Identification of QTLs for Seed Yield and Yield-Related Traits in Brassica Napus Grown with Contrasting Nitrogen Supplies


 Oilseed rape (Brassica napus L.; B. napus) is the main oil crop in China as well as in the world. Nitrogen (N) deficiency significantly reduces the seed yield of B. napus. However, a very few studies involved in the genetic mechanism of seed yield (SY) and SY-related traits of B. napus in response to N deficiency. In this study, plant height (PH), branch number (BN), pod number (PN), seed number (SN), 1000-seed weight (SW) and SY were investigated using a B. napus double haploid (BnaTNDH) population derived from a cross between cultivars ‘Tapidor’ and ‘Ningyou7’ grown at an optimal (ON) and a low N (LN) supplies in three-year field trials. Great variations of SY and related traits were observed in BnaTNDH population under contrasting N supplies. A total of 106 and 110 significant quantitative trait loci (QTLs) were detected for six traits at ON and LN in three field trials, respectively. All of these significant QTLs for the same trait were integrated into 191 consensus QTLs. Nine and eleven consensus QTLs at ON and LN were detected in two or three trials, respectively, and the remaining were environment-specific. One hundred and three unique QTLs were integrated from 191 consensus QTLs, including 29 low N specific QTLs, 35 optimal N specific QTLs and 39 constitutive QTLs. uqA3q was integrated from four consensus QTLs for PN, PH, SN, SY at LN, uqC9f was integrated from consensus QTLs for BN, SY, PN at ON and LN. Both were detected in three trials. This result may help to better understand the genetic mechanism of yield traits in response to low N and promote the breeding of N efficient varieties.


Introduction
Nitrogen (N) is component of nucleic acids, proteins, chlorophyll, alkaloids, vitamins, and hormones, which is essential for plants growth and development (Hawkesford et al., 2012). Oilseed rape (Brassica napus L.; B. napus) is one of the most important oil crops worldwide, which acquires nitrate and ammonium and recycles organic nitrogen (Masclaux-Daubresse et al., 2010). The application rate of N fertilizer in B. napus ranged from 65 to 325 kg / hm 2 in China, which depend on the eld fertility, SY target, varieties and other factors (Zhang et al., 2020). Rational application of N fertilizer can signi cantly promote the seed yield (SY), oil production, protein content and polyunsaturated fatty acid content of B. napus (Gao et al., 2019). On the contrary, irrational fertilization not only decreases the crop yield and quality, but also causes soil acidi cation and eutrophication (Liu et al., 2013b;Guo et al., 2010;Hirel et al., 2011). Breeding N -e cient B. napus cultivars is an important strategy to improve the SY in a sub-optimal N supply and reduce the application of N fertilizers.
SY is a complex trait, which is mainly related to the potential of B. napus for growth and branching after owering which enable the crop to use one yield component to compensate for limitations in another one (Bouchet et al., 2014). SY of B. napus is directly related to pod number per plant, seed number per pod and 1000-seed weight, and also indirectly associated with plant height and branch number (Ding et al., 2012). N de ciency signi cantly decreased SY components such as plant density, branch number, pod number per plant, seed number per pod, except for 1000-seed weight (Cong et al., 2020).
Quantitative trait loci (QTL) analysis based on high-density genetic linkage map can provide basic information on the genetic architecture of quantitative traits (Agrama 2006). Bouchet et al. (2016) mapped 17 low-N speci c QTLs, 18 optimal-N-speci c QTLs for owering days, seed protein content, SY, seed number per pod, 1000-seed weight, oil content and oil/protein in a double haploid (DH) population of B. napus through three-year eld trials, and homologous QTLs for SY were found on A3/C3, A5/C5, A9/C9 chromosomes. Wang et al. (2017) nd that all major QTLs and some stable QTLs for N use e ciency were associated with root morphology traits in B. napus at ON and/or LN. At present, many QTLs have been mapped for SY or N use e ciency in B. napus at ON, but limited QTLs were detected at LN.
In this study, a B. napus DH population (BnaTNDH population) was employed to conduct eld trials at ON and LN for three years. The QTLs for SY and SYrelated traits of B. npaus under contrasting N supplies were identi ed. Some major QTLs in response to low N were obtained.

Materials And Methods
Plant materials and eld trials A BnaTNDH population with 182 lines was used in this study, which was derived from a cross between a European winter type cultivar 'Tapidor' and a Chinese semi-winter type cultivar 'Ningyou7' by microspore culture (Qiu et al., 2006).
Three eld trials were conducted in sandy paddy soil in Qichun county, Hubei Province, China (115°45′N latitude, 30°19′E longitude), during B. napus growing seasons the 2008-2009(Tri.1), 2009(Tri.2) and 2010. Soil properties were as follows: pH (1:1 H 2 O) 4.8, organic matter 34.9 g·kg − 1 , total N 0.22 g·kg − 1 , available N 0.074 g·kg − 1 , Olsen-phosphorus 3.32 mg·kg − 1 , available potassium 42 mg·kg − 1 , and available boron 0.09 mg·kg − 1 . The basal fertilizers included P 38.7 kg·ha − 1 , K 124.5 kg·ha − 1 , ZnSO 4 ·7H 2 O 45 kg·ha − 1 and Borax (Na 2 B 4 O 7 ·10H 2 O) 15 kg·ha − 1 . 60% of 120 and 40 kg·ha − 1 N were applied to create ON and LN conditions before transplantation, and rest of the urea was applied before the overwinter stage. Three replications for 182 BnaTNDH lines and their parents were planted in a randomized complete-plot design with each plot comprising 18 plants, separated by a distance of 0.20 m between plants and 0.28 m between rows. Seeds were sown in a nursery bed in the eld in middle September and seedlings were transplanted 30 d after sowing. Plants were harvested in the following middle May. Standard agricultural practices were followed for eld management.

Measurement of phenotypic traits
In each plot, six individuals from the middle row were used to determine plant height (PH) measured from ground level to the tip of the main in orescence, number of primary branches (BN) measured as the number of primary branches arising from main shoot and seed number per pod (SN) measured as the average number of well lled seeds from 100 well-developed pods sampled from the primary branch in the middle of each plant studied. All representative individuals from each plot were harvested by hand at maturity stage to investigate seed yield per plant (SY) and seed weight of 1,000 seeds (SW). Pod number per plant (PN) was calculated using the following formula: PN = (SY × 1000) / (SW × SN).

Statistical analysis and QTL detection
Data analysis was conducted using SPSS 20.0 (IBM, USA) and Microsoft Excel 2019 (Microsoft, USA). Duncan multiple-range test was used for multiple comparison of different traits between two parents. Three-way ANOVA with F test was used at P < 0.05 level. Different growth environments (years) and N treatments were treated as xed factors, and genotypes were treated as random factor. Correlation analysis was conducted to determine the relationship between the tested traits. The broad-sense heritability (h 2 ) for each trait was calculated at both N levels as follows: h 2 = σ g 2 /(σ g 2 + σ ge 2 /n + σ e 2 /nr), where σ g 2 is the genotypic variance, σ ge 2 is the interaction variance of genotype with environment, σ e 2 is the error variance, n is the number of environments and r is the number of replicates.
The BnaTNDH linkage map contained a total of 2041 molecular marker and the average marker density was from 0.39 to 0.97 per cM . QTLs were detected by composite interval mapping (CIM) using WinQTL cartographer 2.5 software (http://statgen.ncsu.edu/qtlcar/WQTLCart.htm) (Wang et al., 2006). For each trait, QTL threshold (P < 0.05) was estimated from 1,000 permutations (Silva et al., 2012). Biomercator v4.2 was used to integrate consensus QTL and unique QTL (Arcade et al., 2004). The signi cant QTLs for the same trait identi ed in the different trials were integrated into consensus QTLs by meta-analysis. Then the consensus QTLs for different traits that overlapped were integrated into unique QTL. The consensus QTLs detected in at least two trials were considered as major consensus QTLs. Each QTL was denominated as ''q'' (abbreviation of QTL) + trait name + trial number + chromosome name + the serial letter (a,b,c...). For example, qPHON3-A3b denoted the second QTL for plant height on chromosome A3 at ON in Tri.3. Each consensus QTL was denominated as "cq" (abbreviation of consensus QTL) + trait name + chromosome name + the serial letter. For example, cqPHON-A3c indicated the third consensus QTL for PH at ON located on A3. Each unique QTL was denominated as "uq" (abbreviation of unique QTL) + chromosome name + the serial letter. For example, uqA2d indicated the fourth unique QTL on A2.

Results
Differences in the six tested traits between cultivars Tapidor and Ningyou 7, and among BnaTNDH population At ON, SY of Tapidor was signi cantly lower than that of Ningyou7 in the three trials; BN of Tapidor was lower than that of Ningyou7 in Tri.2 and Tri.3; SN of Tapidor was obviously more than that of Ningyou7 in Tri.1; PN of Tapidor was lower than that of Ningyou7 in Tri.3 ( Fig. 1; Table 1). At LN, BN of Tapidor was less than that of Ningyou7 in Tri.1 and Tri.2, and SY of Tapidor was less than that of Ningyou7 in Tri.3 ( Fig. 1; Table 1). There was no signi cant difference in PH between Tapidor and Ningyou7 at two nitrogen supplies in three trials. Table 1 Means and ranges of the seed yield (SY) and SY-related traits in the parental lines and the BnaTNDH population grown at an optimal (ON) and a low N supply (LN) in three eld trials. observed in the BnaTNDH population for the six traits, ranged from 0.51 for BN at LN to 0.84 for SY at ON. In general, a higher h 2 was observed at ON than at LN (Table 1).
In the three trials, all traits showed approximately normal distributions and transgressive segregations at two N supplies (Table 1; Fig. 2). The results of ANOVA showed that environment, N treatment, genotype and the interactions between these factors had signi cant effects on all the tested traits (Table 2). SY was highly positively correlated with PN and SN at both ON and LN across three eld trials (Table 3). There was a weak correlation between PH and BN at both ON and LN across three eld trials. While no signi cant correlation and weak correlation was observed between SY and SW at both ON and LN in Tri.1 and Tri.2, and Tri.3, respectively (Table 3). Table 2 Signi cance of three-way ANOVA analysis of the seed yield and yield-related traits among the BnaTNDH population grown at an optimal and a low N supply in three eld trials. sig. ** ** *** *** *** *** a d.f., degrees of freedom. b sig., signi cance. ns, no signi cance. **P < 0.01, ***P < 0.001. Table 3 Correlation coe cients among seed yield (SY) and SY-related traits in the BnaTNDH population grown at an optimal (above diagonal) and a low N supply (below diagonal) in three eld trials  Among 33 ON-speci c QTLs, uqA2e, uqA3a and uqA9b for two traits were clustered on A2, A3, A9, respectively (Table 5). uqA2e for two traits of PH and SN was located in the interval of 47.1-49.0 cM on A2. uqA3a was integrated from two consensus QTLs, cqPHON A3a and cqBNON A3a, and was clustered in the interval of 0.0 8.3 cM on A3. uqA9b was obtained from two consensus QTLs, cqBNON A9a and cqSNON A9a, and clustered in the interval of 10.9 20.4 cM on A9. Among 27 LN -speci c QTLs, six LN-speci c QTLs for more than two traits were clustered on A2, A3, A4 and A9 chromosomes (Table 5). uqA3h for four traits of PH, PN, SN and SY was located in the interval of 100.6-103.1 cM on A3. uqA9b for three traits of BN, SN and SW was integrated from three consensus QTLs of cqSWLN A9, cqSNLN A9 and cqBNLN A9, and located in the interval of 36.0 38.4 cM on A9. uqA2a, uqA2d, uqA3c and uqA4b were all associated with two traits at LN.
Candidate genes underlying QTLs associated with SY and two major unique QTLs cqSYLN-C9, detected from qSYLN2-C9a and qSYLN3-C9b, was considered to be the major QTL among QTLs associated with SY due to the PVE of qSYLN3-C9b was 15.4% (Table 4, Supplementary Table 1). Two candidate genes, BnaC09g46700D and BnaC09g47860D, were identi ed in the con dence regions of cqSYLN-C9. The orthologues of them in Arabidopsis have been reported in association with glutamate synthase and affecting N assimilation (Hanke et al. 2005, Fontaine et al. 2012). There were not SNPs and InDels in the coding sequence of two candidate genes between Tapidor and Ningyou7. However, there were two SNPs in the promoter of BnaC09g47860D between the two parents (Table 6). BnaC09g46700D Among unique QTLs, uqA3q and uqC9f were detected in three trials and associated with four traits and three traits, respectively. One candidate gene, BnaA03g41350D, was identi ed in the genomic region of uqA3q, whose orthologue gene in Arabidopsis affects triacylglycerol (TAG) biosynthesis in response N de ciency (Yang et al., 2011). There were 22 SNPs and 15 InDels in the promoter but not in the coding sequence between Tapidor and Ningyou7 (Table 6).

Discussion
In the present study, at both N supplies, SW of Ningyou7 were signi cantly higher than that of Tapidor in three trials; at ON, BN of Ningyou7 was signi cantly higher than that of Tapidor in three trials, SY of Ningyou7 was considerably higher than that of Tapidor in Tri.2 and Tri.3 (Table 1). At LN, BN of Ningyou7 was higher than that of Tapidor in Tri.1 and Tri.2, SY of Ningyou7 was signi cantly higher than that of Tapidor in Tri.2 and Tri.3. These were similar to the performance of them in pot culture experiments that SY of Ningyou7 was signi cantly higher than that of Tapidor at both N supplies (Shi et al., 2010).
Ninety one percent of the 191 consensus QTLs for SY and SY-related traits were detected only in one trial (Supplementary Table 1). A large number of environment-speci c QTLs for SY and its related traits are identi ed, indicating the growth environments have important effects on the function of the genes associated with these traits (Shi et al., 2009). Twenty consensus QTLs (10.47%) were detected simultaneously in at least two trials (  Luo et al. (2017) results also showed that QTLs for SY and its related traits at ON supply differed from that at LN supply. These could be attributed to that N de ciency limits dry matter production and decreases other nutrient uptake in plant .
BnaC09g46700D and BnaC09g47860D were predicted to be the candidate genes for cqSYLN-C9. BnaC09g46700D encodes ferredoxin, which is involved in glutamate synthase (GOGAT). BnaC09g47860D encodes glutamate dehydrogenase. There are two isoforms of GOGAT-the NADH-dependent cytosolic isoform (Iry N assimilation) and ferredoxin-dependent plastidic isoform (IIry N assimilation) (Pathak et al., 2008). GOGAT serves as a potential target for improving N uptake e ciency and SY (Karunarathne et al., 2020). The ferredoxin-dependent GOGAT is important in N-carbon metabolomes in rice . The relative expression of the homologous genes of BnaC09g46700D in Arabidopsis thaliana such as At1g10960, At1g60950 and At2g27510 were upregulated under low NO 3 − treatment (Hanke et al., 2005). Moreover, the mutation of At5g18170, At5g07440 and At3g03910, the homologous genes of BnaC09g47860D in Arabidopsis thaliana, changes the primary C and N metabolic activity (Fontaine et al., 2012).
ON and LN-speci c QTL were reported for owering days and SY (Bouchet et al., 2016), and root dry weight (Liu et al., 2009) in B.napus. In this study, there were 103 unique QTLs, including 29 LN -speci c QTLs, 35 ON-speci c QTLs and 39 constitutive QTLs for SY and its related traits (Supplementary Table 2). uqA2h, a constitutive QTL, was associated with SY and SW at ON and SY at LN (Table 5, Supplementary Table 2). Its interval was overlapped the interval of es.A2-30 (Luo et al., 2017), which contained SY and SW at ON.
Among ON-speci c QTLs, there were not QTLs co-located with the QTLs for SY. Among LN-speci c QTLs, there were ve unique QTLs (uqA2i,uqA3m,uqA3q,uqC8c) for SY overlapped with QTLs for SN (Table 5). At ON, the overlapped QTLs between for SY and for SN on A5 and C8 have also been reported in Brassica napus (Bouchet et al., 2014). Moreover, there was a high positive correlation between SY and SN at LN (Table 3).
Pleiotropic QTLs have also been reported for PH and spike length of wheat (Chai et al., 2019), PH and heading date of rice (Liu et al., 2013a). Xu et al. (2014) identi ed two genes in wheat, Rht8 on chromosome 2D and Rht-B1b on chromosome 4B, which have pleiotropic effects for PH, spike length, harvest index and N utilization e ciency. QTLs for SY directly accounted for a small proportion of all identi ed QTLs (Chen et al., 2010;Peng et al., 2011;Luo et al., 2017). The QTLs for some plant architecture related traits, such as BN and PH, are co-located with QTLs for SY Miersch et al., 2016). Plant architecture traits strongly affects light interception and photosynthesis, and plays an important role in total yield and harvest index (Sarlikioti et al., 2011). Among unique QTLs, uqA3q, a LN speci c QTL, and uqC9f, a constitutive QTL, were associated with the overlapped QTLs for PH, SN and SY at LN, and the overlapped QTLs for BN and SY at LN and ON, respectively (Table 5). BnaA03g41350D was predicted to be the candidate gene for uqA3q, and its homologous genes DGAT2 in Arabidopsis thaliana was associated with the triacylglycerol (TAG) biosynthesis. BnaC09g46700D and BnaC09g47860D were identi ed within the interval of uqC9f.
In conclusion, considerable variations of SY and SY related traits were observed among the BnaTNDH population. N de ciency reduced SY and SY-related traits except for SW. Only 20% signi cant QTLs were detected in more than two trials, indicating that different genetic determinants were involved in regulating SY and its related traits at ON and LN. The overlaps of the QTLs for PH and SN with SY were detected in different trials, suggesting that plant architecture had a signi cant effect on SY. Near-isogenic lines should be developed to ne map the major QTLs identi ed in this study such as cqSYLN-C9q and uqA3q. These will be helpful for a better understanding of the molecular mechanism of SY of B. napus under N de ciency and promote the molecular breeding of Ne cient cultivars. Data availability The data sets supporting the results of this article are included within the article and its additional es Code availability Not applicable

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Con ict of Interest Statement The authors declare no con ict of interest.

Figure 1
Phenotyping of Brassica napus cultivars Tapidor (left) and Ningyou7 (right) grown at an optimal N (up) and a low N (down) supply.

Figure 2
Frequency distribution of seed yield (SY) and SY-related traits in the BnaTNDH population grown at an optimal (left) and a low (  Summary of identi ed QTLs, consensus QTLs and unique QTLs for seed yield (SY) and SY-related trait in the BnaTNDH population grown at an optimal and a low N supply. All identi ed QTLs of all the traits were integrated into consensus QTLs, and then consensus QTLs were integrated into unique QTLs. The outermost circle and the second circle represented the genetic map and physical map, respectively. All SNP markers on each chromosome were corresponded to the physical position. From the third circle to the 6th circle, each color of circle stands for one trial, except green ones, which showed the positions of consensus QTLs of each trait at an optimal N or a low N supply. The innermost circle showed the positions of unique QTLs on chromosomes.