Genome-Wide Identiﬁcation and Expression Analysis of nsLTP Gene Family in Rapeseed ( Brassica napus ) Reveals Their Critical Roles in Biotic and Abiotic Stress Responses

: Non-speciﬁc lipid transfer proteins (nsLTPs) are small cysteine-rich basic proteins which play essential roles in plant growth, development and abiotic/biotic stress response. However, there is limited information about the nsLTP gene ( BnLTP ) family in rapeseed ( Brassica napus ). In this study, 283 BnLTP genes were identiﬁed in rapeseed, which were distributed randomly in 19 chromosomes of rapeseed. Phylogenetic analysis showed that BnLTP proteins were divided into seven groups. Exon/intron structure and MEME motifs both remained highly conserved in each BnLTP group. Segmental duplication and hybridization of rapeseed’s two sub-genomes mainly contributed to the expansion of the BnLTP gene family. Various potential cis -elements that respond to plant growth, development, biotic/abiotic stresses, and phytohormone signals existed in BnLTP gene promoters. Transcriptome analysis showed that BnLTP genes were expressed in various tissues/organs with different levels and were also involved in the response to heat, drought, NaCl, cold, IAA and ABA stresses, as well as the treatment of fungal pathogens ( Sclerotinia sclerotiorum and Leptosphaeria maculans ). The qRT-PCR assay validated the results of RNA-seq expression analysis of two top Sclerotinia -responsive BnLTP genes, BnLTP129 and BnLTP161 . Moreover, batches of BnLTPs might be regulated by BnTT1 and BnbZIP67 to play roles in the development, metabolism or adaptability of the seed coat and embryo in rapeseed. This work provides an important basis for further functional study of the BnLTP genes in rapeseed quality improvement and stress resistance. patterns. Our work showed critical roles of the BnLTP family genes in plant growth and development, as well as in response to biotic/abiotic stresses and phytohormone treatments. This study will lay a solid foundation for further functional research of BnLTP genes in quality improvement and stress resistance.


Introduction
Rapeseed (Brassica napus), which provides abundant vegetable oil and substantially potential biodiesel feedstocks, is one of the most important oilseed crops worldwide [1]. The growth of the world's population and the improvement of living standards have increased the global demand for rapeseed oil [2,3]. In addition, the yield and oil quality of rapeseed often suffer from extensive losses, owing to pathogen and biotic/abiotic stresses during rapeseed production worldwide [4][5][6][7]. These situations have increased the demand for the study of the genetic basis of high seed oil accumulation and high stress resistance of rapeseed.
Seeds are an important place for fatty acid synthesis and metabolism, so it is meaningful to have a proper understanding of nsLTPs involved in seed development, metabolism and adaptation. TT1 and bZIP67 are two representative transcription factors (TF) regulating seed coat and seed embryo developmental traits, respectively [32,33]. Therefore, in this study, differentially expressed genes (DEGs) of nsLTP family were identified from the DEG database of transgenic rapeseeds overexpressing BnTT1 and BnbZIP67, and it is speculated that they may be involved in the development and metabolism of seed coat and seed embryo.
The nsLTPs are small, basic proteins that exist in higher plants, which harbor an N-terminal signal peptide and contain an eight-cysteine motif (8CM, C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C) [34]. The nsLTPs function in the cell secretory pathway [16]. The nsLTPs are located in the plasma membrane [35][36][37], extracellular space [38], cell wall [39] and cytoplasm [40]. The nsLTPs have been reported to play important roles in various developmental and physiological processes [21,40,41]. In recent years, the number of plant genomes sequenced has exploded, which facilitates genome-wide analysis of the plant nsLTP gene family in a variety of plant species. The model dicot Arabidopsis has been reported to contain 79 nsLTP genes [42]. Studies showed that there are 77 in rice [42], 63 in maize [42], 58 in sorghum [42], 461 in wheat [43], 70 in barley [44], 63 in B. rapa [45], 89 in cabbage [46], 83 in potato [47], 64 in tomato [48], 51 in Gossypium arboretum [49], 47 in G. raimondii [49], 91 in G. hirsutum [49], 52 in sesame [50] and 64 in Arachis duranensis [51]. To date, there has been no systemic whole-genome analysis of the nsLTP gene (BnLTPs) family in B. napus under various abiotic/biotic stresses, as well as in seed development and germination, and seed coat color formation, although the reference genomes of different cultivars or ecotypes of B. napus have been published. Herein, we systemically identified BnLTPs in B. napus, and then performed the detailed analyses containing phylogenetic relationship, gene structure, MEME motifs, chromosome location, gene duplication and synteny, SSR loci and cis-element in promoters. We also detected their expression patterns in various organs and tissues and in response to biotic/abiotic stresses and phytohormone treatments. This study will lay a solid foundation for further functional research of BnLTP genes as well as their application in improvement of crop quality and stress resistance in B. napus.

Identification of BnLTP Genes
A total of 283 nsLTP genes were identified in the B. napus genome (Table S1). Gene sequences of BnLTP genes varied from 273 bp to 8989 bp in length, and BnLTP mRNAs ranged from 92 bp to 1809 bp in length. BnLTP proteins varied from 90 aa to 400 aa in length. BnLTP proteins all contained conserved 8CM domains ( Figure 1; Table S1). Of the 283 BnLTP proteins, 76 harbored C-terminal GPI anchor signal, whereas it is absent in the remaining 207 BnLTP proteins. The theoretical M W s of BnLTP proteins ranged from 9658.62 Da to 43,916.10 Da. The pI values of BnLTP proteins varied from 3.92 to 11.00. There existed 18-34 aa N-terminal signal peptides in all BnLTP proteins, and thus they all could be substrates for the cell's secretory pathways. In addition, for further evolutionary relationship analysis, a total of 127, 151 and 106 nsLTP genes were identified in B. rapa, B. oleracea and A. thaliana, respectively (Tables S2-S4). remaining 207 BnLTP proteins. The theoretical MWs of BnLTP proteins ranged from 9658.62 Da to 43,916.10 Da. The pI values of BnLTP proteins varied from 3.92 to 11.00. There existed 18-34 aa N-terminal signal peptides in all BnLTP proteins, and thus they all could be substrates for the cell's secretory pathways. In addition, for further evolutionary relationship analysis, a total of 127, 151 and 106 nsLTP genes were identified in B. rapa, B. oleracea and A. thaliana, respectively (Tables S2-S4).

Phylogenetic Relationship Analysis
In total, 667 8CM sequences of nsLTP proteins from B. napus, B. rapa, B. oleracea and A. thaliana (Table S5), were utilized to construct the phylogenetic tree. Evolutionary analysis showed that these 667 nsLTP proteins from four species were separated into seven groups: I, II, III, IV, V, VI and VII ( Figure 2). As shown in Table 1, these seven groups all existed in three Brassica species and A. thaliana. In total, there were 170 nsLTP genes from four species in group I, 118 in group II, 68 in group III, 46 in group IV, 98 in group V, 80 in group VI, and 87 in group VII.  Total  283  127  151  106  I  65  38  40  27  II  49  22  25  22  III  33  11  15  9  IV  21  9  12  4  V  42  19  21  16  VI  35  14  18  13  VII  38  14  20  15

Analysis of Gene Structure and Conserved Motifs
To detect exon-intron organizations of BnLTP genes, their gene structures were illustrated using the GFF3 file from a B. napus genome on the TBtools program ( Figure S1). Of 283 BnLTP genes, 143 contained no intron (Table S1). Of the remaining 140 BnLTP genes containing introns, 64 contained 1 intron, 62 had 2 introns, 5 possessed 3 introns, 4 harbored 4 introns, 2 included 5 introns, 2 contained 7 introns, and 1 had 7 introns. To identify conserved motifs in BnLTP protein sequences, MEME tools were used to perform the prediction and analysis. As shown in Figure S1, there existed 20 conserved MEME motifs in 283 BnLTP proteins.

Phylogenetic Relationship Analysis
In total, 667 8CM sequences of nsLTP proteins from B. napus, B. rapa, B. oleracea and A. thaliana (Table S5), were utilized to construct the phylogenetic tree. Evolutionary analysis showed that these 667 nsLTP proteins from four species were separated into seven groups: I, II, III, IV, V, VI and VII ( Figure 2). As shown in Table 1

Analysis of Gene Structure and Conserved Motifs
To detect exon-intron organizations of BnLTP genes, their gene structures were illustrated using the GFF3 file from a B. napus genome on the TBtools program ( Figure S1). Of 283 BnLTP genes, 143 contained no intron (Table S1). Of the remaining 140 BnLTP genes containing introns, 64 contained 1 intron, 62 had 2 introns, 5 possessed 3 introns, 4 harbored 4 introns, 2 included 5 introns, 2 contained 7 introns, and 1 had 7 introns. To identify conserved motifs in BnLTP protein sequences, MEME tools were used to perform the prediction and analysis. As shown in Figure S1, there existed 20 conserved MEME motifs in 283 BnLTP proteins.

Gene Duplication and Synteny Analysis
To reveal the mechanism underlying the expansion of the nsLTP gene family in B. napus, we detected the types of BnLTP gene duplication. Of 283 BnLTP genes, 191 (67.4%) and 31 (10.9%) genes were derived from segmental duplication and tandem duplication (Table S7). It appears that segmental duplication played important roles in the expansion of the BnLTP gene family. Based on the genome information from B. napus, we analyzed the synteny relationship of BnLTP genes ( Figure 3). For the 283 BnLTP genes, 204 syntenic gene pairs were found in B. napus genome in which 40 (19.6%), 23 (11.2%) and 141 (69.1%) gene pairs were obtained among BnA-BnA subgenomes, BnC-BnC subgenomes and BnA-BnC subgenomes, respectively. Overall, the expansion of the BnLTP gene family might have resulted from segmental duplication and hybridization of its two sub-genomes. (Table S7). It appears that segmental duplication played important roles in the expansion of the BnLTP gene family. Based on the genome information from B. napus, we analyzed the synteny relationship of BnLTP genes ( Figure 3). For the 283 BnLTP genes, 204 syntenic gene pairs were found in B. napus genome in which 40 (19.6%), 23 (11.2%) and 141 (69.1%) gene pairs were obtained among BnA-BnA subgenomes, BnC-BnC subgenomes and BnA-BnC subgenomes, respectively. Overall, the expansion of the BnLTP gene family might have resulted from segmental duplication and hybridization of its two sub-genomes.

Expression of nsLTPs in Different B. napus Tissues
To examine the expression patterns of BnLTP genes, we analyzed the transcription levels of BnLTP genes in 42 various organs/tissues from different development stages, including roots, stems, leaves, buds, anthocauli, sepals, petals, pistils, stamens, anthers, filaments, seeds, funiculus and silique pods using RNA-seq data (Tables S11 and S12). Of the 283 BnLTP genes, 29 were not expressed at any tissues, whereas the remaining genes were expressed in various tissues with the different levels ( Figure 4 and Figure S3), which included organ-specific and constitutive expression patterns. For example, seed-specific genes contained BnLTP017, BnLTP117 and BnLTP210. Of 38 gene clusters, 18 had similar expression patterns among 42 different tissues, and those of the remaining ones were different ( Figure S4). Of 74 duplicated gene groups, 50 harbored similar expression patterns across 42 various organs, and 20 had different expression patterns ( Figure S5). These results showed that members of some duplicated gene groups (56.8%) or gene clusters (52.6%) with the different expression patterns might have undergone functional divergence.
Totally, transcriptome data of 185 BnLTP genes were obtained for eight LMD-obtained tissues from the early globular seed ( Figure S6; Table S13). A total of 50 were expressed in the embryo proper, 90 in the micropylar endosperm, 49 in the peripheral endosperm, 47 in the chalazal endosperm, 36 in the chalazal proliferating tissue, 56 in the chalazal seed coat, 66 in the inner seed coat and 64 in the outer seed coat (FPKM values > 1). In addition, each tissue contained a unique set of BnLTP transcripts. For example, the embryo-specific gene set contained 9 BnLTP genes, and that of micropylar endosperm harbored 34. This result showed that some BnLTP genes might play roles in tissue-specific development processes of the globular seed.
In total, RNA-seq data of 143 BnLTP genes were acquired for three LMD-acquired tissue types of the funiculus ( Figure S7; Table S14). A total of 82, 83 and 58 BnLTP genes were expressed in epidermis, cortex and vasculature (FPKM values > 1), respectively, of which 11, 27 and 10 were tissue-specific in corresponding tissues. This result showed the possible crucial roles of BnLTP genes in tissue-specific development processes of the B. napus funiculus. Totally, transcriptome data of 185 BnLTP genes were obtained for eight LMD-obtained tissues from the early globular seed ( Figure S6; Table S13). A total of 50 were expressed in the embryo proper, 90 in the micropylar endosperm, 49 in the peripheral endosperm, 47 in the chalazal endosperm, 36 in the chalazal proliferating tissue, 56 in the chalazal seed coat, 66 in the inner seed coat and 64 in the outer seed coat (FPKM values > 1). In addition, each tissue contained a unique set of BnLTP transcripts. For example, the embryo-specific gene set contained 9 BnLTP genes, and that of micropylar endosperm harbored 34. This result showed that some BnLTP genes might play roles in tissue-specific development processes of the globular seed.
In total, RNA-seq data of 143 BnLTP genes were acquired for three LMD-acquired tissue types of the funiculus ( Figure S7; Table S14). A total of 82, 83 and 58 BnLTP genes were expressed in epidermis, cortex and vasculature (FPKM values > 1), respectively, of which 11, 27 and 10 were tissue-specific in corresponding tissues. This result showed the possible crucial roles of BnLTP genes in tissue-specific development processes of the B. napus funiculus.

Expression of nsLTPs in Response to Biotic Stresses
Plant nsLTPs have been listed as pathogenesis-related protein-14 family [52], and they have been reported to be involved in plant disease resistance reactions [53,54]. Blackleg disease (caused by L. maculans) and white stem rot (caused by S. sclerotiorum) are the most severe diseases of B. napus. We thus detected the expression response of BnLTPs to these two fungal diseases.

Expression of nsLTPs in Response to Biotic Stresses
Plant nsLTPs have been listed as pathogenesis-related protein-14 family [52], and they have been reported to be involved in plant disease resistance reactions [53,54]. Blackleg disease (caused by L. maculans) and white stem rot (caused by S. sclerotiorum) are the most severe diseases of B. napus. We thus detected the expression response of BnLTPs to these two fungal diseases.
In response to S. sclerotiorum, except for BnLTP016, BnLTP031, BnLTP252 and BnLTP265, the transcriptome data of the remaining 279 BnLTP genes were obtained ( Figure 5; Table S15), of which 152 were expressed (FPKM values > 1). A total of 72 BnLTP genes were S. sclerotiorum-responsive genes (p < 0.05) of which BnLTP033, BnLTP129, BnLTP161 and BnLTP264, might have more critical roles in resistance to white stem rot. In response to L. maculans, except for BnLTP016, RNA-seq data of the remaining 282 BnLTP genes were obtained ( Figure 6; Table S16) of which 174 were expressed (FPKM values > 1). A total of 96 BnLTP genes were L. maculans-responsive genes (p < 0.05) of which BnLTP161 and BnLTP015 might play more important roles in plant defense to blackleg disease.

Expression of nsLTPs in Response to Abiotic Stresses
RNA-seq data of 283 BnLTP genes for heat, drought, NaCl, cold and dehydration stresses were all obtained (Figure 7; Tables S17 and S18), and there were 65, 74, 76, 80 and 67 expressed in various samples from the treatments (FPKM values > 1), respectively. We found that the numbers of BnLTP genes (p < 0.05) responding to heat, drought, NaCl, cold and dehydration were 12, 26, 61, 41 and 32, respectively. The responsive mechanisms were different between different low-temperature conditions. Then, we also examined the expression levels of BnLTP genes in two early-maturing B. napus varieties with different cold tolerance under cold accumulations at chilling (CA) and freezing (FA) temperature and cold shocks at the same temperature (CB and FB) condition. A total of 94, 94, 84 and 90 BnLTP genes were expressed under CA, FA, CB and FB treatments, respectively ( Figure S8; Table S19); 24, 26, 39 and 48 were CA-, FA-, CB-and FB-responsive genes, respectively.
Auxin-responsive elements (TGA-element and AuxRR-core) and abscisic acid-responsive elements (ABRE) were found in the promoters of BnLTP genes, and thus we analyzed their expression patterns under IAA and ABA treatments. Of the 283 BnLTP genes, transcriptome data of 149 and 176 were obtained for IAA and ABA treatment, respectively (Figure 7; Tables S17 and S20). We found that 13 BnLTP genes were IAA-responsive genes, whereas 22 were ABA-responsive genes (p < 0.05). In response to S. sclerotiorum, except for BnLTP016, BnLTP031, BnLTP252 and BnLTP265, the transcriptome data of the remaining 279 BnLTP genes were obtained ( Figure 5; Table S15), of which 152 were expressed (FPKM values > 1). A total of 72 BnLTP genes were S. sclerotiorumresponsive genes (p < 0.05) of which BnLTP033, BnLTP129, BnLTP161 and BnLTP264, might have more critical roles in resistance to white stem rot. In response to L. maculans, except for BnLTP016, RNA-seq data of the remaining 282 BnLTP genes were obtained ( Figure 6; Table  S16) of which 174 were expressed (FPKM values > 1). A total of 96 BnLTP genes were L. maculans-responsive genes (p < 0.05) of which BnLTP161 and BnLTP015 might play more important roles in plant defense to blackleg disease.
Auxin-responsive elements (TGA-element and AuxRR-core) and abscisic acid-responsive elements (ABRE) were found in the promoters of BnLTP genes, and thus we analyzed their expression patterns under IAA and ABA treatments. Of the 283 BnLTP genes, transcriptome data of 149 and 176 were obtained for IAA and ABA treatment, respectively (Figure 7; Tables S17 and S20). We found that 13 BnLTP genes were IAA-responsive genes, whereas 22 were ABA-responsive genes (p < 0.05).

Expression of nsLTPs in B. napus Seed Germination
To explore the roles of BnLTP genes in seed germination trait, we examined their expression patterns during different stages of seed germination of three selected accessions with representative germination rates (high, medium or low; C129, C033 and C032, respectively). During 4 time points, 2, 12, 36 and 72 h after imbibition in these 3 accessions, the numbers of expressed BnLTP genes (FPKM values > 1) increased gradually, ranging from 26 to 135 and those of responsive BnLTP genes also increased gradually ranging from 1 to 46 (Figures 8 and S9; Table S21). This result showed that some BnLTP genes might be important in different seed germination processes.

Expression of nsLTPs in B. napus Seed Germination
To explore the roles of BnLTP genes in seed germination trait, we examined their expression patterns during different stages of seed germination of three selected accessions with representative germination rates (high, medium or low; C129, C033 and C032, respectively). During 4 time points, 2, 12, 36 and 72 h after imbibition in these 3 accessions, the numbers of expressed BnLTP genes (FPKM values > 1) increased gradually, ranging from 26 to 135 and those of responsive BnLTP genes also increased gradually ranging from 1 to 46 (Figures 8 and S9; Table S21). This result showed that some BnLTP genes might be important in different seed germination processes. napus accessions seeds with representative germination rates (high, medium, or low; C129, C033 and C032, respectively) at 0, 12, 24, 48 and 72 h after imbibition.

Expression of nsLTPs in B. napus Brown Seeds and Yellow Mutation
Of the 55,637 unigenes identified in 26 d brown seeds and yellow mutation, 64 BnLTP genes were detected (Table S22). Only three BnLTP genes, BnLTP089, BnLTP110 and BnLTP257 were found to be DEGs between the materials, which were all up-regulated in

Expression of nsLTPs in B. napus Brown Seeds and Yellow Mutation
Of the 55,637 unigenes identified in 26 d brown seeds and yellow mutation, 64 BnLTP genes were detected (Table S22). Only three BnLTP genes, BnLTP089, BnLTP110 and BnLTP257 were found to be DEGs between the materials, which were all up-regulated in yellow mutation. This result showed that they might play important roles in regulating or co-regulating the seed coat color of B. napus.

Expression of nsLTPs in B. napus Overexpressing BnTT1 and BnbZIP67
In total, there existed 40, 30 and 32 BnLTP family DEGs in the mid-stage seeds of transgenic rapeseeds overexpressing pBAN::BnTT1 (BOE), pNapA::BnTT1 (NOE) and pNapA::BnbZIP67 (B67O), respectively (Figure 9a; Tables S23-S25). Some 15 BnLTP DEGs were up-regulated in BOE, 11 in NOE, and 16 in B67O, and meanwhile 25 BnLTP DEGs were down-regulated in BOE, 19 in NOE, and 16 in B67O. In addition, we found that two BnLTP DEGs both were up-regulated in BOE and NOE, and nine were all down-regulated in BOE and NOE, while five were up-regulated in NOE, and they were down-regulated in BOE (Figure 9b). These BnLTP genes might be regulated by BnTT1 or BnbZIP67 and play roles in seed development, secondary metabolism, oil accumulation and stress resistance during seed growth and maturation. yellow mutation. This result showed that they might play important roles in regulating or co-regulating the seed coat color of B. napus.

Expression of nsLTPs in B. napus Overexpressing BnTT1 and BnbZIP67
In total, there existed 40, 30 and 32 BnLTP family DEGs in the mid-stage seeds of transgenic rapeseeds overexpressing pBAN::BnTT1 (BOE), pNapA::BnTT1 (NOE) and pNapA::BnbZIP67 (B67O), respectively (Figure 9a; Tables S23-S25). Some 15 BnLTP DEGs were up-regulated in BOE, 11 in NOE, and 16 in B67O, and meanwhile 25 BnLTP DEGs were down-regulated in BOE, 19 in NOE, and 16 in B67O. In addition, we found that two BnLTP DEGs both were up-regulated in BOE and NOE, and nine were all down-regulated in BOE and NOE, while five were up-regulated in NOE, and they were down-regulated in BOE (Figure 9b). These BnLTP genes might be regulated by BnTT1 or BnbZIP67 and play roles in seed development, secondary metabolism, oil accumulation and stress resistance during seed growth and maturation.

Validation of Selected Sclerotinia-Responsive Genes
To validate the results of RNA-seq expression analysis, two top Sclerotinia-responsive BnLTP genes, BnLTP129 and BnLTP161, were selected to perform the qRT-PCR analysis.

Validation of Selected Sclerotinia-Responsive Genes
To validate the results of RNA-seq expression analysis, two top Sclerotinia-responsive BnLTP genes, BnLTP129 and BnLTP161, were selected to perform the qRT-PCR analysis.

Discussion
The nsLTP proteins belong to the prolamin superfamily with conserved cysteine residues, low molecular mass and a high content of α-helices, which are only found in plants [34,55,56]. The nsLTPs have a hydrophobic cavity that can bind and stabilize various lipid molecules outside membranes [16,17,23,27,57]. An increasing number of studies have shown that plant nsLTP genes play an important role in the response to biotic and abiotic stresses and in plant growth and development [11,18,21,23,25,28,29,31]. To date, with the availability and feasibility of plant reference genomes, the nsLTP gene family has been identified at the genome-wide level in various plant species, including Arabidopsis, Gossypium, sesame, cotton, wheat, potato, tomato, tobacco, barley, maize, cabbage and Chinese cabbage [42][43][44][45][46][47][48][49][50][58][59][60][61]. In this study, 283, 127, 151 and 106 nsLTP genes were identified in B. napus, B. rapa, B. oleracea and A. thaliana genomes, respectively. It appears that the total number of nsLTP genes in allotetraploid B. napus was almost the sum of those in its two diploid parent species, B. oleracea and B. rapa, and three Brassica species contained a higher number of nsLTP genes than that of A. thaliana. This result showed that whole genome triplication of ancestor Brassiceae and allopolyploidization between its two progenitors might have contributed to the expansion of the BnLTP gene family in B. napus. In addition, gene duplication and synteny analysis also further revealed that BnLTP gene expansion was mainly derived from segmental duplication and hybridization of its two sub-genomes, which was also consistent with the previous research results of genome-wide analysis of other gene families in B. napus [62,63].
Numerous studies have showed that some plant nsLTPs were antimicrobial peptides and belonged to the pathogenesis-related protein (PR-14) family members, which possessed extensive antibacterial and antifungal activities [28,29,64]. It has been reported that nsLTP genes played an important role in plant defense to fungal and bacterial pathogens [23,28,29,65]. To date, diverse nsLTPs have been applied in generating plant disease resistance using biotechnological means. For example, overexpression and knockdown of StLTP10 in potato indicated that StLTP10 positively regulated plant resistance to Phytophthora infestans [66]. Cotton GhnsLTPsA10 positively regulated Verticillium wilt and Fusarium wilt resistance in transgenic Arabidopsis and VIGS cotton [11]. Overexpression of AtLTP4.4 in transgenic wheat significantly inhibited the growth of F. graminearum in Bobwhite and RB07 lines in the greenhouse and reduced the size of fungal lesions in in vitro leaf assays [67]. Transgenic Arabidopsis expressing wheat TdLTP4 gene enhanced fungal resistance against Alternaria solani and Botrytis cinerea [68]. Transgenic wheat lines overexpressing TaLTP5 exhibited significantly enhanced resistance to both common root rot (Cochliobolus sativus) and Fusarium head blight (Fusarium graminearum) compared to the untransformed control [69]. Overexpression of the pepper CALTPI gene in Arabidopsis enhanced the resistance against infection by Pseudomonas syringae pv. tomato and Botrytis cinerea [70]. Overexpression of OsLTP1 gene in B. napus enhanced its resistance to S. sclerotiorum [71]. Overexpression of an nsLTPs-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) enhanced resistance to S. sclerotiorum in B. napus, whereas its overexpression in Populus tomentosa also enhanced resistance to fungal pathogens [65,72]. In this study, in response to two fungal pathogens, we identified 72 S. sclerotiorum-responsive BnLTP genes and 96 L. maculans-responsive BnLTP genes. For white stem rot resistance, BnLTP033, BnLTP129, BnLTP161 and BnLTP264, had more critical roles. For blackleg disease resistance, BnLTP161 and BnLTP015 played more roles. Therefore, these BnLTPs could be candidates used to generate the wide broad plant disease resistance.
Many studies have showed that members of the plant nsLTP family played important roles in response to abiotic stress as well as plant hormone signals [68,73,74], which could help plants adapt to changes of environment conditions. For example, a lipid transfer protein variant with a mutant eight-cysteine motif causes photoperiod-and thermo-sensitive dwarfsm in rice [25]. The transgenic plants expressing the pepper CALTPI gene indicated high levels of tolerance to NaCl and drought stresses at various vegetative growth stages [70]. Overexpression of wheat TdLTP4 in Arabidopsis contributed to plant growth under various stress conditions including NaCl, ABA, JA and H 2 O 2 treatments [68]. Previous studies showed that OsLTPL159 was involved in cold tolerance at the early seedling stage in rice, which could be a candidate allele used to improve rice cold tolerance [30]. Overexpression of LTP3 in Arabidopsis conferred enhanced freezing tolerance, whereas lossof-function and overexpression of LTP3 in Arabidopsis both showed its positive regulatory roles in drought tolerance [75]. Drought-Induced LTP (OsDIL) was mainly responsive to abiotic stresses, including drought, cold, NaCl, and stress-related plant hormone ABA [73]. Transgenic rice plants overexpressing OsDIL were more tolerant to drought stress during vegetative development than wild type. The overexpression of NtLTP4 in Nicotiana tabacum increased the resistance to salt and drought stresses [38]. Here, of the 283 BnLTP genes, 12 were heat-responsive genes, 26 were drought-responsive genes, 61 were NaCl-responsive genes, 41 were cold-responsive genes, 32 were dehydration-responsive genes, 13 were IAAresponsive genes and 22 were ABA-responsive genes. It could be speculated that these BnLTP members played critical roles in response to various abiotic stressors, which also might be applied in molecular breeding of high abiotic stress resistance in B. napus.
Numerous studies have showed that plant nsLTPs played diverse roles in various biological processes, including seed development and maturation as well as seed germinations [21][22][23]50]. Overexpression of SiLTPI.23 in Arabidopsis significantly increased seed oil contents by 17-29% compared with the wild type control [50]. OsLTPL36 was reported to play an essential role in seed development and germination in rice [22]. In A. thaliana, the GPI-anchored lipid transfer proteins acted important roles in the development of seed coats and pollen [76]. In this study, we showed that three BnLTP genes, BnLTP089, BnLTP110 and BnLTP257, were DEGs between 26 d brown seeds and yellow mutants. In yellow mutants, their expression levels were all up-regulated, indicating potential roles of these three BnLTP genes in regulating seed coat color. In addition, based on RNA-seq data of 42 various organs/tissues, we found that several BnLTP genes, including BnLTP059 and BnLTP085, displayed seed-specific expression genes, revealing their possible roles in regulating seed development. Each of eight LMD-obtained tissues from the early globular seed possessed a unique set of BnLTP transcripts, which also might show their important roles in tissue-specific development processes of the globular seed. In the seed germination process of three selected accessions, numbers of expressed and responsive BnLTP genes both gradually increased. Meanwhile, overall, at four time points, high germination rate accession material had more expressed and responsive BnLTP genes. This result showed that the expression of these BnLTP genes might result in high germination rate.
In addition, there existed 30-40 BnLTP DEGs in BOE, NOE and B67O, each of which contained the up-regulated and down-regulated BnLTP genes. TT1 and bZIP67 are two critical TFs that regulate seed coat and seed embryo development and metabolism, respectively [32,33]. These BnLTP DEGs might play important roles in the development, metabolism or adaptability of the seed coat and embryo in rapeseed by responding to BnTT1 and BnbZIP67. Meanwhile, it found that the BnLTP family DEGs might have undergone obvious divergence in regulating physiological functions such as seed development, metabolism and adaptation.
Many studies reported that nsLTPs were defined as small basic proteins [16,17,21,23], but some of the nsLTPs identified in this study, were 400 aa in length, which were atypical nsLTP proteins. This study is not the first attempt to classify nsLTPs. Other researchers attempting a systematic analysis of nsLTPs also found these atypical proteins. For example, some of the nsLTPs in A. duranensis were 244 aa in length [51], and some in cabbage were 258 aa in length [46]. Therefore, nsLTPs need to be redefined after the functions of these atypical nsLTPs were validated using biochemistry and molecular biology experiments, and we also believe that the nsLTP community will eventually resolve this conflict in that time. Additionally, it has been shown that some nsLTPs can bind or transfer lipids [13][14][15], and thus this function of BnLTPs will be studied in our future research program.

Phylogenetic Relationship Analysis
The nsLTP protein sequences from B. napus, B. rapa, B. oleracea and A. thaliana were multi-aligned using the MAFFT7 program with default parameters [77], and then the neighbor-joining (NJ) phylogenetic tree was generated using MEGA7 software [78] with the 1000 bootstrap replicates and p-distance model. Sequence logo of 8CM motifs of BnLTP family proteins was analyzed using WebLogo (http://weblogo.threeplusone.com/; accessed on 16 June 2020).

Analysis of Gene Structure and Conserved Motifs
Gene exon-intron structure information of BnLTP genes was retrieved from the GFF3 file in the B. napus genome v5.0, and conserved motif files of BnLTP proteins were generated on MEME suite 5.1.0 (http://meme-suite.org/tools/meme; accessed on 18 June 2020) with default parameters. Finally, the combined image was visualized with TBtools [79].

Chromosome Location, Gene Duplication and Synteny Analysis
The chromosome location information of BnLTP genes was obtained from the GFF3 file in B. napus genome v5.0, and the image was visualized with the TBtools [79]. BnLTP gene clusters were identified if two or more BnLTP genes were clustered together on the chromosome, and meanwhile they were separated by no more than three genes. Gene duplication patterns were classified with MCScanX with default parameters. Duplicate gene pairs were determined if the identity of their coding regions was >85%, and they were clustered together in an evolutionary guide tree generated using Clustal Omega tool (https://www.ebi.ac.uk/Tools/msa/clustalo/; accessed on 9 June 2020), which were also annotated as duplicated gene groups. The syntenic relationships of duplicate gene pairs were visualized using Circos (http://circos.ca/software/; accessed on 29 July 2021).

Analysis of SSR Loci
SSR loci in BnLTP genes or their promoters were checked on MISA-web [80], with default parameters.

RNA Extraction and qRT-PCR Analysis
Total RNA was isolated from seedling leaves of a Sclerotinia-resistant rapeseed cultivar ZS10 at 20 h, 30 h and 40 h after S. sclerotiorum (1980) inoculation, respectively, using an RNAsimple Total RNA Kit (DP419, Tiangen, Beijing, China). First-strand total cDNA was synthesized with 1 µg of total RNA using the PrimeScript Reagent Kit with gDNA Eraser (Takara, Dalian, China). The qRT-PCR assay was performed using FastStart Universal SYBR Green Master (Roche, Mannheim, Germany) on BIO-RAD CFX96 Real-time PCR System (Bio-Rad, Irvine, CA, USA). Three replicates were carried out for each reaction. Two top Sclerotiniaresponsive BnLTP genes, BnLTP129 and BnLTP161, Table S26 were selected to perform the qRT-PCR experiment, and the B. napus ACT7 gene was used as internal control [81].

Chlorophyll Fluorescence Imaging
The plant phenotype imager (device no. 20A00005; a chlorophyll fluorescence imaging system FluorCam7.0; Photon Systems Instruments, Brno, Czech Republic) was used to analyze the fluorescence parameters (Fv/Fm, NPQ and ΦPSII) and chlorophyll fluorescence images of the rapeseed seedling leaves under 20 h, 30 h and 40 h post Sclerotinia infection [82].

Conclusions
In the present study, we performed comprehensive whole-genome mining and analysis of BnLTP family genes in rapeseed, including phylogenetic relationship, exon/intron structure, MEME motifs, synteny analysis, SSR loci, promoter cis-elements and expression