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CN-122012802-A - Molecular marker related to neutral washing fiber content of alfalfa and application thereof

CN122012802ACN 122012802 ACN122012802 ACN 122012802ACN-122012802-A

Abstract

The invention belongs to the technical field of molecular biology, and relates to a molecular marker related to neutral washing fiber content of alfalfa and application thereof. The invention aims to solve the technical problem that the traditional breeding mode has long period. The technical scheme of the invention is a molecular marker related to the content of neutral washing fiber of alfalfa, and specific information is that chromosome is Chr7, position is 52020445, and SNP typing is T/G. The molecular marker and the KASP parting primer can finish genotype screening at the seedling stage, can select plants without waiting for maturity, and greatly shortens the breeding period.

Inventors

  • CHEN LIN
  • ZHANG YUQI
  • MA XINYUE
  • LEI HAIYUE
  • ZHAO LI
  • Xiao Zhengqin

Assignees

  • 中国农业科学院北京畜牧兽医研究所

Dates

Publication Date
20260512
Application Date
20260414

Claims (9)

  1. 1. A molecular marker related to the content of neutral washing fiber of alfalfa is characterized by comprising the specific information of chromosome Chu 7, position 52020445 and SNP typing of T/G.
  2. 2. The molecular marker of claim 1, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID No.1 or SEQ ID No. 2.
  3. 3. The primer combination for KASP detection and typing by using the molecular marker as claimed in claim 1 or 2, wherein the nucleotide sequence is shown in SEQ ID No. 3-5.
  4. 4. A molecular marker detection kit comprises primers shown in SEQ ID No. 3-5.
  5. 5. A molecular marker chip comprises primers shown in SEQ ID No. 3-5.
  6. 6. Use of the molecular marker of claim 1 or 2, the primer combination of claim 3, the kit of claim 4 and/or the molecular marker chip of claim 5 in an optional one of: a. Predicting the neutral washing fiber content of alfalfa; b. identifying and screening alfalfa with different neutral washing fiber contents; c. molecular marker assisted breeding of alfalfa; d. Breeding alfalfa; e. preparing alfalfa breeding products.
  7. 7. A method for screening alfalfa materials with different neutral washing fiber contents or predicting the neutral washing fiber contents of alfalfa is characterized by comprising the following steps of extracting genome DNA of the alfalfa materials to be detected, amplifying molecular markers by using primers shown in SEQ ID No. 3-5, sequencing amplified products, and parting and screening.
  8. 8. The method of claim 7, wherein the amplification process is performed at 94℃for 15min, at 95℃for 20sec, at 65 to 56℃for 60sec, at 10 cycles, and the annealing extension temperature is reduced by-0.8℃for each cycle, at 94℃for 20sec, at 57℃for 60sec, and at 10 cycles.
  9. 9. The method according to claim 7, wherein the typing screening is performed on a material having a low neutral detergent fiber content if the typing result is TT, and on a material having a high neutral detergent fiber content if the typing result is TG or GG.

Description

Molecular marker related to neutral washing fiber content of alfalfa and application thereof Technical Field The invention belongs to the technical field of molecular biology, and relates to a molecular marker related to neutral washing fiber content of alfalfa and application thereof. Background The alfalfa is used as an irreplaceable high-quality coarse fodder in cow cultivation, and the quality of the alfalfa directly influences the dry matter feed intake (DMI) of cows, so that the performance of milk production is determined. In alfalfa quality assessment systems, neutral Detergent Fiber (NDF) is the main component constituting the alfalfa cell wall, and contains cellulose, hemicellulose and lignin, the content of which directly determines the dry matter feed intake of alfalfa. Unlike acid washing fiber (ADF) which mainly affects digestibility, NDF is a primary factor for limiting milk cow feed intake, wherein the higher the NDF content is, the stronger the filling effect of feed in rumen is, and the more easily the milk cow is satiety, so that dry matter intake is limited, and further, the development of milk production potential is inhibited. The NDF content of quality alfalfa is typically controlled below 40% and requires a high proportion of digestible parts and a low proportion of non-digestible parts (e.g., lignin). Therefore, the alfalfa variety with low NDF and high digestibility is bred and becomes a key break for balancing feed intake and energy supply. However, NDF content is a quantitative trait controlled by multiple genes, traditional breeding relies on phenotypic screening, and requires multiple generations of field planting and continuous observation for over 10 years to obtain genetically stable elite lines, and it is difficult to effectively distinguish genotype-environment interactions. The molecular marker assisted selection is to indirectly select in the seedling stage by positioning QTL (quantitative trait locus) related to the NDF content and utilizing the molecular marker, so that the breeding period is shortened to 3-5 years, the seed selection accuracy is remarkably improved, and the technical support is provided for rapidly cultivating the breakthrough alfalfa variety with low NDF and high digestibility. Disclosure of Invention The invention aims to solve the technical problem that the traditional breeding mode has long period. The technical scheme of the invention is a molecular marker related to the content of neutral washing fiber of alfalfa, and specific information is that chromosome is Chr7, position is 52020445, and SNP typing is T/G. Specifically, the nucleotide sequence of the molecular marker is shown as SEQ ID No.1 or SEQ ID No. 2. The invention also provides a primer combination for KASP detection and typing of the molecular marker, and the nucleotide sequence of the primer combination is shown as SEQ ID No. 3-5. The invention also provides a molecular marker detection kit, which comprises primers shown in SEQ ID No. 3-5. The invention also provides a molecular marker chip, which comprises primers shown in SEQ ID No. 3-5. The invention also provides the application of the molecular marker, a primer combination for amplifying the molecular marker, the kit and/or the molecular marker chip in optional one item: a. Predicting the neutral washing fiber content of alfalfa; b. identifying and screening alfalfa with different neutral washing fiber contents; c. molecular marker assisted breeding of alfalfa; d. Breeding alfalfa; e. preparing alfalfa breeding products. The invention also provides a method for screening alfalfa materials with different neutral washing fiber contents or predicting the neutral washing fiber contents of alfalfa, which comprises the following steps of extracting genome DNA of the alfalfa materials to be detected, amplifying molecular markers by adopting the primers shown in SEQ ID No. 3-5, sequencing amplified products, and screening in a parting mode. Specifically, the amplification procedure is as follows, 94 ℃ for 15min, 95 ℃ for 20sec, 65-56 ℃ for 60sec,10 cycles, each cycle annealing extension temperature reduced by-0.8 ℃, 94 ℃ for 20sec,57 ℃ for 60sec,10 cycles. The parting screening standard is that if the parting result is TT, the parting screening is a material with low neutral washing fiber content, and if the parting result is TG or GG, the parting screening is a material with high neutral washing fiber content. The invention has the beneficial effects that through sequencing and analyzing 165 alfalfa germplasm materials, SNP loci which are positioned near 52.0 Mb of alfalfa genome Chr7 chromosome and are obviously related to neutral washing fiber content are obtained. KASP typing primer is developed aiming at the locus, 81 parts of materials are detected, and the result shows that the neutral washing fiber content of the materials with different genotypes is obviously different. The molecular marker and the KASP parting primer can fini