Molecular markers associated with alfalfa taproot length and their applications
By designing KASP primers at SNP site 13162265 on chromosome Chr4 of alfalfa, the problem of difficulty in rapidly screening alfalfa varieties with well-developed root systems in traditional breeding methods was solved, and efficient and accurate breeding was achieved in the seedling stage.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INSTITUTE OF ANIMAL SCIENCES OF CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2026-04-14
- Publication Date
- 2026-07-31
AI Technical Summary
Traditional breeding methods make it difficult to quickly screen and cultivate alfalfa varieties with well-developed root systems without damaging the plants, and the breeding cycle is long and the operation is difficult.
Molecular markers associated with the length of the taproot of alfalfa were developed, specifically the SNP locus 13162265 on chromosome Chr4. KASP detection primers were designed for genotyping and screening, and molecular marker-assisted selection technology was used to identify genotypes in the seedling stage.
It enables rapid and accurate screening and prediction of alfalfa taproot length during the seedling stage, significantly shortening the breeding cycle, improving selection efficiency and accuracy, and eliminating environmental interference.
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Figure CN122060911B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology and relates to molecular markers associated with the length of alfalfa taproot and their applications. Background Technology
[0002] As a perennial leguminous forage crop, alfalfa's root system development directly determines the plant's drought resistance, overwintering ability, and nutrient absorption efficiency, playing a crucial role in ensuring stable and high yields in arid and semi-arid regions. Among root traits, root length is a key indicator of alfalfa's ability to utilize deep soil moisture and nutrients. Longer roots can penetrate deep into the soil to absorb water and nutrients, enhancing the plant's drought and cold resistance. Simultaneously, root nodules fix nitrogen, providing nitrogen reserves for subsequent crops, forming the dual foundation for alfalfa's ecological and forage value. The importance of root length is particularly pronounced in marginal land conditions such as saline-alkali land and arid areas. However, genetic improvement of root length has long faced technical bottlenecks. Traditional breeding methods mainly rely on field excavation and testing, requiring destructive sampling of plants, making it impossible to simultaneously evaluate the root system and propagate seeds on the same plant, resulting in a lengthy breeding cycle and extreme operational difficulties. Molecular marker-assisted selection (MMR) identifies genotypes in seedlings by identifying molecular markers closely linked to root length. This allows for indirect selection of superior root traits without damaging the plant, providing an efficient technical pathway for accelerating the breeding of new alfalfa varieties with well-developed root systems and strong adaptability. Summary of the Invention
[0003] The technical problem to be solved by this invention is to provide a new option for rapidly breeding alfalfa varieties with well-developed root systems.
[0004] The technical solution of this invention is a molecular marker associated with the length of the taproot of alfalfa, with the following specific information: Chromosome: Chr4; Location: 13162265; SNP type: C / G.
[0005] Specifically, the nucleotide sequence of the molecular marker is shown in SEQ ID No. 1 or SEQ ID No. 2.
[0006] The present invention also provides primer combinations for KASP detection and genotyping of the molecular markers, the nucleotide sequences of which are shown in SEQ ID No. 3-5.
[0007] The present invention also provides a molecular marker detection kit, comprising primers shown in SEQ ID No. 3-5.
[0008] The present invention also provides a molecular marker chip, comprising primers shown in SEQ ID No. 3-5.
[0009] The present invention also provides the use of the molecular marker, the primer combination for amplifying the molecular marker, the kit and / or the molecular marker chip in any one of the following: a. Predicting the taproot length of alfalfa; b. Identification and screening of alfalfa with different taproot lengths; c. Molecular marker-assisted breeding of alfalfa; d. Alfalfa breeding; e. Prepare products for alfalfa breeding.
[0010] The present invention also provides a method for screening alfalfa materials with different taproot lengths or predicting the taproot length of alfalfa, comprising the following steps: extracting genomic DNA from the alfalfa material to be tested, amplifying molecular markers using primers described in SEQ ID No. 3~5, sequencing the amplified products, and screening by typing.
[0011] Specifically, the amplification program is as follows: 94℃ for 15 min; 95℃ for 20 sec, 65~56℃ for 60 sec, 10 cycles, with the annealing extension temperature decreasing by -0.8℃ in each cycle; 94℃ for 20 sec, 57℃ for 60 sec, 10 cycles.
[0012] Specifically, the criteria for genotyping and screening are as follows: if the genotyping result is CC, it is a material with a long main root; if the genotyping result is CG or GG, it is a material with a short main root.
[0013] The beneficial effects of this invention are as follows: By sequencing and analyzing 165 alfalfa germplasm materials, this invention obtained a SNP locus located near chromosome 13.16 Mb of the alfalfa genome (Chr4) that is significantly associated with taproot length. KASP genotyping primers were designed for this locus. Based on the designed KASP primers, 78 materials were tested, and the results showed that there were significant differences in taproot length among different genotypes. Using the molecular markers and KASP genotyping primers of this invention, genotype screening can be completed at the seedling stage, without waiting for plant maturity, significantly shortening the breeding cycle. It also eliminates the interference of environmental factors, significantly improving the accuracy and efficiency of target trait selection. The molecular markers of this invention have great application value, enabling pre-selection of taproot length in test materials and molecular-assisted breeding, and have important theoretical and practical significance for the genetic improvement of alfalfa breeding with superior root systems. Attached Figure Description
[0014] Figure 1 1. Identification of sites significantly associated with primary root length.
[0015] Figure 2 KASP marker typing results at Chr4_13162265 site.
[0016] Figure 3 Comparative analysis of primary root length of materials with different allelic genotypes. Detailed Implementation
[0017] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.
[0018] Example 1: Obtaining Significantly Associated Sites The experimental population consisted of 165 alfalfa germplasm materials, including those from different regions and with different phenotypes. Considering the variability among individual plants of the same alfalfa variety, cuttings were taken from the germplasm resource nursery in 2022 and propagated by cuttings in a greenhouse. Each material was transplanted into 9 pots, with 2 clones planted in each pot, for a total of 18 plants. After two rounds of mowing, 6 pots with consistent growth were selected for phenotypic data collection to ensure accuracy. Normal water management was maintained during the experiment. Root phenotype was measured after 40 days of growth. For root phenotype measurement, the plants were gently shaken to remove most of the soil attached to the roots, and then the roots were immersed in water to remove residual soil and fibrous roots. The root system of each material was scanned using the WinRHIZO root analysis system to obtain the taproot length.
[0019] 100 mg of young alfalfa leaves were selected, flash-frozen in liquid nitrogen, and then total plant DNA was extracted using the CWBIO Plant Genomic DNA Extraction Kit (Cowin Biosciences, Taizhou, China) according to the manufacturer's instructions. The concentration and quality of the total DNA were detected using a micro spectrophotometer (Nano Photometer NP80, Germany), and then sequenced on the BGIDNBSEQ next-generation sequencing platform (BGI, Shenzhen, China).
[0020] Each material yielded approximately 30.5 GB of raw data. The raw sequencing data was processed using Trimmomatic (version 0.39) (Bolger et al., 2014) software to remove the adapter and filter out low-quality base sequences. The cleaned sequencing data was then aligned to the haplotype reference genome of 'Zhongmu 1' alfalfa using BWA-MEM. SAMtools (version 1.13) was used to filter multiple alignments and low-quality sequences, resulting in filtered BAM files, which were then sorted. PCR repetitive sequences were marked using the Mark Duplicates function in the Picard toolkit (version 2.23.0). Finally, the processed BAM files were used for variant detection using GATK Haplotype Caller (version 4.2.3.062). The following parameters were set as screening criteria in the SNP filtering strategy: Quality By Depth (QD) less than 2.0, Fisher Strand (FS) greater than 60.0, MQ Rank Sum Test less than -12.5, Read PosRank Sum less than -8.0, SOR (Strand Odds Ratio) greater than 3.0, and Mapping Qualities (MQ) less than 40.0. Subsequently, the selection criteria for a subset of alfalfa SNP markers were defined, including: (1) using VCFtools (version 0.1.16) to remove SNP markers with a deletion rate greater than 20% and a minor allele frequency (MAF) less than 0.05%, thereby creating a basic SNP set containing only biallelic SNP markers; (2) using Plink (version 1.90b6.21) to filter the basic SNP set based on linkage disequilibrium (LD), setting the LD filter window size to 100 SNPs, the window step size to 50 SNPs, and the r2 threshold to 0.2, to obtain a core SNP marker set. The missing genotypes in the LD-filtered SNP marker set were then filled using Beagle software with default parameters. Similarly, VCFtools was used to filter Indel markers, retaining only biallelic markers with a deletion rate less than 20% and a MAF greater than 0.05%.
[0021] GWAS analysis was performed using 2,821,247 high-quality SNP markers. To ensure the accuracy and reliability of the analysis, various software and statistical models were used: TASEEL 5.0 (GLM and MLM) (Bradbury PJ et al., 2007), BLINK (v1.01) (Huang et al., 2019). QQ plots (quantile-quantile plots) are used to measure the reliability of the model. By comparing QQ plots generated by different models, the GLM model in TASSEL 5.0 performed better. Therefore, the GLM model in TASSEL 5.0 was selected to present the final results. Principal component analysis was performed on the SNP data, and the first three principal component variables were used as covariates to correct for population structure. The Manhattan plot visualization was performed using the R package CMplot (Yin et al., 2021). A locus significantly associated with primary root length was found on chromosome 4, with a LOD value of 9.52 and an explainable phenotypic contribution of 7.69% (…). Figure 1 ).
[0022] Example 2: Development of the KASP marker at the Chr4_13162265 locus The Chr4_13162265 locus is located near chromosome 13.16 Mb in the alfalfa genome. The upstream and downstream reference sequences are shown in SEQ ID No. 1 and SEQ ID No. 2. Specific information about the Chr4_13162265 locus is as follows: Chromosome: chr4; Location: 13162265; SNP genotype: G / C.
[0023] SEQ ID No. 1, where lowercase letters represent SNP sites; TAAGCAGCCATAACATTCGTAGTGTTGATTGGAATCTTGTTGTAAATAGAGATTTGAAAATGATATAGCGAAATGGTTCGTCAATGGCTGAGGTAAATACATATCAATCATGTTGCTCACTTTAGTGCAAATTTTTTCTCTCTTGCAGCAAGAGTCTTGCTTTTTCTTTACATCTTGGTATCTTTCCTTGTGTTGCAAGAATgCATTTGAAAGCTCAGTTACTTCTTCCATATTGGAAACTCAGGTTGTGCTAGCATGTAGCACTAGAATAGTGCCTACTGCTAATATAGATCCTCTAATGTGGTTAGCTTTCTTAAAGGTATTGGAGTTTTCTTTTCGTTTTTGCCTCAATCAAATCGAGCAGTATTATCTTAATGATATGCAGTAGAATGTTTCTTATGTCTCAATCAATACTATTGTAGGTTATTGAAGGTGAAAGTGT。
[0024] SEQ ID No.2, where lowercase letters represent SNP sites; TAAGCAGCCATAACATTCGTAGTGTTGATTGGAATCTTGTTGTAAATAGAGATTTGAAAATGATATAGCGAAATGGTTCGTCAATGGCTGAGGTAAATACATATCAATCATGTTGCTCACTTTAGTGCAAATTTTTTCTCTCTTGCAGCAAGAGTCTTGCTTTTTCTTTACATCTTGGTATCTTTCCTTGTGTTGCAAGAATcCATTTGAAAGCTCAGTTACTTCTTCCATATTGGAAACTCAGGTTGTGCTAGCATGTAGCACTAGAATAGTGCCTACTGCTAATATAGATCCTCTAATGTGGTTAGCTTTCTTAAAGGTATTGGAGTTTTCTTTTCGTTTTTGCCTCAATCAAATCGAGCAGTATTATCTTAATGATATGCAGTAGAATGTTTCTTATGTCTCAATCAATACTATTGTAGGTTATTGAAGGTGAAAGTGT。
[0025] KASP primers were designed for the Chr3_76671147 site and flanking sequence using Bacthprimer 3 software. Each KASP marker consisted of two specific primers and one universal primer. Fluorescent adapter sequences were ligated to the 5' end of the specific primers (GAAGGTGACCAAGTTCATGCT is the FAM fluorescent adapter sequence; GAAGGTCGGAGTCAACGGATT is the HEX fluorescent adapter sequence).
[0026] Specific primer 1 (SEQ ID No. 3): GAAGGTGACCAAGTTCATGCTGTATCTTTCCTTGTGTTGCAAGAATG; Specific primer 2 (SEQ ID No. 4): GAAGGTCGGAGTCAACGGATTGTATCTTTCCTTGTGTTGCAAGAATC; Universal primer (SEQ ID No. 5): ACATGCTAGCACAACCTGAGTTTCCA.
[0027] Example 3: Application of the Chr4_13162265 locus Seventy-eight alfalfa germplasm materials were planted (using the same planting method as in Example 1). Then, 100 mg of young alfalfa leaves were selected, flash-frozen in liquid nitrogen, and total plant DNA was extracted using the CWBIO Plant Genomic DNA Extraction Kit (Cowin Biosciences, Taizhou, China) according to the manufacturer's instructions. The concentration and quality of the total DNA were then determined using a Nano Photometer NP80 (Germany).
[0028] KASP marker validation and detection were performed using Douglas Scientific's ArrayTape system. The ArrayTape genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for signal scanning, and INTELLICS for data analysis.
[0029] The PCR system was assembled using NEXAR, and the PCR reaction system is shown in Table 1.
[0030] Table 1 KASP Reaction System .
[0031] Primer C represents a universal primer, while Primer X and Primer Y represent specific primers 1 and 2, respectively. PCR amplification was performed using a SOELLEX instrument. The touch-down PCR amplification conditions were as follows: 94℃ for 15 min; 95℃ for 20 sec, 65~56℃ for 60 sec, 10 cycles, with the annealing extension temperature decreasing by -0.8℃ per cycle; 94℃ for 20 sec, 57℃ for 60 sec, 10 cycles.
[0032] After the PCR reaction was completed, the fluorescence signal of the reaction system was scanned using ARAYA; then, data analysis and genotyping were performed using INTELLICS. A schematic diagram of the marker genotyping is shown below. Figure 2 As shown in the table, red represents the GG allele at this locus, blue represents the CC allele, and purple represents the G / C heterozygous type. The allele types of the 78 germplasm materials at this locus are shown in Table 2. Statistical analysis of the taproot length of the three allele types was performed using a t-test. The results showed that the taproot length of the CC allele was significantly longer than that of the G / C and GG types (P<0.01) (see Table 2). Figure 3 ).
[0033] Table 2. Allelic types of 78 germplasm materials at this locus. .
[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A primer combination for KASP genotyping of a molecular marker associated with Medicago sativa taproot length for use in identifying and selecting Medicago sativa plants with different taproot lengths, characterized in that: The specific information of the molecular marker is as follows: Chromosome: Chr4; Location: 13162265; SNP genotype: C / G, CC genotype indicates alfalfa with a long main root, CG genotype or GG genotype indicates alfalfa with a short main root; The nucleotide sequence of the molecular marker is shown in SEQ ID No. 1 or SEQ ID No. 2, as follows: SEQ ID No. 1, where lowercase letters represent SNP sites; TAAGCAGCCATAACATTCGTAGTGTTGATTGGAATCTTGTTGTAAATAGAGATTTGAAAATGATATAGCGAAATGGTTCGTCAATGGCTGAGGTAAATACATATCAATCATGTTGCTCACTTTAGTGCAAATTTTTCTCTCTTGCAGCAAGAGTCTTGCTTTTTCTTTACATCTTGGTATCTTTCCTTGTGTTGCAAGAATgCATTTGAAAGCTCAGTTA CTTCTTCCATATTGGAAACTCAGGTTGTGCTAGCATGTAGCACTAGAATAGTGCCTACTGCTAATATAGATCCTCTAATGTGGTTAGCTTTCTTAAAGGTATTGGAGTTTTCTTTTCGTTTTTGCCTCAATCAAATCGAGCAGTATTATCTTAATGATATGCAGTAGAATGTTTCTTATGTCTCAATCAATACTATTGTAGGTTATTGAAGGTGAAAGTGT; SEQ ID No. 2, where lowercase letters represent SNP sites; TAAGCAGCCATAACATTCGTAGTGTTGATTGGAATCTTGTTGTAAATAGAGATTTGAAAATGATATAGCGAAATGGTTCGTCAATGGCTGAGGTAAATACATATCAATCATGTTGCTCACTTTAGTGCAAATTTTTCTCTCTTGCAGCAAGAGTCTTGCTTTTTCTTTACATCTTGGTATCTTTCCTTGTGTTGCAAGAATcCATTTGAAAGCTCAGTTA CTTCTTCCATATTGGAAACTCAGGTTGTGCTAGCATGTAGCACTAGAATAGTGCCTACTGCTAATATAGATCCTCTAATGTGGTTAGCTTTCTTAAAGGTATTGGAGTTTTCTTTTCGTTTTTGCCTCAATCAAATCGAGCAGTATTATCTTAATGATATGCAGTAGAATGTTTCTTATGTCTCAATCAATACTATTGTAGGTTATTGAAGGTGAAAGTGT.
2. A kit containing primer combinations for KASP detection and genotyping of molecular markers associated with alfalfa taproot length, used in the identification and screening of alfalfa with different taproot lengths, characterized in that: The specific information of the molecular marker is as follows: Chromosome: Chr4; Location: 13162265; SNP genotype: C / G, CC genotype for alfalfa with long main root; CG genotype or GG genotype for alfalfa with short main root; The nucleotide sequence of the molecular marker is shown in SEQ ID No. 1 or SEQ ID No. 2, as follows: SEQ ID No. 1, where lowercase letters represent SNP sites; TAAGCAGCCATAACATTCGTAGTGTTGATTGGAATCTTGTTGTAAATAGAGATTTGAAAATGATATAGCGAAATGGTTCGTCAATGGCTGAGGTAAATACATATCAATCATGTTGCTCACTTTAGTGCAAATTTTTTCTCTCTTGCAGCAAGAGTCTTGCTTTTTCTTTACATCTTGGTATCTTTCCTTGTGTTGCAAGAATgCATTTGAAAGCTCAGTTACTTCTTCCATATTGGAAACTCAGGTTGTGCTAGCATGTAGCACTAGAATAGTGCCTACTGCTAATATAGATCCTCTAATGTGGTTAGCTTTCTTAAAGGTATTGGAGTTTTCTTTTCGTTTTTGCCTCAATCAAATCGAGCAGTATTATCTTAATGATATGCAGTAGAATGTTTCTTATGTCTCAATCAATACTATTGTAGGTTATTGAAGGTGAAAGTGT; SEQ ID No.2, where the lowercase letters represent SNP sites; TAAGCAGCCATAACATTCGTAGTGTTGATTGGAATCTTGTTGTAAATAGAGATTTGAAAATGATATAGCGAAATGGTTCGTCAATGGCTGAGGTAAATACATATCAATCATGTTGCTCACTTTAGTGCAAATTTTTTCTCTCTTGCAGCAAGAGTCTTGCTTTTTCTTTACATCTTGGTATCTTTCCTTGTGTTGCAAGAATcCATTTGAAAGCTCAGTTACTTCTTCCATATTGGAAACTCAGGTTGTGCTAGCATGTAGCACTAGAATAGTGCCTACTGCTAATATAGATCCTCTAATGTGGTTAGCTTTCTTAAAGGTATTGGAGTTTTCTTTTCGTTTTTGCCTCAATCAAATCGAGCAGTATTATCTTAATGATATGCAGTAGAATGTTTCTTATGTCTCAATCAATACTATTGTAGGTTATTGAAGGTGAAAGTGT。 3. Use according to claim 1 or 2, characterized in that: The nucleotide sequences of the primer combination are shown in SEQ ID No.3 - 5.
4. A method of screening different taproot lengths of Medicago polymorpha, the method comprising: The procedure includes the following steps: extracting genomic DNA from the alfalfa to be tested, amplifying molecular markers using primers shown in SEQ ID No. 3-5, and screening by typing; the typing criteria are as follows: if the typing result is CC, it is alfalfa with a long taproot; if the typing result is CG or GG, it is alfalfa with a short taproot; the specific information of the molecular markers is as follows: chromosome: Chr4; position: 13162265; SNP typing: C / G; the nucleotide sequence of the molecular markers is shown in SEQ ID No. 1 or SEQ ID No. 2, as follows: SEQ ID No. 1, where lowercase letters represent SNP sites; TAAGCAGCCATAACATTCGTAGTGTTGATTGGAATCTTGTTGTAAATAGAGATTTGAAAATGATATAGCGAAATGGTTCGTCAATGGCTGAGGTAAATACATATCAATCATGTTGCTCACTTTAGTGCAAATTTTTCTCTCTTGCAGCAAGAGTCTTGCTTTTTCTTTACATCTTGGTATCTTTCCTTGTGTTGCAAGAATgCATTTGAAAGCTCAGTTA CTTCTTCCATATTGGAAACTCAGGTTGTGCTAGCATGTAGCACTAGAATAGTGCCTACTGCTAATATAGATCCTCTAATGTGGTTAGCTTTCTTAAAGGTATTGGAGTTTTCTTTTCGTTTTTGCCTCAATCAAATCGAGCAGTATTATCTTAATGATATGCAGTAGAATGTTTCTTATGTCTCAATCAATACTATTGTAGGTTATTGAAGGTGAAAGTGT; SEQ ID No. 2, where lowercase letters represent SNP sites; TAAGCAGCCATAACATTCGTAGTGTTGATTGGAATCTTGTTGTAAATAGAGATTTGAAAATGATATAGCGAAATGGTTCGTCAATGGCTGAGGTAAATACATATCAATCATGTTGCTCACTTTAGTGCAAATTTTTCTCTCTTGCAGCAAGAGTCTTGCTTTTTCTTTACATCTTGGTATCTTTCCTTGTGTTGCAAGAATcCATTTGAAAGCTCAGTTA CTTCTTCCATATTGGAAACTCAGGTTGTGCTAGCATGTAGCACTAGAATAGTGCCTACTGCTAATATAGATCCTCTAATGTGGTTAGCTTTCTTAAAGGTATTGGAGTTTTCTTTTCGTTTTTGCCTCAATCAAATCGAGCAGTATTATCTTAATGATATGCAGTAGAATGTTTCTTATGTCTCAATCAATACTATTGTAGGTTATTGAAGGTGAAAGTGT.
5. The method of claim 4, wherein: The amplification procedure is as follows: 94℃ for 15 min; 95℃ for 20 sec, 65~56℃ for 60 sec, 10 cycles, with the annealing extension temperature decreasing by 0.8℃ in each cycle; 94℃ for 20 sec, 57℃ for 60 sec, 10 cycles.