Application of Brassica napus QTL loci in construction of SNP genetic linkage map for traits related to high nitrogen efficient uptake

Abstract

The present application relates to the field of oilseed rape molecular breeding and biotechnology, and particularly relates to application of QTL site of Brassica napus in construction of SNP genetic linkage map for positioning nitrogen efficient absorption related traits. The present application provides application of QTL site of Brassica napus in construction of SNP genetic linkage map for positioning nitrogen efficient absorption related traits, which obtains a large number of SNP markers with gene sequences by determining phenotype data of nitrogen efficient absorption related traits of Brassica napus and high-throughput genotype data, constructs high-density molecular genetic linkage map of Brassica napus by using these marker sites, and positions nitrogen efficient absorption related traits of Brassica napus, obtains markers significantly associated with traits, mines QTL sites associated with nitrogen efficient absorption related traits, and the positioning site is good in stability and wide in application range, and provides basic information for molecular marker assisted breeding of nitrogen efficient absorption of Brassica napus.

Description

Technical Field

[0001] This invention relates to the fields of rapeseed molecular breeding and biotechnology, and in particular to the application of QTL loci in Brassica napus in constructing SNP genetic linkage maps for the localization of traits related to efficient nitrogen fertilizer absorption. Background Technology

[0002] Brassica napus (Brassica oleracea) is a plant belonging to the genus Brassica in the family Brassicaceae. It is a medium to tall plant with abundant branches and leaves, a swollen taproot, and well-developed lateral and fine roots. The leaves resemble those of cabbage, but are bluish-green. The leaves are relatively thick, covered with a waxy powder, and have serrated edges. The basal leaves are semi-erect or prostrate, with prominent petioles. The stem-borne leaves are sessile, with leaf bases that semi-amplexicaul. The petioles are large, deep yellow, and the siliques are relatively large, often perpendicular to the flower axis. The seeds are black and relatively large. It is mainly cultivated in the Yangtze River basin. Brassica napus is a cross-pollinated crop, tolerant of fertilizer, high-yielding, and relatively disease-resistant; however, it has poor cold and drought resistance and does not tolerate poor soil. It is best suited for cultivation in areas with good fertile and well-irrigated conditions.

[0003] Nitrogen is one of the essential macronutrients for plant growth and development, and it is closely related to plant yield. Nitrogen is necessary for rapeseed to maintain photosynthesis and improve its yield and quality. Insufficient or excessive nitrogen fertilizer application in rapeseed can lead to stunted vegetative growth, reduced effective branching, and lighter grains, ultimately resulting in reduced yield. Therefore, real-time and precise management of nitrogen nutrition during the rapeseed growth stage is one of the important management measures to achieve high yield, efficient nutrient utilization, and green production. Rapeseed is an important oilseed crop in my country, but current research on the localization of nitrogen-efficient genes in rapeseed is limited.

[0004] Agronomic traits related to efficient nitrogen fertilizer absorption in Brassica napus are easily influenced by environmental conditions, have a complex genetic basis, and lack a clear correspondence between phenotype and genotype. Phenotypic effects are jointly determined by genotype and environmental effects. Whole-genome-wide association analysis (GWAS) and linkage mapping (QTL) are two main methods for revealing the genetic basis of complex traits. In Brassica napus research, traditional QTL mapping methods, limited to populations of only two parents, result in low resolution of gene regions. While biparental population mapping can be used for gene mapping of specific traits, the rich genetic diversity within a population means that conventional methods cannot capture association loci for multiple different traits. Summary of the Invention

[0005] The purpose of this invention is to provide the application of QTL loci in Brassica napus in constructing SNP genetic linkage maps for the localization of traits related to efficient nitrogen fertilizer absorption.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of QTL loci in Brassica napus in constructing SNP genetic linkage maps for the localization of traits related to efficient nitrogen fertilizer absorption, including:

[0008] S1. Using Xiangyou 15 as the female parent and R210 as the male parent, hybridization was carried out to obtain the F1 generation hybrid population. The F1 generation hybrid population was cultured using microspore culture to obtain the DH population.

[0009] S2. After planting the parent plants and DH population from step S1 in soils with no nitrogen application and low nitrogen application, respectively, sampling and sequencing were performed to obtain 10,952,113 SNP markers. After aligning the 10,952,113 SNP markers with the reference genome and filtering, 42,751 SNP markers were obtained. Preferably, the parent plants and DH population from step S1 were planted in soils with medium to low fertility at two levels: no nitrogen application and low nitrogen application. After sampling and sequencing were performed at the bolting stage, 10,952,113 SNP markers were obtained. After aligning the 10,952,113 SNP markers with the reference genome and filtering, 42,751 SNP markers were obtained.

[0010] S3. Construct a genetic linkage map using the 42,751 SNP markers selected in step S2;

[0011] S4. Identify the phenotypic traits related to high nitrogen fertilizer absorption of the parental lineage and DH population in step S1.

[0012] S5. Analyze the genetic linkage map constructed in step S3 and the phenotypic identification results of nitrogen fertilizer high efficiency absorption related traits in step S4. Use the composite interval mapping method to perform QTL analysis and obtain 14 QTL loci related to nitrogen fertilizer high efficiency absorption phenotypic traits.

[0013] Of these, one is located on chromosome A07 and named qBSD.A07-1, two are located on chromosome A09 and named qBSD.A09-1 and qSWPP.A09-1 respectively, two are located on chromosome A10 and named qBSD.A10-1 and qAWDP.A10-1 respectively, two are located on chromosome A03 and named qEPP.A03-1 and qEPP.A03-2 respectively, and one is located on chromosome C02 and named qBSD. One QTL locus, qBSD.C03-1, is located on chromosome C03; one on chromosome C04, qEPP.C04-1; one on chromosome C05, qSWPP.C05-1; two on chromosome C09, qAWDP.C09-1 and qNAE.C09-1; and one on chromosome A08, qNAE.A08-1. The locations and confidence intervals of each QTL locus are shown in the table below.

[0014]

[0015]

[0016] Preferably, during the filtering process, the removed SNP sites include segregation sites; the retained SNP sites include: (1) sites that are homozygous and inconsistent with the parents; (2) sites that are sequenced at a parental sequencing depth >10 and a progeny sequencing depth >1; and (3) sites that have a deletion rate <0.2.

[0017] Preferably, the nitrogen fertilizer high-efficiency absorption related phenotypic traits include stem base diameter, number of siliques per plant, grain weight per plant, aboveground dry matter weight per plant, and nitrogen fertilizer absorption efficiency.

[0018] Preferably, the sampling time is during the bolting stage.

[0019] Preferably, the total amount of nitrogen fertilizer with low nitrogen level is 3 kg / mu, based on the mass of N.

[0020] The present invention discloses the following technical effects:

[0021] The main objective of this invention is to obtain a large number of SNP markers with gene sequences by measuring phenotypic data and high-throughput genotypic data related to efficient nitrogen fertilizer absorption in Brassica napus. Using these marker loci, a high-density molecular genetic linkage map of Brassica napus was constructed, and the localization study of the nitrogen fertilizer absorption-related traits in Brassica napus was carried out. Markers with significant marker-trait associations were obtained, and QTL loci associated with nitrogen fertilizer absorption-related traits were discovered. The localization loci have good stability and wide applicability, providing basic information for molecular marker-assisted breeding of efficient nitrogen absorption in Brassica napus. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figures 1-3 The frequency distribution of traits related to efficient nitrogen fertilizer absorption in rapeseed DH population is shown in the figure. N0 represents no nitrogen treatment, N1 represents low nitrogen treatment, BSD represents stem base diameter, EPP represents the number of siliques per plant, SWPP represents the grain weight per plant, ADWP represents the aboveground dry matter weight per plant, and NAE represents nitrogen fertilizer absorption efficiency.

[0024] Figure 4 A map showing the distribution of SNP markers on chromosomes;

[0025] Figure 5 This is a distribution map of bin markers on chromosomes;

[0026] Figure 6 A map showing the location of QTLs related to nitrogen fertilizer use efficiency on chromosomes;

[0027] Figure 7 A diagram showing the location of chromosome A07 in the stem base using crude QTL mapping without nitrogen treatment;

[0028] Figure 8 A diagram showing the location of chromosome A09 in the crude QTL mapping at the stem base under nitrogen-free treatment;

[0029] Figure 9 A diagram showing the location of chromosome A10 in the crude QTL mapping at the stem base under nitrogen-free treatment;

[0030] Figure 10 A diagram showing the location of chromosome CO2 in the crude QTL mapping at the stem base under low nitrogen treatment;

[0031] Figure 11 A diagram showing the location of chromosome C03 in the stem base using crude QTL mapping under low nitrogen treatment;

[0032] Figure 12 The location of chromosome A03 for QTL mapping of the number of siliques per plant under nitrogen-free treatment;

[0033] Figure 13 Location of chromosome A03 for QTL mapping of silique number per plant under low nitrogen treatment;

[0034] Figure 14 Location of chromosome C04, used to map the QTL mapping of silique number per plant under low nitrogen treatment;

[0035] Figure 15 A diagram showing the location of chromosome A09 in the QTL mapping of grain weight per plant under nitrogen-free treatment;

[0036] Figure 16 A map showing the location of chromosome C05 in the QTL mapping of grain weight in a single plant under low nitrogen treatment;

[0037] Figure 17 QTL mapping chromosome location diagram for aboveground dry matter of a single plant without nitrogen treatment;

[0038] Figure 18 QTL mapping of chromosome A10 for the aboveground dry matter of a single plant under low nitrogen treatment;

[0039] Figure 19 QTL mapping of chromosome C09 for aboveground dry matter of a single plant under low nitrogen treatment

[0040] Figure 20 Map showing the location of chromosome A08 for QTL mapping of nitrogen fertilizer uptake efficiency under low nitrogen treatment;

[0041] Figure 21 Map showing the location of chromosome C09 for QTL mapping of nitrogen fertilizer uptake efficiency under low nitrogen treatment;

[0042] Figures 22-31 For constructing a genetic linkage map;

[0043] In the diagram, Position in 7 is located on chromosome A07, Position in 9 on chromosome A09, Position in 10 on chromosome A10, Position in 12 on chromosome C02, Position in 13 on chromosome C03, Position in 3 on chromosome A03, Position in 14 on chromosome C04, Position in 15 on chromosome C05, and Position in 19 on chromosome C09. Detailed Implementation

[0044] Example 1

[0045] 1. Materials and Methods

[0046] 1.1 Materials

[0047] Two rapeseed varieties with significant differences in nitrogen fertilizer absorption efficiency, Xiangyou 15 (high nitrogen fertilizer absorption efficiency, female parent) and R210 (low nitrogen fertilizer absorption efficiency, male parent, variety right number CNA20090934.8), were used as parents to obtain F1 through artificial hybridization. 180 DH populations were then obtained through microspore culture. Xiangyou 15 was provided and preserved by Hunan Agricultural University, and R210 was provided and preserved by the Crop Research Institute of Jiangxi Academy of Agricultural Sciences.

[0048] 1.2 Field Trials

[0049] In October 2020, the parent lines and DH line population were planted at the Gao'an Experimental Base of the Jiangxi Academy of Agricultural Sciences. Experiments were conducted on experimental plots with moderate to low soil fertility. Two nitrogen treatments were established: no nitrogen application and low nitrogen application. The low nitrogen treatment involved applying nitrogen fertilizer at sowing, seedling stage, and early flowering stage. The total nitrogen fertilizer application rate was 3 kg / mu (approximately 0.067 hectares) based on pure nitrogen mass, with 1.8 kg / mu as basal fertilizer, 0.6 kg / mu at the seedling stage, and 0.6 kg / mu at the early flowering stage, applied in three applications. A randomized block design was used, with two parallel trials for each treatment. Each plot consisted of three rows with 12 plants per row, a row spacing of 33.0 cm, and a plant spacing of 10 cm. Field management was the same as conventional production to ensure consistent external growth environments for all samples. Data was collected after maturity.

[0050] 1.3 Trait Examination

[0051] After maturity, the stem base diameter, number of siliques per plant, grain weight per plant, aboveground dry matter weight per plant, and nitrogen fertilizer absorption efficiency of the parent plants and DH line populations under nitrogen-free and low-nitrogen treatments were investigated. The stem base diameter was obtained by measuring the average of 6 typical plants with vernier calipers. The number of siliques per plant was obtained by manually counting the average number of effective siliques of 6 typical plants. The grain weight per plant was obtained by manually weighing the average grain weight of 6 typical plants. The aboveground dry matter weight per plant was obtained by manually weighing the average aboveground dry weight of 6 typical plants. The nitrogen fertilizer absorption efficiency was calculated using Equation 1.

[0052] Formula 1: Plant nitrogen accumulation / (soil nitrogen supply + nitrogen application) × 100%.

[0053] 1.4 Genetic linkage map and QTL analysis

[0054] Based on the Illumina NovaSeq sequencing platform, paired-end (PE) sequencing was performed on samples from the parents (Xiangyou 15 and R210) and the DH population. The samples were obtained from leaves after bolting, yielding 10,952,113 SNP markers. After data quality control, alignment with the reference genome (the sequence of the reference genome was obtained from [https: / / www.ncbi.nlm.nih.gov / genome / ?term=rape]), and filtering, 42,751 SNP markers were obtained, which were used to construct a high-density SNP genetic linkage map. During filtering, SNP sites removed included partial segregation sites (P<0.05); retained SNP sites included: (1) sites that were homozygous and inconsistent with the parents; (2) sites with a parent sequencing depth >10 and a progeny sequencing depth >1; (3) sites with a deletion rate <0.2. The high-quality data obtained after filtering were aligned to the reference genome using the bwa(0.7.12-r1039)(Li, H., and R. Durbin et al., 2009) mem program (https: / / www.ncbi.nlm.nih.gov / genome / ?term=rape); and picard was used. The 1.107 software (http: / / www.psc.edu / index.php / user-resources / software / picard) was used to sort the SAM files and convert them into BAM files; GATK (version 3.8) software was used to detect SNPs; 42,751 SNPs were selected, and 18,718 bin markers were constructed using QTL IciMapping v4.1 software.

[0055] Genetic linkage maps were constructed using MSTmap and JoinMap 4.0 software. QTL analysis was performed using Composite Interval Mapping (CIM) in Windows QTLCartographer 2.5 software. 1000 permutation tests were performed for each trait, and a significance level of 0.05 was used to determine the presence of QTLs.

[0056] 2 Results and Analysis

[0057] 2.1 Phenotypic variation of nitrogen fertilizer efficiency absorption related traits in both parents and DH population

[0058] The phenotypic variation of nitrogen fertilizer efficiency-related traits in both parents and the DH population is shown in Table 1. The correlation coefficients of nitrogen fertilizer efficiency-related traits in the rapeseed DH population are shown in Table 2. The frequency distribution diagram of nitrogen fertilizer efficiency-related traits in the rapeseed DH population is shown in Table 2. Figures 1-3 .

[0059] Table 1. Changes in traits related to efficient nitrogen fertilizer absorption in rapeseed under two nitrogen levels in parental and DH populations.

[0060]

[0061]

[0062] Note: N0 is the no nitrogen treatment, N1 is the low nitrogen treatment (3 kg / mu), BSD is the stem base diameter, EPP is the number of siliques per plant, SWPP is the grain weight per plant, ADWP is the aboveground dry matter weight per plant, and NAE is the nitrogen fertilizer absorption efficiency. The same applies to the following table.

[0063] Table 2 Correlation coefficients of nitrogen fertilizer efficiency in rapeseed DH population

[0064]

[0065] As shown in Table 1, under different nitrogen levels, the differences in stem base diameter, number of siliques per plant, grain weight per plant, and aboveground dry matter weight between the two parents were all highly significant, indicating a highly significant difference in nitrogen fertilizer absorption efficiency between the two parents. The average values ​​of all traits in the DH population were between those of the two parents. Except for the maximum value of SWPP, which was lower than that of Xiangyou 15, the maximum and minimum values ​​of all traits in the DH population exceeded those of the parents, showing a clear over-parental segregation phenomenon. Table 2 shows that the correlations among traits varied across treatments, with most traits exhibiting significant or highly significant positive correlations. Figures 1-3 It can be seen that the nitrogen fertilizer absorption efficiency-related traits of the DH population are all continuously distributed. Under different nitrogen level treatments, the stem base diameter, number of siliques per plant, grain weight per plant, and aboveground dry matter weight per plant of the DH population are all normally distributed. The nitrogen fertilizer absorption efficiency of the DH population is partially normally distributed, which is suitable for QTL analysis.

[0066] Correlation analysis showed that, under the two nitrogen levels, stem base diameter, grain weight per plant were not significantly correlated with nitrogen fertilizer absorption efficiency, but the number of siliques per plant and the aboveground dry matter of per plant were significantly positively correlated with nitrogen fertilizer absorption efficiency.

[0067] 2.2 Construction of high-density genetic linkage maps

[0068] DH populations were constructed using two germplasms, Xiangyou 15 and R210, which showed significant differences in nitrogen-efficient utilization. Resequencing of 180 DH lines and their parents yielded 42,751 SNPs. Using QTL IciMapping v4.1 and R / onemap software, 4,302 bins were obtained, with a total length of 2959.89 cM. The average linkage group length was 155.78 cM, and the average marker interval was 0.69 cM. Figure 4 ).

[0069] 2.3 QTL Analysis of Related Traits for High-Efficiency Nitrogen Fertilizer Absorption in Rapeseed

[0070] QTL analysis was performed on traits related to efficient nitrogen fertilizer absorption using QTL Cartographer 2.5 software. A total of 14 QTLs were detected, located on chromosomes A03, A07, A08, A09, A10, CO2, CO3, CO4, C05, and C09, respectively. Details of the QTLs are shown in Table 3. The phenotypic variation that a single QTL can explain ranges from 5.24% to 18.22% (Table 3).

[0071] Table 3. QTLs of nitrogen fertilizer efficiency absorption detected at two nitrogen levels.

[0072]

[0073]

[0074] 2.3.1 Stem base thickness QTL: Five QTLs were located for stem base thickness at both nitrogen levels, explaining 5.24%–18.22% of the phenotypic variation. The major QTL sites were located at A10 and CO3, explaining 18.22% and 16.36% of the phenotypic variation, respectively. The additive effects of the QTL sites at A10 and CO3 at both nitrogen levels were less than 0 (Table 3), indicating that the additive effects originated from the parent R210.

[0075] 2.3.2 QTLs related to the number of siliques per plant: Three QTLs related to the number of siliques per plant were located at two nitrogen levels. The major QTL locus was located on chromosome A03, explaining 12.66% of the phenotypic variation. The effect values ​​of the two QTL loci on A03 were -8.28 and -9.51, respectively, and the effect value of the QTL locus on C04 was 9.31 (Table 3). This indicates that the additive effect of the former two originated from the parent R210, and the additive effect of the latter originated from the parent Xiangyou 15.

[0076] 2.3.3 Single-plant grain weight QTLs: Two QTLs were located for single-plant grain weight at two nitrogen levels, located at A09 and C05, which explained 7.99%-8.45% of the phenotypic variation, respectively. The additive effects of the two QTL sites were both greater than 0 (Table 3), that is, the additive effects were both derived from the parent Xiangyou 15.

[0077] 2.3.4 Single-plant aboveground dry matter weight QTLs: No single-plant aboveground dry matter weight-related QTLs were located at the N0 level, but two single-plant aboveground dry matter weight-related QTLs were located at the N1 level, located at A10 and C09, explaining 5.85% and 7.95% of the phenotypic variation, respectively. The additive effects of the QTL sites at A10 and C05 were 2.49 and 2.14, respectively (Table 3), indicating that the additive effects originated from the parent Xiangyou 15.

[0078] 2.3.5 Nitrogen fertilizer uptake efficiency QTLs: Two nitrogen fertilizer uptake efficiency QTLs were located at A08 and C09, explaining 5.98%-8.52% of the phenotypic variation, respectively. The additive effects of the QTL sites at A08 and C09 were both greater than 0 (Table 3), meaning that the additive effects originated from the parent Xiangyou 15.

[0079] 3. Conclusion

[0080] 3.1 Construction of a high-density genetic linkage map

[0081] DH populations were constructed using two germplasms, Xiangyou 15 and R210, which showed significant differences in nitrogen-efficient utilization. 180 DH lines and their parents were selected from each population to form mapping populations. DNA was extracted from each population, and library construction and high-throughput sequencing were performed according to the resequencing protocol. After data quality control, alignment with a reference genome (the reference genome sequence was obtained from [https: / / www.ncbi.nlm.nih.gov / genome / ?term=rape]), and filtering, 42751 SNPs were obtained. Figure 4 ), using QTL IciMapping v4.1 software, 18718 bin markers were constructed ( Figure 5 Based on 18,718 bin markers, linkage groups were performed using the software R / onemap, and the Kosambi method was used to sort the markers within each linkage group. Correction was then performed based on the physical location of the markers. The final figure above shows a total of 4,302 markers with a total length of 2,959.89 cM, an average linkage group length of 155.78 cM, and an average interval between markers of 0.69 cM (Table 4). Figures 22-31 ).

[0082] Table 4. Statistics on Chain Group Information

[0083]

[0084]

[0085] 3.1 QTL mapping of traits related to efficient nitrogen fertilizer absorption in rapeseed

[0086] Under different nitrogen levels, the differences in stem base diameter, number of siliques per plant, grain weight per plant, and aboveground dry matter weight between the two parents were all highly significant, indicating a highly significant difference in nitrogen fertilizer absorption efficiency between the two parents. The average values ​​of all traits in the DH population were between those of the two parents, except that the maximum value of grain weight per plant (SWPP) was less than that of Xiangyou 15. The maximum and minimum values ​​of all traits in the DH population exceeded those of the parents, showing a clear over-parental segregation phenomenon. The nitrogen fertilizer absorption efficiency-related traits in the DH population showed a continuous distribution. Under different nitrogen levels, the stem base diameter, number of siliques per plant, grain weight per plant, and aboveground dry matter weight of the DH population all showed a normal distribution, while the nitrogen fertilizer absorption efficiency of the DH population showed a skewed normal distribution. Figures 1-3 It is suitable for QTL analysis.

[0087] Correlation analysis showed that the identified phenotypic traits—stem base diameter, number of siliques per plant, grain weight per plant, and aboveground dry matter weight per plant—were all negatively correlated with nitrogen fertilizer uptake efficiency (NAE) under the no-nitrogen treatment, and none of them reached a significant level. However, under the low-nitrogen treatment, they all reached a highly significant positive correlation.

[0088] Using QTL IciMapping v4.1 software, complete interval mapping was employed to perform QTL analysis on traits related to efficient nitrogen fertilizer uptake. A total of 14 QTLs were detected, located on chromosomes A03, A07, A08, A09, A10, CO2, CO3, CO4, C05, and C09. The phenotypic variation explained by a single QTL ranged from 5.24% to 18.22%. Figure 6 ).

[0089] QTL mapping for stem base thickness: Five QTLs were mapped to stem base thickness at both nitrogen levels, explaining 5.24%–18.22% of the phenotypic variation. Major QTL loci were located on chromosomes A10 and CO3, explaining 18.22% and 16.36% of the phenotypic variation, respectively. The additive effects of the QTL loci on chromosomes A10 and CO3 at both nitrogen levels were less than 0. Figures 7-11 (Table 3) indicates that the additive effects all originate from the parent R210.

[0090] QTL mapping for silique number per plant: Three QTLs related to silique number per plant were mapped at two nitrogen levels. The major QTL locus was located on chromosome A03, explaining 12.66% of the phenotypic variation. The effect sizes of the two QTL loci on chromosome A03 were -8.28 and -9.51, respectively, while the effect size of the QTL locus on chromosome C04 was 9.31. Figures 12-14 Table 3 shows that the additive effects of the first two are derived from the parent R210, while the additive effect of the latter is derived from the parent Xiangyou 15.

[0091] QTL mapping of single-plant grain weight: Two QTLs were mapped to single-plant grain weight at two nitrogen levels, located on chromosomes A09 and C05, explaining 7.99%-8.45% of phenotypic variation, respectively. The additive effect of both QTL loci was greater than 0. Figure 15 and Figure 16 Table 3 shows that the additive effects all originated from the parent Xiangyou 15.

[0092] QTL mapping for aboveground dry matter per plant: No QTLs related to aboveground dry matter per plant were mapped under the nitrogen-free treatment (N0). Under the low-nitrogen treatment (N1), two QTLs related to aboveground dry matter per plant were mapped, located on chromosomes A10 and C09, explaining 5.85% and 7.95% of the phenotypic variation, respectively. The additive effects of the QTL loci on chromosomes A10 and C05 were 2.49 and 2.14, respectively. Figures 17-19 Table 3 shows that the additive effects all originated from the parent Xiangyou 15.

[0093] Nitrogen fertilizer uptake efficiency QTL mapping: Two nitrogen fertilizer uptake efficiency QTLs were mapped, located on chromosomes A08 and C09, explaining 5.98%-8.52% of phenotypic variation, respectively. The additive effect of the QTL loci on chromosomes A08 and C09 was greater than 0. Figure 20 and Figure 21 Table 3 shows that the additive effects all originated from the parent Xiangyou 15.

[0094] In summary, this invention utilizes two rapeseed varieties with significantly different nitrogen fertilizer absorption efficiencies—Xiangyou 15 (high nitrogen fertilizer absorption efficiency) and R210 (low nitrogen fertilizer absorption efficiency)—to obtain F1 strains. A DH population containing 180 lines was then obtained through microspore culture. The parents and the DH population were planted under two nitrogen application levels: no nitrogen and low nitrogen. Phenotypic surveys and QTL analyses were conducted on stem base diameter, number of siliques per plant, aboveground dry matter weight per plant, and nitrogen fertilizer absorption efficiency, which are closely related to the absorption efficiency of rapeseed under the two nitrogen application levels. The results showed that 14 QTLs were detected under the two nitrogen application levels, located on chromosomes A03, A07, A08, A09, A10, CO2, CO3, CO4, C05, and C09, with each QTL explaining 5.24%–18.22% of the phenotypic variation. These QTLs will provide useful information for the genetic improvement of efficient nitrogen absorption in rapeseed.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of Brassica napus QTL loci in constructing a SNP genetic linkage map for positioning traits related to high-efficiency nitrogen uptake, characterized in that, The application is: S1. Hybridizing Xiangyou 15 as the female parent and R210 as the male parent to obtain an F1 hybrid population, culturing the F1 hybrid population by microspore culture to obtain a DH population; S2. Planting the parents and the DH population in step S1 in soils without nitrogen and in soils with low nitrogen, sampling and sequencing, obtaining 10952113 SNP markers, aligning the obtained 10952113 SNP markers with a reference genome, filtering to obtain 42751 SNP markers; the removed SNP sites in the filtering include bias separation sites; the retained SNP sites are: (1) retaining sites with parental homozygous and inconsistent; (2) retaining sites with a parental sequencing depth > 10 and a progeny sequencing depth > 1; (3) retaining sites with a deletion rate < 0.2; S3. Constructing a genetic linkage map by using the 42751 SNP markers screened in step S2; S4. Phenotyping the parents and the DH population in step S1 for nitrogen-efficient uptake related traits; S5. Analyzing the genetic linkage map constructed in step S3 and the phenotyping results of the nitrogen-efficient uptake related traits in step S4, performing QTL analysis by composite interval mapping to obtain 14 QTL sites related to the phenotypes of the nitrogen-efficient uptake related traits; the phenotypes of the nitrogen-efficient uptake related traits are stem base diameter, single plant pod number, single plant grain weight, single plant aboveground dry matter weight, and nitrogen uptake efficiency; Among them, one is located on chromosome A07, named qBSD.A07-1 , two are located on chromosome A09, named qBSD.A09-1 and qSWPP.A09-1 , two are located on chromosome A10, named qBSD.A10-1 and qAWDP.A10- 1 , two are located on chromosome A03, named qEPP.A03-1 and qEPP.A03-2 , one is located on chromosome C02, named qBSD.C02-1 , one is located on chromosome C03, named qBSD.C03-1 , one is located on chromosome C04, named qEPP.C04-1 , one is located on chromosome C05, named qSWPP.C05-1 , two are located on chromosome C09, named qAWDP.C09-1 and qNAE.C09-1 , one is located on chromosome A08, named qNAE.A08-1 ; the position and confidence interval of each QTL site are shown in the following table: 。 2. Use according to claim 1, characterized in that, The sampling time is the bolting period.

3. Use according to claim 1, characterized in that, The total amount of nitrogen fertilizer at the low nitrogen level is 3 kg / acre in terms of the mass of N.

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