of a substance of sequence 3 and/or sequence 4 in the genome of the melon to be tested;
the product has any one of the following functions 1) to 3):
1) identifying or assisting in identifying the seed coat color genotype of the melon to be detected;
2) breeding a melon variety carrying a yellow seed coat genotype;
3) breeding a melon variety with white seed coat as a progeny;
the color of the seed coat is yellow or white.
3. A method for identifying or assisting in identifying the seed coat color genotype of a melon to be detected comprises the following steps:
detecting whether the genome of the melon to be detected contains a sequence 3 and/or a sequence 4, if the genome of the melon to be detected only contains the sequence 4, determining that the seed coat color genotype of the melon to be detected is a yellow seed coat genotype A2A 2; if the genome of the melon to be detected contains the sequence 3, the seed coat color genotype of the melon to be detected is a white seed coat genotype A1A1 or a white seed coat genotype A1A 2;
the genotype A2A2 of the yellow seed coat is that 2 homologous chromosomes of the melon genome all contain a sequence 4;
the genotype A1A1 of the white seed coat is that 2 homologous chromosomes of a melon genome all contain a sequence 3;
the genotype A1A2 of the white seed coat is that 1 homologous chromosome of the melon genome contains a sequence 3, and the other homologous chromosome contains a sequence 4;
the color of the seed coat is yellow or white;
the step of detecting whether the genome of the melon to be detected contains the sequence 3 and/or the sequence 4 is to amplify the melon to be detected by using a set of primers and detect an amplification product;
if the amplification product only contains a 117bp fragment, the genome of the melon to be detected only contains a sequence 4;
if the amplification product contains a 131bp fragment, the genome of the melon to be detected contains a sequence 3;
the primer set consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.
4. A method for identifying or assisting in identifying the seed coat color of the offspring of the muskmelon to be detected comprises the following steps:
detecting whether the genome of the melon to be detected contains a sequence 3 and/or a sequence 4, if the genome of the melon to be detected only contains the sequence 4, determining that the seed coat color of the progeny of the melon to be detected is yellow or the candidate is yellow; if the genome of the melon to be detected contains the sequence 3, the seed coat color of the melon progeny to be detected is not yellow or the candidate is not yellow;
the color of the seed coat is yellow or white;
the step of detecting whether the genome of the melon to be detected contains the sequence 3 and/or the sequence 4 is to amplify the melon to be detected by using a set of primers and detect an amplification product;
if the amplification product only contains a 117bp fragment, the genome of the melon to be detected only contains a sequence 4;
if the amplification product contains a 131bp fragment, the genome of the melon to be detected contains a sequence 3;
the primer set consists of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.
5. A method for breeding a melon variety carrying a yellow seed coat genotype or a melon variety with a yellow seed coat as a progeny is characterized in that: the method of claim 3 is used for identifying and breeding the melon to be tested with the seed coat color genotype of yellow seed coat genotype A2A 2.
6. A method for breeding melon varieties carrying white seed coat genotypes or breeding melon varieties with white seed coats as offspring seeds is characterized in that: the method of claim 3 is used for breeding the melon to be tested, the color genotype of the seed coat of which is the white seed coat genotype A1A1 or A1A 2.
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