Fine Mapping of a Pleiotropic Locus (BnUD1) Responsible for the Up-Curling Leaves and Downward-Pointing Siliques in Brassica napus

Leaves and siliques are important organs associated with dry matter biosynthesis and vegetable oil accumulation in plants. We identified and characterized a novel locus controlling leaf and silique development using the Brassica napus mutant Bnud1, which has downward-pointing siliques and up-curling leaves. The inheritance analysis showed that the up-curling leaf and downward-pointing silique traits are controlled by one dominant locus (BnUD1) in populations derived from NJAU5773 and Zhongshuang 11. The BnUD1 locus was initially mapped to a 3.99 Mb interval on the A05 chromosome with a BC6F2 population by a bulked segregant analysis-sequencing approach. To more precisely map BnUD1, 103 InDel primer pairs uniformly covering the mapping interval and the BC5F3 and BC6F2 populations consisting of 1042 individuals were used to narrow the mapping interval to a 54.84 kb region. The mapping interval included 11 annotated genes. The bioinformatic analysis and gene sequencing data suggested that BnaA05G0157900ZS and BnaA05G0158100ZS may be responsible for the mutant traits. Protein sequence analyses showed that the mutations in the candidate gene BnaA05G0157900ZS altered the encoded PME in the trans-membrane region (G45A), the PMEI domain (G122S), and the pectinesterase domain (G394D). In addition, a 573 bp insertion was detected in the pectinesterase domain of the BnaA05G0157900ZS gene in the Bnud1 mutant. Other primary experiments indicated that the locus responsible for the downward-pointing siliques and up-curling leaves negatively affected the plant height and 1000-seed weight, but it significantly increased the seeds per silique and positively affected photosynthetic efficiency to some extent. Furthermore, plants carrying the BnUD1 locus were compact, implying they may be useful for increasing B. napus planting density. The findings of this study provide an important foundation for future research on the genetic mechanism regulating the dicotyledonous plant growth status, and the Bnud1 plants can be used directly in breeding.


Introduction
Leaf morphology affects dicotyledonous plant photosynthetic activities and the accumulation of dry matter. The slight up-curling of leaves may increase the overall light energy use efficiency and improve plant yields. Leaf shape formation is a complex developmental process involving leaf primordium formation, polarity establishment, and cell differentiation. The curled leaf phenotype is caused by mutations to genes related to leaf development [1,2], including genes encoding the transcription factors Class III HOME-ODOMAIN LEUCINE-ZIPPER (HD-Zip III) [3][4][5], KANADI (KAN) [6][7][8], WUSCHEL RELATED HOMEOBOX (WOX) [9], and TB1-CYC-PCF (TCP) [10]. These transcription factors modulate leaf polarity establishment and cause leaves to curl upward by regulating the expression of leaf asymmetry development-related genes, including ASYMMETRIC LEAVES 1 (AS1) and AS2 [11]. Plant hormone biosynthesis and signal transduction is Leaf morphological mutations in B. napus (e.g., upward and downward curling and wrinkling) are related to photosynthetic efficiency and affect the yield. Previous research on leaf curling revealed the associated loci and genes, including a dominant locus (BnDWF/DCL1) [36], semi-dominant genes (sca and ds-4) [37,38], and the genes (BnUC1, BnUC2, and BnUC3) [39][40][41] responsible for the up-curling of B. napus leaves. Analyses of gene functions demonstrated that abnormalities in some leaf flatness-related traits are caused by the deficient expression of genes encoding proteins mediating phytohormone (e.g., auxin and brassinolide) biosynthesis or signaling [13,[42][43][44]. Defects in genes directly affecting phytohormone regulatory pathways usually result in severe morphological changes that lead to abnormal plant development, with potential implications for breeding. However, identifying loci associated with leaf morphological variations may help developmental biologists and breeders attempting to develop new varieties with enhanced photosynthetic activities.
In the present study, we analyzed a B. napus cv. NJAU5773 mutant (Bnud1) that had downward-pointing siliques and up-curled leaves using a genetic and molecular approach. We fine mapped a dominant locus (BnUD1), identified candidate genes for the mutated pleiotropic traits on the basis of sequencing and gene expression experiments, and explored the effects of BnUD1 on agronomic traits. Our findings may serve as the foundation for the breeding of B. napus lines with potentially improved morphological characteristics and may be useful for elucidating the genetic mechanism underlying leaf and silique development.

Performance of the Bnud1 Mutant
Brassica napus NJAU5773 (named Bnud1 in this study) has up-curling leaves at the seedling stage, which is in contrast to the normal flat leaves of the canola variety Zhongshuang 11 (ZS11) (Figure 1a). Additionally, NJAU5773 has downward-pointing flowers that develop into downward-pointing siliques (Figure 1b,c). Plants with up-curling leaves and downward-pointing siliques exhibit semi-dwarfism in a segregating population derived from NJAU5773. To examine the Bnud1 mutant, near isogenic lines (ZS11-UD1 and ZS11) were developed via marker-assisted selection.

Inheritance of the Up-Curling Leaf and Downward-Pointing Silique Traits
In the current study, NJAU5773 was reciprocally crossed with canola variety ZS11, Figure 1. Performance of the parents NJAU5773 and ZS11 for constructing mapping population. (a) shows the leaves of the parent NJAU5773 with up-curling leaves (right) and the parent ZS11 with flat leaves (left) at the seeding stage. (b) shows the flowers of the parent NJAU5773 with downward-pointing flowers (right) and the parent ZS11 with normal flowers (left) at the flower stage. (c) shows the siliques of the parent NJAU5773 with downward-pointing silique (right) and the parent ZS11 with upright silique (left) at the maturity stage.

Inheritance of the Up-Curling Leaf and Downward-Pointing Silique Traits
In the current study, NJAU5773 was reciprocally crossed with canola variety ZS11, which has a sequenced genome (http://brassicadb.org/brad/, accessed on 15 January 2023), to generate F 1 plants and progeny populations for genetic analyses. Investigations involving segregating populations, which included F 2 , and successively backcrossed populations indicated that the segregation ratio (i.e., plants with up-curling leaves and downward-pointing siliques vs. wild-type plants) was consistent with the Mendel inheritance ratio (as suggested by the Chi-square test) (Table 1). Accordingly, the up-curling leaf and downward-pointing silique traits appear to be controlled by a dominant locus (i.e., BnUD1).

Mapping of the BnUD1 Locus
To map the BnUD1 locus, a BC 6 F 2 population consisting of 308 plants was derived from the cross between the two parents (NJAU5773 and ZS11). Thirty plants with up-curling leaves and downward-pointing siliques and 30 plants with flat leaves and upright siliques were selected from the BC 6 F 2 population to scan the BnUD1 locus via a bulked segregant analysis (BSA) approach. The two pooled samples were sequenced, with 30.06× sequence coverage for plants with the BnUD1 locus and 21.65× sequence coverage for plants lacking the BnUD1 locus. The clean reads were aligned to the B. napus cv. ZS11 reference genome (ZS11-v20200127; http://cbi.hzau.edu.cn/cgi-bin/rape/download_ext, accessed on 15 January 2023). This helped to identify the single nucleotide polymorphisms (SNPs) between the two pools. A total of 444,519 SNPs and 79,892 small insertion/deletions (InDels) were detected (Table S1). The ∆SNP index was calculated and the following two segments of the A05 chromosome were considered as candidate BnUD1-harboring regions: 7,094,487-11,086,188 bp (approximately 3.99 Mb) and 37,261,266-37,670,607 bp (approximately 0.41 Mb) ( Figure S1; Table S2).

Gene Cloning
The homologous segment sequences in the fine mapping interval were downloaded from the B. napus cv. ZS11 genome database. The genes in the interval were annotated on the basis of the A. thaliana genome to identify the candidate genes associated with the up-curling leaf and downward-pointing silique traits. The interval harbored 11 annotated genes ( Table 2). To analyze the candidate genes in the BnUD1 locus, we cloned the 11 genes from the two parents of the mapping populations (NJAU5773 and ZS11). The subsequent alignment of these sequences indicated that BnaA05G0157900ZS, BnaA05G0158100ZS, and BnaA05G0158300ZS differed between the two parents ( Figure S2), whereas the other examined genes within the mapping interval were identical in the two parents. The comparison of the BnaA05G0157900ZS sequences revealed that the encoded protein in NJAU5773 has three amino acid (AA) substitutions, of which the Gly-to-Ala mutation (G45A) occurred in the trans-membrane region, the Gly-to-Ser mutation (G122S) was detected in the PMEI domain, and the Gly-to-Asp mutation (G394D) was present in the pectinesterase domain ( Figure S2a). In addition, a 573 bp segment was inserted into the pectinesterase domain of BnaA05G0157900ZS gene in the Bnud1 mutant. Next, we developed an InDel marker (BnA05ID1) specific for this insertion (Table S3). The marker co-segregated with the up-curling leaf and downward-pointing silique phenotypes in the segregating populations ( Figure 4). These sequence alterations may affect leaf and silique trait formation in the Bnud1 mutant.  Figure S2a). In addition, a 573 bp segment was inserted into the pectinesterase domain of BnaA05G0157900ZS gene in the Bnud1 mutant. Next, we developed an InDel marker (BnA05ID1) specific for this insertion (Table S3). The marker co-segregated with the up-curling leaf and downward-pointing silique phenotypes in the segregating populations ( Figure 4). These sequence alterations may affect leaf and silique trait formation in the Bnud1 mutant.  The protein encoded by BnaA05G0158100ZS in the Bnud1 mutant included one substitution (Val-to-Asp) at AA position 146, which is in the RING domain of a RING/U-box The protein encoded by BnaA05G0158100ZS in the Bnud1 mutant included one substitution (Val-to-Asp) at AA position 146, which is in the RING domain of a RING/U-box superfamily protein ( Figure S2b). This substitution may lead to a change in function. The examination of BnaA05G0158300ZS in the Bnud1 mutant detected three AA substitutions, but they were not located in the IENR2 domain encoded specifically by this gene ( Figure S2c). The results of the sequence analyses suggested that BnaA05G0157900ZS and BnaA05G0158100ZS are the most likely candidate genes in the BnUD1 locus.

Candidate Gene Analysis
To determine whether the gene mutations are associated with the mutant traits, we further examined the gene functions by conducting a bioinformatic analysis as well as gene expression experiments using a pair of isogenic lines with the ZS11 genetic background. The BnaA05G0157900ZS gene is homologous to AT1G53840, which encodes PECTIN METHYLESTERASE 1 (PME1). The PMEs can catalyze the specific demethylesterification of HG to form load-bearing Ca 2+ crosslinks that affect the texture and rigidity of the cell wall [29][30][31]. Thus, they are reportedly involved in various biological processes, including cell wall expansion, seed germination, and hypocotyl elongation [45][46][47]. The overexpression of PMEI-encoding genes in wild-type A. thaliana plants leads to the production of curled leaves, a convoluted shoot, and downward-pointing siliques [34,35]. In the present study, the four sequence changes in the Bnud1 mutant BnaA05G0157900ZS gene were located in the gene-coding region or intron region. On the basis of the previously reported potential function of the protein encoded by BnaA05G0157900ZS, these sequence mutations are probably responsible for the observed mutated traits.
The mutation in the Bnud1 mutant BnaA05G0158100ZS gene was located in the sequence encoding the RING/U-box domain. This gene is homologous to AT1G53820, which encodes the biological stress-responsive RING/U-box superfamily member ARABIDOPSIS TOXICOS EN LEVADURA 60 (AL60). This gene has not been linked to substantial alterations to leaf and silique morphological characteristics [48][49][50][51][52]. The BnaA05G0158300ZS gene in Bnud1 has three mutations that are not within the sequence encoding the conserved IENR2 domains. Moreover, BnaA05G0158300ZS is homologous to AT1G53800, which was annotated as a gene involved in sarcomeric titin assembly during cardiac myofibrillogenesis in animals [53,54]. However, AT1G53800 homologs in plants have not been investigated.
The expression levels of the three mutated genes in the mapping interval were determined by performing a quantitative real-time polymerase chain reaction (qRT-PCR) analysis of leaf samples from the four plants with the Bnud1 mutated traits and the four plants with the wild-type traits in the BC 6 F 2 population. Both BnaA05G0157900ZS and BnaA05G0158100ZS were expressed at significantly lower levels in the plants with the Bnud1 mutated traits than in the plants with the wild-type traits ( Figure 5). In contrast, the BnaA05G0158300ZS expression level did not differ between the two plant types. This implied that BnaA05G0158300ZS is not associated with the formation of mutated traits ( Figure 5). These results suggested that BnaA05G0157900ZS and BnaA05G0158100ZS may be responsible for the up-curling leaf and downward-pointing silique traits of the Bnud1 mutant.
plants have not been investigated.
The expression levels of the three mutated genes in the mapping interval were determined by performing a quantitative real-time polymerase chain reaction (qRT-PCR) analysis of leaf samples from the four plants with the Bnud1 mutated traits and the four plants with the wild-type traits in the BC6F2 population. Both BnaA05G0157900ZS and BnaA05G0158100ZS were expressed at significantly lower levels in the plants with the Bnud1 mutated traits than in the plants with the wild-type traits ( Figure 5). In contrast, the BnaA05G0158300ZS expression level did not differ between the two plant types. This implied that BnaA05G0158300ZS is not associated with the formation of mutated traits ( Figure 5). These results suggested that BnaA05G0157900ZS and BnaA05G0158100ZS may be responsible for the up-curling leaf and downward-pointing silique traits of the Bnud1 mutant.

Agronomic Traits
To evaluate the effects of the BnUD1 locus on plant agronomic traits, 30 plants with the Bnud1 mutated traits and 30 plants with the wild-type traits were randomly sampled from the BC 6 F 2 population derived from the cross between NJAU5773 and the recurrent parent ZS11. The values for some of the agronomic traits, including plant height, branch height, main inflorescence length, number of first effective branches, and 1000-seed weight, were significantly lower for the plants with up-curling leaves and downward-pointing siliques than for the plants with flat leaves and upright siliques ( Table 3). The other agronomic traits, including stem diameter, siliques of the main inflorescence, total siliques per plant, and silique length, did not differ between the plants with and without the BnUD1 locus (Table 3). Thus, the BnUD1 locus appeared to negatively affect the plant stature, resulting in a compact architecture. Additionally, the number of seeds per silique was significantly higher for the plants with the BnUD1 locus than for the plants without the BnUD1 locus, reflecting the positive effects of the BnUD1 locus on the seed yield.

Determination of the Chlorophyll Content and Photosynthetic Efficiency
Compared with the plants with the wild-type traits at the seedling stage, the leaf chlorophyll (Chl) a, Chl b, and total Chl contents as well as the Chl a/b ratio were significantly higher for the plants with the Bnud1 mutated traits selected from the BC 6 F 2 population derived from the cross between the NJAU5773 and the recurrent parent ZS11 (Table 4). This result indicated that the Bnud1 mutated traits were associated with increases in the leaf Chl content. The leaf net photosynthetic rate, stomatal conductance, and concentration of intercellular CO 2 were significantly higher in the plants with the BnUD1 locus than in the plants without the BnUD1 locus. However, the leaf transpiration rate did not differ between the plants with the BnUD1 locus and the plants without the BnUD1 locus (Table 5). Hence, the BnUD1 locus may lead to increased photosynthetic efficiency.

Discussion
Leaves and siliques are important photosynthesis-related organs that influence the agronomic value of crops. Up-curling leaves and downward-pointing siliques are typically the result of the heteromorphic development of plant organs. However, the genetic mechanism underlying the formation of up-curling leaves is generally unrelated to the genetic basis of silique formation.
Mutations that alter the ability of PMEs to catalyze the demethylesterification of the cell wall HG may modify cell wall components, resulting in up-curling leaves and silique and stem morphological abnormalities [29,34,35]. In this study, a PME gene contributing to the up-curling leaf and downward-pointing silique traits was identified (Figure 2 and Figure S2a). More specifically, the marker analysis revealed that mutations in the PME gene BnaA05G0157900ZS may help to explain the formation of downwardpointing siliques and up-curling leaves (Figures 3 and 4). Another candidate gene (BnaA05G 0158100ZS) for the mutated traits encodes a RING/U-box-containing protein associated with biological stress responses ( Table 2). Although this gene may be part of a complex genetic regulatory system, whether it is directly associated with the mutated traits is unclear.
The mutant NJAU5773, which was originally identified in a breeding population and obtained via consecutive generations of selfing, has three mutated genes in the mapping interval ( Figure S2). When the two candidate genes for the BnUD1 locus-associated traits were used as queries to screen for the corresponding gene sequences in the B. napus pangenome database (http://cbi.hzau.edu.cn/bnapus/), the mutated candidate genes were undetectable in other genomes. The segment harboring the BnUD1 locus is located in the A sub-genome of B. napus, similar to the homologous segment in the A genome of B. rapa. However, the candidate genes differed from the corresponding homologous genes. Therefore, the candidate gene mutations are unique to NJAU5773 and may be the product of natural evolutionary events.
Moderately up-curled leaves and downward-pointing siliques can theoretically increase light transmittance and the light saturation point, thereby increasing the overall photosynthetic efficiency. The increased planting density associated with appropriately up-curled leaves and downward-pointing siliques may also positively affect the harvest index [73,74]. Thus, the up-curling leaf trait should be explored in more detail. To date, three loci related to the up-curling leaf trait have been identified in B. napus (i.e., BnUC1, BnUC2, and BnUC3) [39][40][41]. In present work, we found that the Bnud1 mutant had an increased photosynthetic efficiency at seedling stage, and relatively small plant architecture ( Figure 1; Table 5). The BnUD1 locus is expected to be applied to increase the efficiency of leaf photosynthetic activities and decrease plant height (Tables 3 and 5), but the underlying mechanisms will need to be characterized in future investigations. Unlike the other loci mediating the up-curling of leaves, the BnUD1 locus also controls the formation of downward-pointing siliques and increases in the number of seeds per silique (Table 3). However, the reason for the increase in the number of seeds per silique is unclear. We speculate that the influx of carbon is enhanced in the downward-pointing siliques. Alternatively, the increase in seed production may be the result of increased pollination efficiency.

Plant Materials
The double-low B. napus (oilseed rape) line NJAU5773 with up-curling leaves (before the budding stage) and downward-pointing siliques obtained from our germplasm and canola variety ZS11 provided by Nanjing Agricultural University were used as the parents to produce the F 1 population. The F 1 individuals were selfed to generate F 2 mapping populations and backcrossed with the recurrent parent ZS11 (female) to construct the mapping populations. The selfed and backcrossed populations were examined to calculate the segregation ratio of plants with up-curling leaves and downward-pointing siliques to plants with flat leaves and upright siliques. The BC 5 F 3 and BC 6 F 2 populations were used for the preliminary and fine mapping of the BnUD1 locus. The plants with up-curling leaves and downward-pointing siliques and the plants with flat leaves and upright siliques in the BC 6 F 2 population were used for the BSA, qRT-PCR, and analyses of the Chl content, photosynthetic efficiency, and agronomic traits.
All materials were grown on the research farm of Nanjing Agricultural University (Nanjing, China). Plants were cultivated in 2.5 m rows, with 15 plants per row and 0.4 m between rows.

Genetic Analysis
To determine the number of genes controlling the up-curling leaf and downwardpointing silique phenotype of the Bnud1 mutant, all generations, including the F 1 , F 2 , and BC 1 populations and the derived lines (BC 1 -BC 6 , BC 5 F 3 , and BC 6 F 2 ), were grown in the field. Leaf and silique morphology were assessed at the seedling and maturity stages, respectively. Chi-square tests were performed using the segregation data in each population to analyze the genetic regulation of the up-curling leaf and downward-pointing silique traits.

Bulked Segregant Analysis
To map the BnUD1 locus, a BC 6 F 2 population was developed from the cross between the Bnud1 mutant with up-curling leaves and downward-pointing siliques and wild-type plants with flat leaves and upright siliques. Genomic DNA was extracted from young leaves using cetyl-trimethylammonium bromide (CTAB). The BC 6 F 2 family population was identified according to a BSA. Equal amounts of DNA from 30 plants with up-curling leaves and downward-pointing siliques and 30 wild-type plants from the BC 6 F 2 population were pooled to form the Bnud1 mutant trait bulk (UDB) and the wild-type trait bulk (WTB), respectively. The polymorphisms between the bulks (UDB and WTB) were screened using InDel markers from a previous study [75].
To efficiently develop linked markers in the target region in B. napus, a BSA sequencing (BSA-seq) experiment was performed. Genomic DNA from the different bulks was subjected to a whole-genome sequencing analysis. Short-insert (350-450 bp) sequencing libraries were constructed from approximately 2 µg parental genomic DNA using the TruSeq ® DNA Sample Preparation Kit (Illumina, San Diego, CA, USA). The quantified libraries were sequenced on the HiSeq 3000 platform (Illumina) to produce 150 bp pairedend reads. The InDels and SNPs were called as previously described [76,77]. The de novo assembled ZS11 genome was used as the reference for calculating the SNP index of UDB and WTB. The ∆SNP index was calculated by subtracting the SNP index for UDB from the SNP index for WTB.

Mapping of the BnUD1 Locus
The BSA-seq results for the mapping interval were used to identify 5345 SNPs/ 1275 InDels and 721 SNPs/199 InDels covering 3.99 Mb and 0.41 Mb intervals between UDB and WTB ( Figure S1; Table S2). Next, 103 differential InDel sites (at positions 37,261,266-37,670,607 bp and 7,094,487-11,086,188 bp of A05) were used to develop molecular markers that could narrow the mapping interval. The InDel markers were used to analyze the BC 5 F 3 and BC 6 F 2 populations to fine map the BnUD1 locus (Table S3). Finally, a fine linkage map for the locus associated with the up-curling leaf and downward-pointing silique traits was constructed using the JoinMap 4.1 software and the polymorphic InDel markers [78]. The primers used are listed in Table S3.
The PCR conditions for the molecular marker experiments were as follows: 94 • C for 5 min; 35 cycles of 94 • C for 30 s, annealing temperature of each InDel marker for 30 s, and 72 • C for 30 s; 72 • C for 10 min.

Identification of Genes in the Mapping Interval and Comparative Sequencing
The sequences of the fine mapping interval on the B. napus cv. ZS11 A05 chromosome were downloaded from the Brassicaceae database (http://brassicadb.org/brad/, accessed on 15 January 2023) and the B. napus pan-genome information online resource (http://cbi. hzau.edu.cn/bnapus/, accessed on 15 January 2023) to identify the genes in the mapping interval. The genes detected in the mapping interval were annotated on the basis of B. napus cv. ZS11 annotated genes.
Extracted DNA was digested using RNase I (Takara, Dalian, China) to remove RNA. The RNA-free DNA samples were genotyped by genome sequencing. Total RNA was extracted from the leaves at the seedling stage using the RNAprep Pure Plant Kit (BioTeke, Beijing, China). First-strand cDNA was synthesized from the RNA using a reverse transcription kit (Takara, Tokyo, Japan). All 11 genes identified in the mapped interval were cloned from the two parents with gene-specific primers designed using the Primer Premier 5.0 software [79] (Tables S4 and S5). The PCR amplifications were performed as previously described [80]. The amplified fragments were inserted into the pEASY-Blunt Cloning Kit vector (TransGen, Beijing, China) and sequenced. The resulting sequences were aligned using Clustal X1.83 software [81].

Quantitative Real-Time PCR Analysis
A qRT-PCR analysis was conducted to compare the expression levels of the three mutated genes in the mapping interval between the plants with the Bnud1 mutated traits and the plants with the wild-type traits in the BC 6 F 2 population. Leaves were collected from four plants at the five-leaf stage and then used to prepare the cDNA template for the qRT-PCR analysis, which was completed with gene-specific primers (Table S6). The gene expression levels were normalized against the expression of an BnActin gene (i.e., housekeeping gene) ( Table S6). The qRT-PCR was performed using the SYBR Green Realtime PCR Master mix and the CFX96-2 PCR system (Bio-Rad, Hercules, CA, USA) and the relative expression levels were analyzed as described previously [82]. Relative expression levels were calculated according to the 2 −∆∆Ct method, with the actin gene serving as the internal control. Four biological replicates were used.

Agronomic Trait Analysis
To investigate the effects of the BnUD1 locus on plant agronomic traits, 30 plants with the Bnud1 mutated traits and 30 plants with the wild-type traits were randomly selected from the BC 6 F 2 population. The examined agronomic traits included plant height, branch height, main inflorescence length, stem diameter, number of first effective branches, number of siliques on the main inflorescence, total number of siliques per plant, silique length, seeds per silique, and 1000-seed weight. The mean values for all agronomic traits were compared between the plants with Bnud1 mutated traits and the plants with wild-type traits by t-tests.

Determination of the Chlorophyll Content and Photosynthetic Efficiency
Fifteen homozygous plants with the Bnud1 mutated traits and 15 plants with the wild-type traits were randomly selected from the BC 6 F 2 population at the seedling stage to measure the Chl contents. Specifically, Chl was extracted from 0.2 g fresh leaves using 50 mL 80% acetone, after which the Chl content was determined using the Alpha-1500 spectrophotometer (LASPEC, Shanghai, China). The leaf Chl a, Chl b, and total Chl contents were measured as previously described [83,84].
Six plants with the BnUD1 locus and six plants without the BnUD1 locus were randomly selected from the BC 6 F 2 population at the seedling stage for an analysis of photosynthetic efficiency. The photosynthetic characteristics of the plants were determined using the Li-Cor 6400 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA), with the built-in light source set at 1000 µmol photons m −2 s −1 at 23 • C as previously described. All measurements were completed between 9:00 a.m. and 11:00 a.m. [84].

Conclusions
A new mutant B. napus plant (NJAU5773) with up-curling leaves and downwardpointing siliques was identified in our B. napus germplasm. Inheritance studies showed that the up-curling leaf and downward-pointing silique traits were controlled by one dominant