Identification and Candidate Gene Analysis of Brcl1, a Novel Gene Confers a Leaf Curled Phenotype in Brassica rapa L.

Abstract

Leaf shape is an important determinant of photosynthesis, yield and quality in plants. In this study, we obtained a curled leaf mutant, cl1, from an ethyl methanesulfonate (EMS)-induced mutagenesis population. It was designated the Brcl1^YS locus. Bulk segregant RNA sequencing combined with recombinant screening identified the candidate interval responsible for Brcl1^YS in a 97.5 kb region on chromosome A02. Twelve genes were identified within the candidate region. Sequence differences and co-separation verification confirmed that BraA02g017030.3C was the most promising candidate gene underlying the Brcl1^YS locus. It is homologous to Arabidopsis AT1G66350 (RGL1), which has been shown to act as a negative regulator of the gibberellin pathway. Combined with cell morphology observation, it is speculated that the loss of function of Brcl1^YS results in differences in cell development, ultimately leading to changes in leaf morphology. The results will contribute to the understanding of the molecular mechanisms underlying leaf curling in B. rapa.

1. Introduction

Brassica rapa is cultivated worldwide as an important crop, primarily for its leafy vegetables, such as Chinese cabbage and Pak-choi. It is also grown partly for fodder and oilseed production, including turnip rape and Yellow Sarson [1]. Leaf shape reflects a number of trade-offs, including light capture, gas exchange and temperature buffering [2]. Light absorption is the main driver of photosynthesis in domesticated plants and is one of the main determinants of crop yield [3,4,5]. Moderate leaf curling can enhance plant resilience by increasing light absorption and reducing water loss under adverse conditions [6,7]. This contributes to improved photosynthetic efficiency and grain yield. Thus, the molecular basis of leaf curling could provide a theoretical basis for growth and yield in B. rapa.

Leaf shape is influenced by various factors, including the external environment, internal gene expression levels, and hormone levels [8,9]. Several genes have been reported to be associated with leaf development, including KANADI, Class III HOMEODOMAIN LEUCINE-ZIPPER (HD-Zip-III), WUSCHEL RELATED HOMEOBOX (WOX), TB1-CYC-PCFs (TCPs) and other transcription factors involved in the establishment of leaf polarity [10,11,12,13]. It was also shown that hormonal factors are important in the process of leaf development. The uneven distribution of auxin (IAA) in the plant can cause leaf curling [14]. Meanwhile, the mutation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) gene family could cause leaf curling and dwarfism in Arabidopsis, Brassica napus, and Vaccinium uliginosum L. [15,16,17]. AUXIN RESPONSE FACTORS (ARFs) could interact with HD-ZIP IIIs and KANADI to regulate leaf development, and the loss of ARF3 function in Arabidopsis lead to leaf curling [18]. These studies have shown that deficient expression of genes encoding proteins that mediate phytohormone biosynthesis or signaling are responsible for abnormalities in some leaf flatness traits [19,20]. However, as a significant hormone regulating plant growth, the role of gibberellin (GA) in leaf curling has not been reported.

GA was involved in almost the whole process of plant growth, including seed germination, flowering and fruit development [21]. Through mass genetic screening and analysis in various plants, DELLA proteins act as key oppressors in the GA signaling pathway, which belongs to the GRAS gene family and includes GAI (GA-insensitive), RGA (Repressor of ga1-3), RGL1 (RGA-like1), RGL2 (RGA-like2) and RGL3 (RGA-like3) [22,23]. DELLA proteins are highly influential, as they can interact with many hormones and environmental response factors to regulate plant growth and development [24]. Recent reports found that DELLA proteins positively regulate seed size by enhancing cell division in Arabidopsis [25], and they can interact with NAC though the GA signaling to promote secondary cell wall formation in cotton [26]. As a member of the DELLA protein subfamily, over expression of RGL1 decreased the content of GA and regulated seed germination, leaf expansion, flowering, stem elongation and flower development [27]. In maize, changes in local GA level could regulate the leaf morphology by affecting cell division [28]. Therefore, RGL1 may regulate the content of hormones or cell division to control leaf morphology.

In the present study, we identified an EMS mutant, Brcl1^YS, which was phenotypically characterized by significant leaf curling compared to the recipient parent, Yellow Sarson (YS). We fine-mapped the locus responsible for the leaf curling trait using single nucleotide polymorphisms (SNPs) and other molecular markers. We identified the candidate gene for the trait based on gene sequencing, candidate gene co-separation and expression analyses. Our results will provide clues for further positional cloning and functional research on Brcl1^YS and provide a good basis for elucidating the molecular mechanism underlying the leaf curl trait in B. rapa.

2. Results

2.1. Characteristics of Brcl1^YS

Each ten individuals, from 15 to 50 days, in the similar growth stage, were utilized to detect variations between the two parents, Brcl1^YS and YS. Compared with the YS, the leaves of Brcl1^YS were up-curled and crinkled, whereas YS had normal, flat leaves (Figure 1a,b). In addition, the Brcl1^YS had shorter stature (Figure 1c). The plant height, stem width, leaf length and leaf width were further determined after 45 days. All the four indicators, including plant height (YS with 8.97 ± 0.76 cm and Brcl1^YS with 4.98 ± 0.44 cm), stem width (YS with 0.74 ± 0.02 cm and Brcl1^YS with 0.62 ± 0.06 cm), leaf length (YS with 8.38 ± 0.53 cm and Brcl1^YS with 6.13 ± 0.26 cm), and leaf width (YS with 5.38 ± 0.22 cm and Brcl1^YS with 3.35 ± 0.19 cm), showed significant differences between the two parents, YS and Brcl1^YS (Table 1).

2.2. Genetic Analysis of Curled Leaf in Mutant Brcl1^YS

The phenotype of both reciprocal F₁ showed no difference with the curled parent, Brcl1^YS. There were no significant differences observed between the F₁ and reciprocal F₁ individuals, and both of them showed curled leaves, similar to the curled parent, Brcl1^YS.

The segregation ratio of the subsequent F₂ population was in accordance with the expected Mendelian segregation ratio of 3:1 (Curled vs. Normal leaves) (Table 2). It can be concluded that the curled leaf was controlled by a pair of dominant genes in B. rapa.

2.3. Analysis of Physiological Indices

Our study evaluated the photosynthetic rate (Pn), the intercellular CO₂ concentration (Ci), the transpiration rate (E), stomatal conductance (Gs) and the photosynthetic pigment content (chlorophyll a, chlorophyll b, chlorophyll, and carotenoid) to identify the difference between Brcl1^YS and YS (Table 3). Compared with YS, the Pn of Brcl1^YS was significantly increased, while the E, Ci and Gs were significantly decreased. Additionally, the photosynthetic pigment content of Brcl1^YS was significantly higher than that of YS. These results indicate that the mutant Brcl1^YS photosynthesis and resilience were enhanced.

2.4. Histological Analysis

The leaf of Brcl1^YS and YS were used to identify the cell morphology. The result showed that the vascular bundle cells of the mutant Brcl1^YS had a slight difference with YS (Figure 2a,b), but the Brcl1^YS leaves showed obvious curls from the vascular bundle center, and the curled degree of the leaves was about 103° (Figure 2c,d). Compared with the YS, the palisade cell and spongy mesophyll cells were smaller and arranged irregularly. Furthermore, the size of the epidermal cells was significantly different, as the cells were smaller in the curved surface but larger in the ventral surface in Brcl1^YS (Figure 2e,f).

2.5. Bulked Segregant RNA Sequencing (BSR-Seq) Analysis

To identify the candidate interval for Brcl1^YS, 45,494,770 and 46,962,128 high quality reads were mapped to the B. rapa reference genome V3.0 from the N-pool and C-pool, respectively. In total, compared with the reference genome, 197,180 SNPs and 210,789 InDels were identified in the BSR-seq data, respectively. Each of these variation loci were used to calculate the ED⁵. The results showed that the candidate region of the Brcl1^YS includes two regions, starting at 8,426,511 and ending at 9,253,907 on Chr. A02; the other region started at 16,103,255 and ended at 17,842,043 on Chr. A10 (Figure 3).

2.6. Fine Mapping of the Brcl1^YS Gene

According to the result of BSR-seq, we uniformly developed 10 pairs of InDel primers within two candidate intervals (Table S1).

Linkage analysis showed that the first interval in Chr. A02 (8,426,511–9,253,907) was linked to the Brcl1^YS locus. The F₂ recessive homozygous, including 717 individuals, was used to narrow down the candidate interval. Indel79, Indel83, Indel86, Indel87, SNP15, SNP3 and SNP2 were located on one side of Brcl1^YS, and SNP14 and SNP1 on the other side of Brcl1^YS. SNP2 and SNP14 were tightly linked to Brcl1^YS, with genetic distances of 0.20 cM and 0.42 cM, respectively. Brcl1^YS was mapped to a physical candidate interval of 97.5 kb (Figure 4).

2.7. Sequence Variations and Co-Segregation Verification of Candidate Genes

According to the Brassica rapa database (http://brassicadb.cn/#/Download/, accessed on 6 January 2021) and the homologous genes of Arabidopsis in TAIR (https://www.arabidopsis.org/, accessed on 6 January 2021), 12 genes (BraA02g017010.3C-BraA02g017120.3C) were located in the candidate interval in total (Table S2).

The results showed that the full length of BraA02g017030.3C was 1524 bp, including one exon and 508 amino acids. The Brcl1^YS promoter sequence remained unchanged when compared to YS, while the CDS sequence contained five non-synonymous SNP mutations. Asparagine changed to serine at position 1364 due to a G-base mutation to an A-base mutation that was consistent with the EMS mutation features (Figure 5). Co-separation primer found that this mutation exists in the fifteen recessive F₂ individuals (Figure 6) thus we concluded that the BraA02g017030.3C was the most likely candidate gene.

2.8. Expression Pattern Analysis of BraA02g017030.3C

To analyze the expression levels of BraA02g017030.3C, RNA was extracted from different organs (root, stem, leaf, flower) and leaves from different development periods (the cotyledon, the first, third and sixth true leaf) in YS and Brcl1^YS, respectively. The results showed that the BraA02g017030.3C is expressed in each organ, with the highest expression observed in the first true leaf (Figure 7).

3. Discussion

The leaf is the main photosynthetic organ and its morphology is an important agronomic trait in breeding the ideal plant. Moderate leaf curling could enhance resistance to adversity while increasing the light area and reducing water loss [8]. Since the leaf is the edible part of B. rapa, leaf curling is a significant factor influencing the yield and quality [29]. Thus, the molecular basis of leaf curl might not only set a theory basis for the growth of B. rapa but also offer general insights into improving the resistance to abiotic stress.

Previous studies showed that appropriate leaf curling is beneficial to improve the photosynthetic efficiency of plants and increasing the effective accumulation of photosynthetic products, but severe leaf curling could inhibit plant growth [30,31]. OsZHD1 and OsACL1 were important genes for rice leaf curling. Severe leaf curling reduced the fertility rate and delayed the heading date. Rice CLD1/SRL1 also modulated leaf rolling. The phenotypic characterization of the mutant found that leaf curling improved the content of chlorophyll [32,33]. In our study, we also detected the content of chlorophyll, Pn, Ci, E and Gs; the leaf curling mutant had higher chlorophyll and Pn, but Ci, E and Gs lower than normal leaf YS. The results indicate that the leaf curling trait locus may be useful to improve photosynthetic efficiency. The results are consistent with previous studies on B. napus [34,35]. Thus, breeding B. rapa varieties with moderate leaf curling could potentially improve the yield by improving the photosynthesis and stress resistance.

Functional and structural annotations showed that BraA02g017030.3C encodes the DELLA protein RGL1, which plays an inhibitor of GA transduction pathway and inhibited cell proliferation and expansion [25,36]. Recent utilization of CRISPR/Cas9 gene editing has resulted in the acquisition of a loss-of-function mutation in BraRGL1 due to two amino acid changes in the GRAS domain. The flower bud differentiation and bolting time of BraRGL1 mutants were significantly accelerated [37]. This finding is consistent with the observed phenotype of this material. However, whether there is functional redundancy of RGL1 and whether it is related to leaf curling still needs to be verified by further experiments. Most studies show that RGL1 is a significant factor in seed germination [38]. Meanwhile, the AtWRKY45 and AtWRKY75 transcription factors also interacted with RGL1 to positively regulate age-triggered leaf senescence [39,40]. In addition, RGL1 could combine with AUXIN RESPONSE FACTOR 7 (ARF7) and Aux/INDOLE-3-ACETIC ACID 9 (IAA9) to influence the IAA and GA signaling pathways, ultimately regulating cambial activity in poplar [41]. In Arabidopsis, a loss-of-function rgl1 line had reduced GA₄ expression and exhibited GA-independent activation of seed germination, leaf expansion, flowering, stem elongation, and floral development. Additionally, exogenous GA₃ spraying could not rescue the phenotype [27]. In our study, we found that leaf curling, caused by changes in cells, may be influenced by GA or IAA signaling, and Brcl1^YS may play a role in this process. However, the function of Brcl1^YS in leaf curling has not been clear.

In summary, we employed BSR-seq based on F₂ populations to identify the causal gene underlying Brcl1^YS, which is responsible for curled leaves in B. rapa, as further validated using co-segregation analysis. The data on Pn, Ci, E, Gs and photosynthetic pigment content indicate that the mutation of Brcl1^YS has the potential to enhance the photosynthesis and stress resistance of B. rapa (Table 3). Sequence and expression pattern analysis of BraA02g017030.3C had a nonsynonymous mutation and was mainly expressed in the first true leaf. Therefore, BraA02g017030.3C is the most likely candidate gene. Combined with cell morphology observation, it is speculated that the loss of function of Brcl1^YS results in differences in cell development, ultimately leading to changes in leaf morphology.

4. Materials and Methods

4.1. Plant Materials and Phenotypic Evaluation

Rapid Cycling Brassica rapa (RcBr) is resistant to bolting and vernalization-dependent. The B. rapa curled leaf mutant1 (Brcl1) was derived from the RcBr with a flat blade by EMS mutagenesis. The seeds were provided by Scott Woody (University of Wisconsin). Subsequently, an individual from the cross between Brcl1 and Yellow Sarson (YS) was selfed to develop the F₂ segregating population. Ten individuals in the F₂ population exhibiting a rolled leaf phenotype were selected for three successive backcrossing with the recurrent parent, YS, following one generation of selfing. Individuals exhibiting a rolled leaf phenotype were selected and designated as Brcl1^YS, which were expected to be near-isogenic lines (NILs) for the recurrent parent, Yellow sarson. A cross between Brcl1^YS and YS was conducted to generate the new NIL-F₂ population. The analysis was conducted on a total of 500 F₂ individuals, which had been cultivated under greenhouse conditions since September 2018. Concomitantly, conventional linkage analysis was carried out on 1283 F₂ individuals in March 2019. All plants were sown directly into 10 cm pots without additional vernalization. Phenotypic analysis was conducted at the Shenyang Agricultural University Experiment Station, Shenyang, China, at the following geographic coordinates: 41.8 °N, 123.4 °E.

A phenotypic investigation of the Brcl1^YS and YS was conducted utilizing digital display vernier calipers. Four indices were employed to assess the dissimilarities between Brcl1^YS and YS, including plant height, stem size, leaf width and leaf length. Once the plants had reached approximately 45 days of age, the mutant Brcl1^YS and YS, which exhibited similar growth trends, were selected for further analysis. The mean of 10 individuals was employed to ascertain the disparity in phenotypic data via Student’s t-test for each index, utilizing SPSS v17.0 (IBM Corp., Armonk, NY, USA).

4.2. Genetic Analysis

To ascertain the genetics of curled leaves in B. rapa, phenotypic characterization was carried out for each generation (Brcl1^YS, YS, F₁, F₂), and the segregation ratios of the F₂ populations were determined by using the Chi-square test [42].

4.3. Photosynthetic Index and Chlorophyll Content Determination

To determine the growth differences between Brcl1^YS and YS, measurements were made of the photosynthetic rate (Pn), the intercellular CO₂ concentration (Ci), the transpiration rate (E) and stomatal conductance (Gs) using a Li-6400 portable photosynthesis system (LI-COR Biosciences, Lincoln, NE, USA). On a sunny day between 08:00–12:00 h, the photosynthetic index for Brcl1^YS and YS was measured at the same growth stage after the plant’s fourth true leaf had fully formed. Photosynthetic indices were measured using a modified method [43]. There were three duplicates carried out, each including six plants. Ten Brcl1^YS and YS leaves were mixed with the solution using a 0.5 cm diameter punch and left in the dark for 24 h. The method of chlorophyll and carotenoid contents were conducted according to previous studies [44].

4.4. Paraffin Section

Before being paraffin sectioned, fresh leaves of Brcl1^YS and YS were fixed for 24 h in formalin-glacial acetic acid (FAA). The leaves were then pumped outside to dry naturally. The samples were dehydrated for two hours using varying alcohol concentrations (30–95%). Then, overnight at 4 °C, they were incubated in 100% alcohol before being penetrated with xylene. Then, paraffin embedding and sectioning were conducted using a microtome (LeicaRM2016, Leica, Wetzlar, Germany). Using an optical microscope (Nikon ECLIPSE 80i, Tokyo, Japan), the stem and leaf shapes were observed. Using the NIS-Element SF3.2 software’s capture option, the photo was taken, the adjusted image was chosen, and it was saved (Nikon, Tokyo, Japan).

4.5. BSR-Seq Analysis

In order to perform BSR-Seq analysis, two pools were formed by mixing equal amounts of RNA from 50 normal plants (N pool) and 50 curled plants (C pool), based on the phenotypic scores of 500 F₂ individuals recorded in September 2018.

Following the construction of the cDNA libraries, sequencing was performed using the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) and the data analysis was handled by Personalbio in Shanghai, China. Trimmomatic v0.30 (Illumina, CA, USA) was used to eliminate low-quality bases in order to produce clean data, ensuring data accuracy [45]. Using Hisat2 (Johns Hopkins University, Baltimore, MD, USA), the clean data were compared to the B. rapa reference genome V3.0 (BRAD; http://brassicadb.cn/#/Download/, accessed on 12 March 2019) [46]. Samtools v0.1.18 and Bcftools v0.1.19 (Trust Sanger Institute, Cambridgeshire, UK) were used to detect single nucleotide variation (SNV) [47,48]. Based on an mpileup file produced by samtools v0.1.18 (Trust Sanger Institute, Cambridgeshire, UK) for BSR-seq, the Euclidean distance (ED) value was computed. The ED value of each unique SNV site was raised to the power of five, or ED⁵, in order to remove background noise [49]. The top 1% of ED⁵ values were used as a threshold in the screening process to identify the candidate area for the curled leaf phenotype. This was then compared with multiple SNV sites spread across various chromosomes.

4.6. DNA Extraction, Marker Development, Linkage Map Construction

Total DNA was extracted using the CTAB technique with a few minor modifications [50]. The amplification and PCR reaction volumes followed the earlier instructions [44]. Primer Premier 5.0 software (Premier Biosoft, San Francisco, CA, USA) was used to construct the primers for the InDel markers. The BSR-seq database was used to identify sequence variations in the candidate interval, which were utilized to design SNP or InDel markers. Join Map 4.0 was used to create a genetic linkage map [51]. Using Kosambi’s function [52], the genetic map distances (cM) were derived from the recombination frequencies. Table S1 displays the details of the primer.

4.7. Candidate Gene Prediction and Co-Separation Verification

The Brassica rapa database (BRAD; http://brassicadb.cn/#/Download/, accessed on 6 January 2021) and the Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org/, accessed on 6 January 2021) provided the gene information found in the candidate interval. The exact primers for co-separation and full-length sequencing amplification were designed using Primer Premier 5.0 (Table S1). The PCR products were purified using a Gel Extraction Kit (CWBIO, Beijing, China) and then ligated into a pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing at Sangon Biotech (Shanghai, China). Using the DNAMAN program (Lynnon Biosoft, San Ramon, CA, USA), the sequences were aligned. For co-separation verification, fifteen F₂ homozygous recessive individuals were used.

4.8. The Expression Pattern of the Candidate Gene

The candidate gene’s level of expression was assessed using qRT-PCR. The different organs (root, stem, leaf, flower) and leaves from different development periods (the cotyledon, the first true leaf, third true leaf and sixth true leaf) were used to extract the total RNA of Brcl1^YS and YS using an RNA extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China). The RNA was then subjected to reverse transcription to produce cDNA. Quantitative real-time PCR (qPCR) was performed using the cDNA template in a 25 µL reaction volume [44]. The relative expression level was analyzed with the 2^−ΔΔCT technique [53]. The cycle threshold (Ct) values were determined by averaging three separate biological replicates. Every sample underwent three separate technical replicate evaluations. The QuantStudio™ Real-Time PCR Software V1.3 (ABI, Los Angeles, CA, USA) was utilized to analyze the data. Gene-specific primers were designed using Primer Premier 5.0 (Table S1). The Actin gene (encoding beta actin) formed the reference gene [54].

5. Conclusions

BraA02g017030.3C was the most promising gene conferred to Brcl1^YS, which is homologous with Arabidopsis RGL1, belonging to DELLA protein and involved in the GA signal pathway. These results could facilitate our understanding of the mechanisms underlying leaf morphogenesis and provide a genetic resource for B. rapa improvement.

Author Contributions

Y.W. and G.Q. planned and designed the research. L.W. and H.L. performed the majority of the research and conducted data analysis. Y.S. and S.H. supervised the experiments and participated in data analysis. Y.L. and Z.L. participated in candidate gene cloning. W.W., C.L. and R.J. performed expression analysis of the target genes. L.W. and G.Q. wrote the manuscript with input from other coauthors. Y.W. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the results are included in this article. Additional relevant materials are available upon reasonable request from the corresponding author. The raw data from Bulked segregant RNA sequencing have been deposited at the NCBI Sequence Read Archive (SRA) repository under the accession numbers SRR29689162 and SRR29689163.

Acknowledgments

Special thanks for improving the English of the manuscript go to Bing Fang, Department of Foreign Language Teaching, Shenyang Agricultural University.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Tables

Figure 1. Phenotypic characterization of YS and Brcl1YS. (a) The orthograph of YS and Brcl1YS, scale bars = 1 cm; (b) the leaf of YS and leaves at different period of Brcl1YS, scale bars = 1 cm; (c) the flowering stage of YS and Brcl1YS, scale bars = 2 cm.

Figure 2. Cell observation of Brcl1YS and YS. (a,b) The vascular bundle of Brcl1YS and YS: scale bars = 50 μm; (c,d) Cross-section of leaf blade of Brcl1YS and YS: scale bars = 200 μm, the arrow represents the angle between the leaf and the vascular bundle center; (e,f) The leaf cells of Brcl1YS and YS: scale bars = 50 μm. Note: Vb—Vascular bundle; Ec—Epidermic cell; Pc—Palisade cell; Sm—spongy mesophyll.

Figure 3. BSR-Seq analysis. The BSR-Seq based distribution of SNPs on chromosomes. The x-axis shows the 10 B. rapa chromosomes and the y-axis shows the ED5 values of the filtered SNPs; the dashed line is the threshold of the top 1%.

Figure 4. Fine mapping of the Brcl1YS locus. (a) The Brcl1YS locus was black-colored in an interval of 1.04 cM between SNP markers SNP1 and SNP15 on chromosome A02. (b) The candidate region was finely mapped in the physical interval of 97.5 kb between SNP2 and SNP14 markers.

Figure 5. Candidate gene sequence analysis. The full length of BraA02g017030.3C between YS and Brcl1YS. The red boxes was five non-synonymous SNP mutations between YS and Brcl1YS, *: represents termination codon.

Figure 6. Candidate gene validation by co-separation of Brcl1YS. The mutant locus of YS, Brcl1YS and recessive F2 individuals.

Figure 7. The expression pattern of BraA02g017030.3C in the two parents. (a) The expression level of BraA02g017030.3C in different organs (root, stem, leaf, flower) detected by qRT-PCR; (b) the expression level of BraA02g017030.3C in different leaf development periods (the cotyledon, the first true leaf, the third true leaf and the six true leaf) detected by qRT-PCR. The error bars represent the standard errors from three replications; * represents p [less than] 0.05; ** represents p [less than] 0.01.

Table 1

Measurements of phenotype of YS and Brcl1^YS.

	Plant Height (cm)	Stem Width (cm)	Leaf Length (cm)	Leaf Width (cm)
YS	8.97 ± 0.76	0.74 ± 0.02	8.38 ± 0.53	5.38 ± 0.22
Brcl1 YS	4.98 ± 0.44 **	0.62 ± 0.06 **	6.13 ± 0.26 **	3.35 ± 0.19 **

** indicates significant difference at the 0.01 level.

Table 2

Genetic analysis of Brcl1^YS.

PopulationEmployed	Pedigree	TotalPlants	CurledLeaves	NormalLeaves	ExpectedRatio	X2
Brcl1 YS		50	50	0
YS		50	0	50
F1	YS×Brcl1YS	50	50	0
RF1	Brcl1YS×YS	50	50	0
F2-A	(YS×Brcl1YS) ⊗	500	381	119	3:1	0.40
F2-B	(YS×Brcl1YS) ⊗	1283	953	330	3:1	0.35

⊗ represent selfed.

Table 3

Photosynthetic index determination of YS and Brcl1^YS.

	Pn(μmol m−2 s−1)	E(mmol m−2 s−1)	Ci(μmol mol−1)	Gs(mmol m−2 s−1)	Chla	Chlb	Chl	Car
					(mg g−1 FW)
YS	10.75 ± 0.77	5.13 ± 0.87	351.88 ± 27.01	0.35 ± 0.73	1.57 ± 0.80	0.52 ± 0.27	2.09 ± 0.34	0.20 ± 0.07
Brcl1 YS	12.69 ± 0.74 *	4.05 ± 0.86 *	315.26 ± 30.29 **	0.23 ± 0.11 *	1.85 ± 0.42 *	0.79 ± 0.18 *	2.34 ± 0.59 *	0.44 ± 0.14 *

* indicate significant difference at the 0.05 level; ** indicate significant difference at the 0.01 level.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26020732/s1.

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