Identification and application of rape low-temperature resistance associated molecular marker

Abstract

The invention discloses identification of a molecular marker associated with low-temperature resistance of rape and application of the molecular marker, a major gene locus of an oil content character located in a C06 chromosome of the rape is screened out, and two molecular markers LTRSWU6C06M1 and LTRSWU6C06M2 which are closely linked with the major gene locus are developed. Whether an SWU6 derivative line contains the major QTL site or not is detected through the two molecular markers, then a low-temperature resistant line is rapidly screened for rape breeding, the assistant breeding selection target is clear, and the cost is saved.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular marker technology, specifically relating to the identification and application of molecular markers associated with low-temperature resistance in rapeseed. Background Technology

[0002] Rapeseed, as my country's largest oilseed crop, accounts for over 50% of domestic oilseed production. It plays a crucial role in ensuring the effective supply of edible vegetable oils and feed protein, improving food structure, and promoting the development of the livestock and processing industries. Rapeseed does not compete with major grain crops for land, and its potential for yield and acreage development is enormous. The Yangtze River Basin is a major rice and rapeseed growing area in my country, accounting for approximately 30% of the global planting area and total output. The rice-rapeseed rotation system in the Yangtze River Basin leads to crop rotation conflicts, resulting in delayed rapeseed sowing. Late sowing makes rapeseed more susceptible to the effects of low temperatures. Statistical data shows that rapeseed yield reductions due to low-temperature damage in the middle and lower reaches of the Yangtze River range from 15% to 30%, with severe areas experiencing yield losses exceeding 50%. Autumn and winter cold damage slows rapeseed seedling growth, reduces growth volume, and makes them vulnerable to frost damage, affecting their safe overwintering and ultimately leading to yield decline. Therefore, improving rapeseed's low-temperature resistance has become key to the genetic improvement of early-maturing rapeseed in double-cropping rice areas and late-sown rapeseed in japonica rice producing areas.

[0003] Studies have shown that low temperatures affect multiple stages of rapeseed growth and development. During the germination stage, low-temperature stress leads to a gradual decrease in seed germination rate, germination potential, germination index, and vigor index. Reactive oxygen species (ROS) within rapeseed seeds significantly increase, inducing elevated activity of intracellular antioxidant enzymes SOD, CAT, POD, and APX to mitigate ROS damage to cells. In the seedling stage, low-temperature stress inhibits biomass accumulation, particularly root growth and development. Furthermore, while the dry-to-fresh weight of rapeseed seedlings decreases, the dry-to-fresh ratio is generally higher than that of normal seedlings, indicating dehydration, reduced respiration, and slower nutrient absorption in seedlings under low temperatures.

[0004] my country has a solid foundation in rapeseed research and breeding. However, because most important agronomic traits exhibit quantitative genetic characteristics, they are easily influenced by environmental conditions, resulting in poor selection effectiveness. In recent years, breakthroughs in genomics research and the successful application of molecular breeding technologies, such as marker selection, transgenics, and molecular design of varieties, have significantly improved crop genetic breeding levels. Compared with conventional breeding, this technology can determine the presence of target genes by analyzing molecular markers closely linked to them and perform precise mapping. Simultaneously, marker-assisted selection reduces blind selection, shortens breeding cycles, and greatly improves selection efficiency. Currently, there are few reports on QTL mapping for low-temperature resistance in rapeseed. This invention aims to screen major-effect QTLs with a positive effect on low-temperature resistance in rapeseed through genetic analysis and QTL mapping for marker-assisted selection breeding of low-temperature resistance in rapeseed. Summary of the Invention

[0005] The purpose of this invention is to provide two molecular markers associated with low-temperature resistance in rapeseed and their application in the identification or breeding of low-temperature resistance in rapeseed.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The method for identifying major gene loci for low-temperature resistance in rapeseed is as follows: a) Hybridize rapeseed varieties SWU6 (resistant to low temperature) and J904 (not resistant to low temperature), and self-pollinate the F1 generation to produce the segregating F2 generation; b) Plant the parent plants and F2 generation segregating populations, and select 30 plants from each of the extreme materials that are resistant to low temperature (i.e., single plants that survive after late sowing and do not turn purple) and those that are not resistant to low temperature (i.e., single plants that turn purple or even fail to survive after late sowing). c) The leaves of the extreme materials from the F2 generation segregating population were divided into R pools and S pools, and their DNA was extracted and mixed in equal amounts to construct a mixed pool; the parents and mixed samples were resequencing the whole genome using high-throughput sequencing technology. d) Sequencing data quality control and filtering: Trimmomatic software was used to filter and control the data, and samtools mpileup was used to detect SNPs and InDel. e) Use the software ANNOVAR to annotate SNPs / Indels for genomic loci and mutation types: The SNP-index algorithm and the ED algorithm were used to perform marker association analysis to screen for candidate regions that were detected in common. Finally, a major gene locus for the low-temperature tolerance trait of rapeseed was screened in the scaffoldC06 42900001bp-47370001bp interval.

[0007] Further, InDel molecular markers LTRSWU6C06M1 and LTRSWU6C06M2, linked to the major gene locus for low-temperature tolerance in rapeseed, were developed. The nucleotide sequence of the LTRSWU6C06M1 molecular marker is shown in SEQ ID NO. 1, and the nucleotide sequence of the LTRSWU6C06M2 molecular marker is shown in SEQ ID NO. 2. Rapeseed containing either LTRSWU6C06M1 or LTRSWU6C06M2 molecular markers exhibits low-temperature tolerance, while rapeseed without these molecular markers exhibits low-temperature intolerance.

[0008] Two pairs of primers were developed for the aforementioned InDel molecular markers. The primer sequences for the LTRSWU6C06M1 molecular marker are shown in SEQ ID NO. 5 and 6, and the primer sequences for the LTRSWU6C06M2 molecular marker are shown in SEQ ID NO. 7 and 8. The low-temperature resistance of rapeseed can be determined based on the length of the PCR amplified bands. If the primers for the LTRSWU6C06M1 molecular marker amplify only a 1151 bp band, or the primers for the LTRSWU6C06M2 molecular marker amplify only a 1492 bp band, it indicates that the rapeseed is low-temperature resistant. If the primers for the LTRSWU6C06M1 molecular marker amplify only a 465 bp band, or the primers for the LTRSWU6C06M2 molecular marker amplify only a 577 bp band, it indicates that the rapeseed is not low-temperature resistant.

[0009] Compared with the prior art, the present invention has the following advantages and effects: In traditional breeding methods, phenotypic identification requires waiting until the seeds are harvested, planted into seedlings, and observed in low-temperature incubators or in fields after sowing, growing under low-temperature winter conditions. This method is highly susceptible to environmental influences. Therefore, breeding rapeseed for low-temperature tolerance is not only time-consuming but also difficult and costly. This invention, for the first time, locates the major QTL locus for low-temperature tolerance in rapeseed resources and further develops two molecular markers, LTRSWU6C06M1 and LTRSWU6C06M2, closely linked to the major gene locus. By detecting molecular markers associated with low-temperature tolerance, the level of low-temperature resistance can be predicted, allowing for elimination at the seedling stage. This not only saves production costs but also greatly improves selection efficiency, enabling rapid screening of low-temperature resistant lines for rapeseed low-temperature tolerance breeding. Attached Figure Description

[0010] Figure 1 Phenotypes of the F2 population obtained from the parents SWU6 and J904 and the F1 hybrid seeds.

[0011] Figure 2 Distribution of ΔSNP-index on the genome between the two pools.

[0012] Figure 3 ED distribution across the genome between the two pools.

[0013] Figure 4 Gel electrophoresis image of LTRSWU6C06M1 labeled in F2 generation lines. Lanes 1, 4, and 9 represent cryogenic lines.

[0014] Figure 5 Gel electrophoresis image of LTRSWU6C06M2 labeled in F2 generation lines. Lanes 1, 4, and 9 represent cryogenic lines. Detailed Implementation

[0015] Example 1: Discovery of the major gene locus LTRSWU6C06 for low-temperature tolerance in rapeseed 1. Construction and phenotypic investigation of the F2 segregating population of the low-temperature tolerant rapeseed line combination SWU6 and the low-temperature intolerant rapeseed line J904. The population used in this embodiment was the F2 generation, a hybrid offspring of the cold-resistant SWU6 and the cold-intolerant J904 rapeseed line (a publicly disclosed biological material). The parental lines, F1, and F2 representative types (plant size, leaf color (purple or green)) could be directly observed with the naked eye. The F2 segregating population exhibited three phenotypes: SWU6-like, SWU6×J904 heterozygous, and J904-like. Based on these different phenotypes, the F2 generations were named F2-SWU6, F2-J904, and F2-SWU6×J904 (…). Figure 1 As shown in the figure, the field phenotype of F2-SWU6 is similar to that of the late-sown parent SWU6, with green leaves and stems. The phenotype of F2-J904 is similar to that of the late-sown parent J904, with stunted plant development, severe purple discoloration of leaves or stems, and even death. The field phenotype of F2-SWU6×J904 is intermediate in color between the two parents, and the plant size is similar to that of the parent SWU6. Based on the F2 generation segregating population phenotype, the ratio of resistant: moderately resistant: non-resistant is approximately 1:2:1, which is a qualitative trait.

[0016] 2. Leaf sampling and DNA extraction from parental lines and F2 segregating populations of materials resistant or non-resistant to low temperatures. Based on phenotypic selection, leaves were taken from both cold-tolerant and cold-intolerant materials in the F2 population. DNA was extracted from the leaves of the parents and the extreme F2 materials using the CTAB method. The specific steps are as follows: A. Take an appropriate amount of leaf sample and immediately place it in a frozen mortar. Add liquid nitrogen and grind it into powder. Quickly transfer it into a 50ml centrifuge tube, add the preheated extraction solution (0.2M Tris-Cl, 0.25M NaCl, 25mM EDTA, 0.5% SDS, pH 7.5) in a 60℃ water bath, mix well, and place in a 60℃ water bath for 40 min. B. Remove the centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1, V / V), slowly invert the centrifuge tube up and down 30-50 times to mix thoroughly, and centrifuge at 1300×g for 10 minutes. C. Transfer the supernatant to another centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1, V / V), and extract again; take the supernatant and add 0.6 times the volume of ice-cold isoamyl alcohol, slowly invert the centrifuge tube until flocculent precipitate forms; let stand for 30 min, pick out the precipitate, wash 2-3 times with 70% alcohol, wash once with anhydrous ethanol, dry, and dissolve in sterile water at 65℃ for 20 min; D. Add an equal volume of chloroform:isoamyl alcohol (24:1, V / V) again and extract again; take the supernatant, add 0.1 times NaAc (3mol / L, pH 5.2), mix well, and slowly add 2 volumes of ice-cold anhydrous ethanol. Let stand for 5 min, then slowly rotate the centrifuge tube until flocculent precipitate appears. Pick out the precipitate and transfer it to a 1.5 ml centrifuge tube. Wash 2-3 times with 70% ethanol, wash once with anhydrous ethanol, dry, and dissolve in sterile water. Take 30 DNA samples each from cryogenic extreme materials and cryogenic extreme materials respectively, and mix equal volumes according to the DNA concentration of each sample to form two pools.

[0017] 3. Genome resequencing and data analysis of SWU6, J904 and F2 extreme materials DNA from mixed pools of SWU6, J904, and F2 extreme materials was resequencing and analyzed as follows: (1) Trimmomatic software was used for data filtering and quality control. The parameters were: LEADING:3, TRAILING:3, SLIDINGWINDOW:4:15, MINLEN:120. Genome alignment: Clean data was aligned to the rapeseed double 11 genome (http: / / cbi.hzau.edu.cn / bnapus / ) using BWA. The alignment results were deduplicated using samtools (rmdup). After quality control filtering, the Q30 was above 96%, the aligned data covered 32×-35× of the genome, and 86% of the genome regions had a sequencing depth ≥4×.

[0018] (2) SNP detection was performed using the samtools tool mpileup to screen for loci with a depth ≥ 6 and a quality ≥ 20. SNP detection and filtering were performed on each sample, and a total of 1,385,173 SNP loci that were homozygous within the parental line and segregated between the parents were identified.

[0019] (3) SNP-index algorithm for trait-label association analysis: The SNP-index algorithm used a sliding window method with a window size of 1 Mb and a step size of 10 Kb to calculate the SNP-index (SNP frequency) and frequency difference between the two mixed pools. The analysis results are shown in Figure 2 The ΔSNP-index distribution on chromosome C06 is as follows: gray dots represent the SNP frequency of each SNP locus, pink and green dots represent the SNP difference frequency of each window, light blue represents the 95% confidence level, and dark blue represents the 99% confidence level. Window values ​​greater than the threshold (99%) at the 99% confidence level are selected as candidate intervals. Overlapping windows are merged to obtain preliminary localization of trait-related regions: scaffoldC06: 27690001bp-36830001bp; 40890001bp-49870001bp.

[0020] (5) ED algorithm for BSA association analysis: For the SNP sites screened in (2) above, the Euclidean distance of each site in the two progeny pools is calculated. ED is an abbreviation for Euclidean distance, which uses the distance difference to reflect the linkage strength between the marker and the target region by calculating the frequency distance between each mutant in different pools. In the ED algorithm analysis, the candidate intervals were detected with a threshold of 0.475 (99th percentile): scaffoldC06: 27690001bp-36830001bp, 40890001bp-49870001bp ( Figure 3 In the ED algorithm analysis, a threshold of 0.673 (99.5th percentile) was used to filter the data, resulting in a further narrowed interval scaffoldC06: 42900001bp-47370001bp, with a total length of approximately 4.5 Mb. This sequence was named LTRSWU6C06.

[0021] 4. Identification of the markers for the correlation intervals of low-temperature resistance traits Sequence alignment of the scaffoldC06 region (42900001bp-47370001bp) of the two parental materials SWU6 and J904 revealed that the major gene locus is located on chromosome C06 of SWU6. The InDel markers linked to this locus are LTRSWU6C06M1 and LTRSWU6C06M2, both exhibiting insertion / deletion variations in both materials. In the LTRSWU6C06M1 marker, compared to the cryogenically intolerant material J904, the cryogenically tolerant material SWU6 has an inserted 686 bp sequence, the nucleotide sequence of which is shown in SEQ ID NO.1. In the LTRSWU6C06M2 marker, the cryogenically tolerant material SWU6 has an inserted 915 bp sequence, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0022] Two pairs of primers were developed for the LTRSWU6C06M1 and LTRSWU6C06M2 markers. The primer sequences for the LTRSWU6C06M1 marker are shown in SEQ ID NO.5 and 6, and the primer sequences for the LTRSWU6C06M2 marker are shown in SEQ ID NO.7 and 8. After PCR amplification, the primers were genotyped by agarose gel electrophoresis for marker-assisted selection breeding.

[0023] Example 2: Application of molecular markers LTRSWU6C06M1 and LTRSWU6C06M2 in low-temperature resistance breeding DNA was extracted from the leaves of seedlings of the F2 generation after crossing SWU6 with the cultivar Zhongshuang 11. Specific primers for the molecular markers LTRSWU6C06M1 and LTRSWU6C06M2 were used for amplification. The PCR reaction system consisted of a 20 μl system containing 1 μl DNA template (50 ng / μl), 1 μl each of forward and reverse primers (10 μmol each), 0.5 μl dNTPs, 0.5 μl Taq polymerase (5 U / μl), 2 μl MgCl2, and 14 μl ddH2O. The reaction time and temperature were as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 45 s, 62℃ annealing for 45 s, 72℃ extension for 30 s, repeated 30 times; followed by a final extension at 72℃ for 5 min. PCR products were detected by 2% agarose gel electrophoresis.

[0024] The F2 segregating population was amplified using two labeled specific primers, and the low-temperature resistance of rapeseed was determined based on the length of the PCR amplification bands. Figure 4As shown, the specific primers labeled LTRSWU6C06M1 amplified only one 1151 bp band (SEQ ID NO. 3), indicating that the rapeseed seedling has strong low-temperature resistance; the specific primers labeled LTRSWU6C06M1 amplified only one 465 bp band, indicating that the rapeseed seedling is not resistant to low temperatures; the specific primers labeled LTRSWU6C06M1 amplified both 1151 bp and 465 bp bands, indicating that the rapeseed seedling is heterozygous and has moderate low-temperature resistance. Similarly, if... Figure 5 As shown, the specific primers labeled TRSWU6C06M2 amplified only one 1492 bp band (SEQ ID NO.4), indicating that the rapeseed seedling has strong low-temperature resistance; if the specific primers labeled TRSWU6C06M2 amplified only one 577 bp band, it indicates that the rapeseed seedling has no low-temperature resistance; if the specific primers labeled TRSWU6C06M2 amplified both 1492 bp and 577 bp bands, it indicates that the rapeseed seedling is heterozygous and has moderate low-temperature resistance. The above molecular marker identification results are consistent with the phenotypic identification results.

Claims

1. The application of molecular markers in the identification or breeding of low-temperature resistance in rapeseed, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1 or 2.

2. The application according to claim 1, characterized in that, Rapeseed containing the molecular marker shown in SEQ ID NO.1 or 2 exhibits low-temperature tolerance, while rapeseed without the molecular marker shown in SEQ ID NO.1 or 2 exhibits low-temperature intolerance.

3. A specific primer for detecting the molecular marker of claim 1, characterized in that, The primer sequences are shown in SEQ ID NO.5 and 6, or SEQ ID NO.7 and 8.

4. The application of the primers described in claim 3 in the identification or breeding of low-temperature resistance in rapeseed, characterized in that, Primers shown in SEQ ID NO. 5 and 6 amplified a 1151 bp band in low-temperature resistant rapeseed and a 465 bp band in low-temperature intolerant rapeseed; primers shown in SEQ ID NO. 7 and 8 amplified a 1492 bp band in low-temperature resistant rapeseed and a 577 bp band in low-temperature intolerant material.

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