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CN-117947203-B - KASP molecular marker related to adjustment of content of erucic acid, oleic acid, linoleic acid and linolenic acid in rapeseed oil and application thereof

CN117947203BCN 117947203 BCN117947203 BCN 117947203BCN-117947203-B

Abstract

The application relates to the technical field of molecular genetic breeding, in particular to a KASP molecular marker related to regulating the content of erucic acid, oleic acid, linoleic acid and linolenic acid in rapeseed oil and application thereof. The molecular marker is positioned at the position 1114 bp of the BnaA08.HDG4 gene cds sequence, the position is G or A, the base is G, the BnaA08.HDG4 of the sample is judged to be a mutant sequence, the genotype is defined as GG, the locus is judged to be an original sequence if the base is A, the genotype is defined as AA, the GG corresponds to low oleic acid, low linoleic acid, low linolenic acid and high erucic acid, and the AA is opposite. The KASP molecular marker can record and analyze fluorescent signals generated in the PCR process through a computer, realizes the monitoring of mutation sites, has high consistency of detection results and phenotypes, can realize the rapid and accurate detection of the novel nucleotide mutation sites, and provides an accurate, rapid and effective detection method for selecting and adjusting the varieties containing erucic acid, oleic acid, linoleic acid and linolenic acid in rapeseed oil.

Inventors

  • FU YING
  • ZHANG QUANFENG
  • QIAN LUNWEN
  • YAO MIN
  • YU HUASHENG
  • Ni xiyuan
  • ZHANG YAOFENG
  • SHI JIANGHUA
  • WANG YUNLIU

Assignees

  • 浙江省农业科学院

Dates

Publication Date
20260508
Application Date
20240122

Claims (7)

  1. 1. The application of a reagent for detecting molecular markers related to the content of erucic acid, oleic acid, linoleic acid and linolenic acid in brassica napus seeds or a kit containing the reagent in screening brassica napus varieties is characterized in that the molecular markers are positioned at 1114bp positions of a BnaA08.HDG4 gene cds sequence, are named as A1114G sites, correspond to the 115 rd positions of SEQ ID NO. 1 or SEQ ID NO. 2 and are G or A, the base is G, bnaA08.HDG4 of a sample is judged to be a mutation sequence, the genotype is defined as GG, the site is judged to be an original sequence if the base A, and the genotype is defined as AA; GG genotype rape corresponds to low oleic acid, low linoleic acid, low linolenic acid, high erucic acid content rape, and AA genotype rape is the opposite.
  2. 2. The use according to claim 1, wherein the reagent is a KASP detection reagent comprising two specific primers and one universal primer.
  3. 3. The use according to claim 2, wherein the nucleotide sequences of the two specific primers are shown in SEQ ID NO. 3 and SEQ ID NO. 4 and the nucleotide sequence of one universal primer is shown in SEQ ID NO. 5.
  4. 4. A method for screening brassica napus varieties, comprising the steps of: Extracting rape genome DNA, carrying out PCR amplification by using a primer, and detecting a molecular marker in an amplified product so as to screen rape varieties; The molecular marker is related to the content of erucic acid, oleic acid, linoleic acid and linolenic acid in brassica napus rapa, and is positioned at 1114bp position of the BnaA08.HDG4 gene cds sequence, named as A1114G locus, corresponding to SEQ ID NO 1 or SEQ ID NO 2 115 position which is G or A, wherein the base is G, bnaA08.HDG4 of the sample is judged to be a mutant sequence, the genotype is defined as GG, if the base is A, the locus is judged to be an original sequence, and the genotype is defined as AA; GG genotype rape corresponds to low oleic acid, low linoleic acid, low linolenic acid, high erucic acid content rape, and AA genotype rape is the opposite.
  5. 5. The method according to claim 4, wherein the extracted rape genome is detected by agarose electrophoresis and Nanodrop2100, respectively, the agarose electrophoresis shows a single DNA band, A260/280 is between 1.8 and 2.0, A260/230 is between 2.0 and 2.2, and the diluted DNA concentration is 20 ng/. Mu.L.
  6. 6. The method according to claim 4, wherein the PCR reaction system is configured such that 2.5. Mu.l of the rape sample DNA template, 2 XSPRASTMINX 2.5. Mu.l, and KASP assayMix 0.07. Mu.l are used.
  7. 7. The method according to claim 4, wherein the PCR reaction conditions are 94℃for 15min, 94℃for 20sec,61-55℃for 1min, 0.6℃for 10 cycles of annealing temperature reduction per cycle, and 94℃for 20sec,55℃for 1min for 26 cycles.

Description

KASP molecular marker related to adjustment of content of erucic acid, oleic acid, linoleic acid and linolenic acid in rapeseed oil and application thereof Technical Field The invention relates to the technical field of molecular genetic breeding, in particular to a KASP molecular marker related to regulating the content of erucic acid, oleic acid, linoleic acid and linolenic acid in rapeseed oil and application thereof. Background Rape Brassica napus (genome AACC, 2n=38) is the world second largest oil crop, and is also an important oil crop in our country. Along with the improvement of living standard and the improvement of dietary structure, the demand of people for edible rapeseed oil is growing increasingly, and the cultivation of high-quality rapeseed oil becomes one of important targets of rape breeding. The quality of the nutritional and health oil of rapeseed is mainly determined by the fatty acid composition. Compared with other vegetable oils, rapeseed oil contains rich unsaturated fatty acids, mainly consisting of monounsaturated oleic acid and polyunsaturated linoleic acid and linolenic acid, and the optimal ratio is 2:1. Among them, oleic Acid (Oleinic Acid, C18:1) and Linoleic Acid (C18:2) are considered to be very healthy and nutritious to the human body. However, the three unsaturated double bonds in the extracted Linolenic Acid (C18:3) are easy to oxidize, so that the thermal stability of frying and the storage time of oil products are reduced. Erucic acid (Erucic Acid, C22:1) has long been recognized as causing health problems in humans and livestock and is difficult to digest due to its long chain, while modern canola is selected for its erucic and sulfuric acid content based on "double low" seeds, and therefore, reducing erucic and linolenic acid content has been an important goal of canola production. In arabidopsis, the genetic basis of the fatty acid biosynthesis and modification pathways of seeds is well described. In order to better understand the genetic mechanisms of rape seed fat composition and biosynthesis, many potential QTLs have been found in the parental isolates over the last decades, which are associated with seed quality traits. These QTLs include oil content, protein content, sulfan content and fatty acid composition. In recent years, whole genome association analysis (GWAS), also known as linkage mapping based on linkage disequilibrium, has been aimed at identifying genetic variations associated with traits. A large number of QTLs or genes from natural populations have been mined and have higher resolution and higher cost effectiveness. Thus GWAS has been successfully demonstrated as a powerful tool. Molecular markers are a powerful tool for crop breeding due to the accuracy, rapidness and high efficiency, and the technology has been substantially utilized in various crops due to the advantages of early selection, environmental protection, accuracy, rapidness and high efficiency. The types of molecular markers are numerous, and markers that have been used substantially in crops at present include early RFLP (Restriction fragment length Polymorphism, restriction FRAGMENT LENGTH polymorphs, RFLP), RAPD (random amplified Polymorphism, random AmplifiedPolymorphism DNA, RAPD), AFLP (amplified fragment length Polymorphism, AMPLIFIED FRAGMENT LENGTH polymorphs, AFLP), the most widely used SSR (simple repeated sequence, simple Sequence Repeat, SSR), CAPS (cut amplified polymorphic sequence, CLEAVEDAMPLIFIED PolymorphicSequence, CAPS) markers. However, these methods have limitations such as low throughput and high cost. KASP (competitive allele-specific PCR, kompetitive ALLELE SPECIFIC PCR) is a technique proposed by LGC (government chemist laboratories) in the uk for accurate double allele determination of SNPs and InDels at specific sites in the genome. The KASP molecular marker has the advantages of high stability, high accuracy, low cost, rapidness, high efficiency and the like, and particularly has the most obvious application characteristics when the sample size is large and SNP sites are few. Chinese patent application of invention (publication No. CN110760608A, publication No. 2020-02-07) filed by the applicant discloses two pairs of novel high oleic acid allelic mutations (BnAfad a and BnCfad a) of the BnFAD gene in brassica napus. The nucleotide sequences of the genes are shown in SEQ ID NO. 1 and SEQ ID NO. 2. The invention also provides two pairs of specific KASP molecular markers KASP-421 and KASP-1073 which can rapidly screen the novel high oleic acid allelic mutation sites, and the markers are used for single plant genotype identification and high oleic acid strain screening. The two pairs of KASP molecular markers provided by the method are developed from SNP variation of exon regions of the BnFAD gene of rape, the specificity of the screening result is higher, and the detection result is more accurate and reliable. The KASP molecular marker provided by the met