Molecular markers closely related to the leaf cutin traits of brassica and their application

Abstract

The application discloses a molecular marker closely related to a rapeseed leaf waxiness character and application thereof, wherein the nucleotide sequence of the molecular marker WAX1097-1300 is shown as SEQ ID NO. 3 or SEQ ID NO. 4, the nucleotide sequences of the primer pair of the molecular marker are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and in breeding, only the material with 205bp single band is reserved as a homozygous wax powder material, only the material with 212bp single band is reserved as a homozygous non-wax powder material, and the material with both 205bp and 212bp bands is reserved as a heterozygous non-wax powder material, so that the efficiency and accuracy of the next round of selection can be improved, the workload of screening and identification in the later stage is reduced, and the breeding process is accelerated.

Description

TECHNICAL FIELD

[0001] The present application relates to a molecular marker closely related to leaf wax trait, in particular to a molecular marker closely related to Brassica napus leaf wax trait and application thereof. BACKGROUND

[0002] The surface of most terrestrial plants is covered with a layer of wax, which is the first barrier between the aboveground part of the plant and the outside environment. Plant cuticle wax has a variety of physiological and ecological functions, such as preventing the deposition of water-soluble harmful substances on the plant surface, resisting insect and pathogen invasion, resisting ultraviolet radiation, reducing non-stomatal water loss in plant tissues, preventing low temperature frost injury and mechanical damage, etc. In addition, cuticle wax also affects plant development, mainly in the influence on plant epidermis morphology, such as abnormal development of trichomes, abnormal differentiation of stomata and organ fusion, etc. Preuss (A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signaling during fertilization [J]. Genes & Development, 1993, 7(6): 974-985) and others have shown that the wax on the surface of pollen is involved in the signal recognition between pollen and stigma and is related to plant fertility.

[0003] In recent years, more and more researches on plant cuticle wax have been conducted, and some wax-related genes have been cloned from wheat, Arabidopsis, rice, Thellungiella salsuginea, etc. In the research of Brassica napus, there are still few reports on genes controlling the wax trait of Brassica napus leaves, so the mining and utilization of wax genes are of great significance to Brassica napus production.

[0004] Although a number of genes controlling the wax trait of Brassica napus leaves have been identified in the present study, there is still a lack of molecular markers with practical application value in breeding. Through literature search, there is no research report on molecular markers of Brassica napus leaf wax trait and their application in China, and no related patent technology is disclosed or used. SUMMARY

[0005] The present application provides a molecular marker closely related to the wax trait of Brassica napus leaves and its application, which solves the problems of large workload and long cycle in the identification of Brassica napus leaf wax trait in conventional breeding methods, significantly improves the efficiency of identifying whether there is wax powder in Brassica napus leaves, and speeds up the breeding improvement.

[0006] In order to achieve the above-mentioned purpose, the present application provides a molecular marker closely related to the leaf wax trait of Brassica napus, the nucleotide sequence of which is shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0007] Another object of the present application is to provide a molecular marker primer pair closely related to the leaf wax trait of Brassica napus, the nucleotide sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0008] Another object of the present application is to provide the use of the molecular marker or the molecular marker primer pair in controlling the presence or absence of wax powder on the leaves of Brassica napus.

[0009] Another object of the present application is to provide the use of the molecular marker or the molecular marker primer pair in the selection breeding of the leaf wax trait of Brassica napus.

[0010] Preferably, the leaf wax trait of Brassica napus is determined according to the sequence or fragment length of the product amplified by PCR using the primer pair: only the product with the nucleotide sequence shown in SEQ ID NO. 3 or the specific band with a fragment length of 205 bp indicates that the leaves of Brassica napus have wax powder; only the product with the nucleotide sequence shown in SEQ ID NO. 4 or the specific band with a fragment length of 212 bp indicates that the leaves of Brassica napus do not have wax powder; and the simultaneous presence of the products with the nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 or the specific bands with fragment lengths of 205 bp and 212 bp indicates that the plant is a heterozygous genotype, and the leaves of Brassica napus do not have wax powder.

[0011] Preferably, the process comprises:

[0012] (1) using single-strand DNA of Brassica napus as a template;

[0013] (2) performing PCR amplification using the primer pair according to claim 1;

[0014] (3) separating the amplification product by polyacrylamide gel electrophoresis, and determining the leaf wax trait of Brassica napus according to the electrophoretic bands.

[0015] Preferably, the Brassica napus plant with only the product with the nucleotide sequence shown in SEQ ID NO. 3 or the specific band with a fragment length of 205 bp is selected for breeding.

[0016] The molecular marker closely related to the leaf wax trait of Brassica napus and the use thereof according to the present application solve the problems of large workload and long cycle in the population identification of the leaf wax trait of Brassica napus in conventional breeding methods, and have the following advantages:

[0017] The application detects that a region significantly associated with leaf waxiness is located at 32.94-33.60 Mb on A09 chromosome (threshold value is 0.4, P<0.01), the region spans 0.66 Mb, and the InDel marker WAX1097-1300 closely related to the leaf waxiness is developed, which significantly improves the efficiency of identifying whether the rapeseed leaf has wax powder or not and speeds up the breeding improvement.

[0018] In breeding, by identification and screening through functional molecular markers, the material only appearing 205 bp single band is reserved as the homozygous wax powder material, the material only appearing 212 bp single band is reserved as the homozygous non-wax powder material, and the material appearing both 205 bp and 212 bp bands is reserved as the heterozygous non-wax powder material, which can improve the efficiency and accuracy of the next round of selection, reduce the workload of later screening and identification, and speed up the breeding process. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the BSA analysis result of the oilseed rape leaf waxiness trait resequencing of the application.

[0020] Figure 2 It is a schematic diagram of the detection of the molecular marker WAX1097-1300 of the application in the rapeseed with wax powder and without wax powder.

[0021] Figure 3 It is the sequence of the amplification product of the molecular marker WAX1097-1300 of the application. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0023] Experimental Example 1: Screening of molecular markers closely related to the waxiness of rapeseed leaves

[0024] The method for obtaining the molecular markers closely related to the waxiness of rapeseed leaves is described as follows.

[0025] (I) Population construction and phenotype identification

[0026] F1 generation is generated by crossing rapeseed "Zhongshuang 11" and "7J002", and F1 generation is planted in the field after selfing. F1 generation is configured for two consecutive years, and F2 population in 2017 and F2 population in 2018 are obtained. The mature period of the plant is observed by naked eye to determine whether the rapeseed leaf has wax powder or not.

[0027] (II) Resequencing BSA analysis

[0028] Eleven wax and eleven non-wax plants of the 2017 F2 population were selected to construct wax and non-wax sub-pool, respectively, and were sent to Wuhan Geneseeq Company for re-sequencing together with the wax parent Zhong 11 and the non-wax parent 7J002. BSA analysis strategy was adopted, and the average value of Δ(SNP-index) was calculated by sliding window (2 Mb window size, 20 Kb step) to determine the candidate interval. An interval significantly associated with leaf wax trait was detected on chromosome A09 between 32.94-33.60 Mb (threshold value was 0.4, P<0.01), which spanned 0.66 Mb (see Figure 1 ).

[0029] (III) Candidate marker analysis:

[0030] All sequences in the 0.66 Mb interval were queried in the Brassica napus reference genome Darmor.v4.1, and the InDel marker WAX1097-1300 closely related to the leaf wax trait was developed using the software PrimerPremier 5.

[0031] (IV) Detection of the marker:

[0032] DNA was extracted from the wax parent Zhong 11 and the non-wax parent 7J002, and PCR amplification was performed using the molecular marker WAX1097-1300, and the amplification primers were as follows:

[0033] The forward primer sequence (SEQ ID NO. 1) was:

[0034] 5'-TCCTTGTACCCCAACAAACCA-3';

[0035] The reverse primer sequence (SEQ ID NO. 2) was:

[0036] 5'-ACAGTGATCATGAGCAACACTC-3'.

[0037] After 10% polyacrylamide gel electrophoresis separation of the PCR amplification products, two specific bands of 205 bp (SEQ ID NO. 3) and 212 bp (SEQ ID NO. 4) were obtained (see Figure 2 , the first lane was marker, the second lane was the wax parent Zhong 11 (7H001), the third lane was the non-wax parent 7J002, the fourth lane was F1, which showed non-wax, and the fifth to nineteenth lanes were 15 single plants with leaf wax in the 2017 F2 population, the twentieth to twenty-ninth lanes were 10 single plants without leaf wax in the 2017 F2 population, and the thirtieth to thirty-ninth lanes were 10 single plants without leaf wax in the 2017 F2 population).

[0038] PCR amplification was performed in the double 11 with wax powder parent, 7J002 without wax powder parent, F1 plants and 35 F2 population plants. The amplification products were separated by 10% polyacrylamide gel electrophoresis. It was found that the double 11 with wax powder parent and 15 F2 plants with wax powder only appeared a single band of 205 bp; the 7J002 without wax powder parent and 10 F2 plants without wax powder only appeared a single band of 212 bp; F1 (showing no wax powder) and 10 F2 plants without wax powder appeared two bands of 205 bp and 212 bp, which were determined to be hybrid genotype plants.

[0039] Application of the molecular marker closely related to the wax trait of the rape leaf in rape breeding

[0040] The F2 population in 2018 contained 1199 single plants, of which 314 single plants showed wax powder on the leaves, and 885 single plants showed no wax powder on the leaves. The marker developed in the experimental example 1 was used for identification, and the steps were as follows:

[0041] (1) The DNA of the single plant of the rape was used as a template;

[0042] (2) The forward and reverse primers were used for PCR process, and the PCR reaction system was shown in Table 1;

[0043] Table 1 is the PCR reaction system

[0044]

[0045]

[0046] The PCR amplification program was as follows: 94℃ 5min, [94℃ 30s, 60℃ 30s (ΔT=-0.5℃), 72℃ 30s] x 10 cycles, [94℃ 30s, 55℃ 30s, 72℃ 30s] x 25 cycles, 72℃ 5min, 16℃ pause. After running, it was stored at 4℃.

[0047] (3) The PCR amplification products were separated by 10% polyacrylamide gel electrophoresis, and the results were as follows:

[0048] There were 306 single plants only appearing a single band of 205 bp, and the leaves showed wax powder traits;

[0049] There were 287 single plants only appearing a single band of 212 bp, and the leaves showed no wax powder traits;

[0050] There were 601 single plants appearing two bands of 205 bp and 212 bp, and the leaves showed no wax powder traits, which were hybrid plants.

[0051] (4) Select 10 single plants (leaves containing wax powder) appearing 205bp single band, 5 single plants (leaves without wax powder) appearing 212bp single band, and 5 single plants (leaves without wax powder) appearing both 205bp and 212bp specific bands in step (3) above, and self-cross to produce F 2:3 generations respectively, and plant in the field to investigate the phenotype, it is found that the phenotype of the first two types of materials does not separate, and is consistent with the phenotype above; the 5 single plants appearing both bands have F 2:3 generations respectively, and plant in the field to investigate the phenotype, it is found that the phenotype of the first two types of materials does not separate, and is consistent with the phenotype above; the 5 single plants appearing both bands have F

[0052] The above identification results show that, in breeding, by functional molecular marker identification and screening, the material appearing only 205bp single band is reserved as the pure hybrid material with wax powder, the material appearing only 212bp single band is reserved as the pure hybrid material without wax powder, and the material appearing both 205bp and 212bp bands is reserved as the hybrid material without wax powder, which can improve the efficiency and accuracy of the next round of selection, reduce the workload of later screening and identification, and accelerate the breeding process.

[0053] Although the content of the present application has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present application. After reading the above content, various modifications and substitutions of the present application will be apparent to those skilled in the art. Therefore, the protection scope of the present application should be defined by the appended claims.

Claims

1. A molecular marker closely associated with the leaf waxiness trait in Brassica napus, characterized in that, The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 3 or SEQ ID NO.

4.

2. The molecular marker of claim 1 is applied to control whether the rape leaf has wax powder or to select breeding of the wax trait of the rape leaf.

3. Use of a primer pair for detecting the molecular marker according to claim 1 in selecting breeding of rapeseed leaf waxiness trait, characterized in that, The nucleotide sequences of the primer pair of the molecular marker are shown as SEQ ID NO. 1 and SEQ ID NO.

2.

4. Use according to claim 3, characterized in that, The wax trait of the rape leaf is determined according to the sequence or fragment length of the product obtained by PCR amplification with the primer pair: Only the product with the nucleotide sequence shown as SEQ ID NO. 3 or the specific band with the fragment length of 205 bp appears, and the rape leaf has wax powder; Only the product with the nucleotide sequence shown as SEQ ID NO. 4 or the specific band with the fragment length of 212 bp appears, and the rape leaf has no wax powder; Both the products with the nucleotide sequences shown as SEQ ID NO. 3 and SEQ ID NO. 4 or the specific bands with the fragment lengths of 205 bp and 212 bp appear, and the rape leaf has no wax powder.

5. Use according to claim 3, characterized in that, The process of the application comprises: (1) using single rape DNA as a template; (2) using the primer pair of claim 1 to perform PCR amplification; (3) separating the amplification product by polyacrylamide gel electrophoresis, and determining the wax trait of the rape leaf according to the electrophoresis band.

6. Use according to claim 3, characterized in that, The rape plant with only the product with the nucleotide sequence shown as SEQ ID NO. 3 or the specific band with the fragment length of 205 bp appearing is selected for breeding.

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