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Article

Genome-Wide Identification and Evolution of Receptor-Like Kinases (RLKs) and Receptor like Proteins (RLPs) in Brassica juncea

by
Hua Yang
1,2,
Philipp E. Bayer
1,
Soodeh Tirnaz
1,
David Edwards
1 and
Jacqueline Batley
1,*
1
School of Biological Sciences, University of Western Australia, Crawley, WA 6009, Australia
2
School of Agriculture and Food Sciences, University of Queensland, St Lucia, QLD 4067, Australia
*
Author to whom correspondence should be addressed.
Biology 2021, 10(1), 17; https://doi.org/10.3390/biology10010017
Submission received: 27 October 2020 / Revised: 21 December 2020 / Accepted: 21 December 2020 / Published: 30 December 2020
(This article belongs to the Section Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:

Simple Summary

Plants have evolved defence mechanisms to protect themselves against microbial pathogens. The identification of genes underlying quantitative trait loci is extremely challenging in complex polyploid genomes. In this research, we identify and characterise two types of resistance genes; RLKs (receptor like kinases) and RLPs (receptor like proteins) in Indian mustard (Brassica juncea), one of the major crops in India and an important member of the Brassicaceae family, which can be linked to QTL for disease resistance. The outcome provides a valuable resource for facilitating the identification of functional resistance genes which can be employed by breeders toward the production of resistant cultivars.

Abstract

Brassica juncea, an allotetraploid species, is an important germplasm resource for canola improvement, due to its many beneficial agronomic traits, such as heat and drought tolerance and blackleg resistance. Receptor-like kinase (RLK) and receptor-like protein (RLP) genes are two types of resistance gene analogues (RGA) that play important roles in plant innate immunity, stress response and various development processes. In this study, genome wide analysis of RLKs and RLPs is performed in B. juncea. In total, 493 RLKs (LysM-RLKs and LRR-RLKs) and 228 RLPs (LysM-RLPs and LRR-RLPs) are identified in the genome of B. juncea, using RGAugury. Only 13.54% RLKs and 11.79% RLPs are observed to be grouped within gene clusters. The majority of RLKs (90.17%) and RLPs (52.83%) are identified as duplicates, indicating that gene duplications significantly contribute to the expansion of RLK and RLP families. Comparative analysis between B. juncea and its progenitor species, B. rapa and B. nigra, indicate that 83.62% RLKs and 41.98% RLPs are conserved in B. juncea, and RLPs are likely to have a faster evolution than RLKs. This study provides a valuable resource for the identification and characterisation of candidate RLK and RLP genes.

1. Introduction

In plants, cell surface receptors play an important role in perceiving self-derived or non-self-derived extracellular signals, where the communication between the extracellular matrix and the cell interior happens [1,2]. Plant receptor-like kinases (RLKs) and receptor-like proteins (RLPs) constitute two major classes of cell-surface receptors. Generally, the plant cell surface receptors participate in innate immunity, stress responses and a wide variety of developmental processes [3,4,5,6,7,8,9]. A typical structure of RLKs is composed of a single-pass transmembrane domain and a cytoplasmic kinase domain [10,11]. RLPs are structurally similar to RLKs. The only difference is that the RLP carries a short cytoplasmic tail instead of the intracellular kinase domain present in RLKs [9,12]. Due to the kinase domain, the RLKs can trigger signalling on their own, whereas RLPs require the formation of a functional complex with an RLK to activate downstream signalling [13,14,15]. The first RLP was identified in Solanum lycopersicum; defined as Cf-9, a leucine-rich repeat RLP (LRR-RLP), which confers resistance to the effector Avr9 of the fungus Cladosporium fulvum [16]. Whereas, the first report of a RLK, ZmPK1, was isolated from Zea mays, and belongs to the S-domain class, functioning in specific self-pollen recognition [17].
RLKs and RLPs can be classified into sub-families on the basis of the type of extracellular domain they carry. The largest subfamily of RLK and RLP families carry a leucine-rich repeat (LRR) domain and are defined as LRR-RLK and LRR-RLP [3,4]. These two sub-families, LRR-RLKs and LRR-RLPs, are known to participate widely in plant defence and development. In plants, both LRR-RLKs and LRR-RLPs have been observed to contain significant rates of duplication, loss or retention [10,12,18,19,20,21,22].
Further sub-families of RLKs and RLPs carry the Lysin motif (LysnM) and are known as LysM-RLK and LysM-RLP. These sub-families of proteins play an important role in innate immunity and/or symbiosis. In Arabidopsis thaliana, the LysM-containing chitin elicitor RLK1 (CERK1) has been found to be essential for chitin perception and also functions in defence response against fungal pathogens [23,24,25,26]. In contrast, LysM-RLKs in legumes coordinate with rhizobial bacteria to control symbiotic processes, through perceiving secreted bacterial lipochitooligosaccharides (Nod factor, NF) signals [27,28,29,30,31].
Brassica juncea, commonly known as Indian mustard, is an allotetraploid plant species with AABB genome produced by natural interspecific hybridisation between the diploid species B. nigra (BB) and B. rapa (AA) [32,33]. It is widely planted, as it is known to contain many beneficial agronomic traits compared to B. napus, such as increased heat and drought tolerance, high blackleg and shatter resistance and early vigour [34,35,36,37,38,39,40]. The B. juncea reference genome, 922 Mb in length, is currently available [33]. This provides an opportunity to characterise the LRR-RLK and LRR-RLP genes in B. juncea and understand their genomic distribution.
Whilst a genome-wide study of the evolution and distribution of nucleotide binding site-leucine-rich repeat (NLR) resistance genes have been conducted in B. juncea [41], the majority of RLK (LysM-RLK and LRR-RLK) and RLP (LysM-RLP and LRR-RLP) genes remain uncharacterised in B. juncea. Here, we present the identification, characterisation and distribution of the RLKs and RLPs subfamilies in the B. juncea genome. Comparative analyses are also performed to identify the orthologous genes and define collinear relationships between B. juncea and its related diploid progenitors, B. rapa and B. nigra. The outcomes provide information that facilitate the further investigation of resistance gene evolution and distribution in B. juncea, which ultimately can assist breeders for resistance improvement.

2. Materials and Methods

2.1. Brassica Reference Genomes

The Brassica genomes used in this study, include B. juncea T84-66 v1.5 (genome: AABB, 779.5 Mbp) [33], B. nigra DH YZ12151 scaffold version (genome: BB) [33] and B. rapa Chiifu-401-42 v2.5 (genome: AA, 370.2 Mbp) [42]. Their genome sequence, annotation and protein sequences were obtained from the website http://brassicadb.org/brad/.

2.2. Genome Wide Identification of RLK and RLP Genes

Candidate proteins of RLK and RLP families were retrieved using the RGAugury pipeline [43]. Firstly, BLASTp analysis was performed to screen potential RGA candidates in Brassica species against the database RGAdb with an E-value cut-off of E−5. Secondly, specific domains in RLKs (LRR, LysM, TM and STTK domains) and RLPs (LRR, LysM and TM domains) were detected through InterProScan and Phobius. Finally, the genes were classified into RLKs and RLPs on the basis of their unique domain structures. Brassica RLKs were grouped into three-types: LRR-RLK, LysM-RLK and RLK-other-receptor; while RLPs contained two types, namely LRR-RLP and LysM-RLP. In this study, we focused on LysM and LRR types of RLKs and RLPs.

2.3. Genomic Distribution of RLK and RLP Genes

RLKs and RLPs were mapped to their corresponding chromosomes on the basis of their physical positions in the Brassica genomes. Gene clusters were determined using the physical positions of the genes which were situated on the same chromosome. In this study, a gene cluster was defined as a chromosome region harbouring three or more genes within 200 kb, according to previous literature [44]. Clusters formed by tandem duplications were defined as a tandem cluster. RLK and RLP clusters were detected, according to the kinase domain.

2.4. Gene Duplication Analysis of RLK and RLP Genes

Gene duplication analysis in this study was performed on the basis of previously reported criteria, with over 70% in both the coverage and identity of alignment during BLASTp comparison [45,46,47]. Gene duplicates located within a 5 Mb region on the same chromosome were considered as tandemly duplicated, while those situated beyond 5 Mb were defined as segmentally duplicated [48,49,50]. The duplication events observed within the B. juncea A or B sub-genomes were defined as intra-genomic duplications. In contrast, inter-genomic duplications referred to the duplications between the A and B sub-genomes.

2.5. Ortholog and Paralog Analysis

Paralogous copies were defined as the duplicated genes which occupied two different positions in the same genome [51,52]. Paralogous genes were confirmed by the BLASTp comparison of all the predicted RLKs and RLPs proteins against each other with E-value of E-20 [45]. However, orthologous genes, which were observed in two different species originating from a common ancestor, were confirmed when the E-value was smaller than E-45 during BLASTp comparison of all the predicted RLK or RLP proteins against each other, with over 70% of similarity and coverage [51,52].

2.6. Multiple Alignment and Phylogenetic Analysis

Multiple sequence alignment of RLK and RLP proteins were performed separately in Geneious v7.1.9 with the CLUSTAL W method (Biomatters, Auckland, New Zealand) [53]. The neighbour-joining method with 1000 bootstrap iterations was used to construct a phylogenetic tree with aligned proteins in Geneious v7.1.9 (Biomatters, Auckland, New Zealand) [53].

3. Results

3.1. Genome-Wide Identification of RLK and RLP Genes in B. juncea

A total of 493 candidate RLK genes, accounting for 0.613% of the predicted coding genes, were identified in the B. juncea genome, which is over twice as many as the RLP family of 228 genes (0.283%) (Table 1). Only nine LysM-RLKs and two LysM-RLPs were observed in B. juncea, suggesting that the LysM subfamily was much smaller than the LRR subfamily, which contained 484 LRR-RLKs (98.17% of RLKs) and 226 LRR-RLPs (99.12% of RLPs), respectively (Table 1).

3.2. Genomic Distribution of RLK and RLP Genes in B. juncea

Physical mapping was performed to detect the distribution of candidate RLK and RLP genes across the B. juncea genome (Figure 1, Figure 2, Figure 3 and Figure 4; Tables S1 and S2). In total, 92.90% of RLKs (458 genes) and 92.98% of RLPs (212 genes) were physically mapped, according to their corresponding positions in the genome. The remaining genes (35 RLKs and 16 RLPs) could not be assigned to any chromosome in B. juncea.
In B. juncea, the assigned RLKs were distributed almost evenly between the A and B genomes, with 236 (51.53%, five LysM-RLKs and 231 LRR-RLKs) and 222 (48.47%, three LysM-RLKs and 219 LRR-RLKs) (Table S1). The number of RLP genes (117) in the B genome was slightly higher than in the A genome, which contained 95 genes (Table S2). All of the assigned candidate genes were found to be distributed across all the 18 chromosomes in B. juncea (Figure 1, Figure 2, Figure 3 and Figure 4). LRR-RLKs and LRR-RLPs could be observed on each chromosome, while LysM-RLKs were only present on some chromosomes (A04–A06, A08, B01 and B04). However, only one LysM-RLP was situated on chromosome A06 and B03 (Figure 1, Figure 2, Figure 3 and Figure 4; Table S2).

3.3. RLK and RLP Gene Clustering in B. juncea

In this study, a gene cluster was defined as a chromosomal region harbouring three or more genes within 200 kb. Gene cluster analysis was performed for RLKs and RLPs. Accordingly, 13.54% (62 genes) of the assigned RLKs were located in gene clusters, while only 25 RLPs (11.79%) were observed in RLP clusters (Tables S1 and S2). Most of the assigned RLKs and RLPs represented a single-gene locus in B. juncea. In addition, un-clustered RLKs and RLPs were located across the genome of B. juncea (Figure 1, Figure 2, Figure 3 and Figure 4). Some of the un-clustered RLKs and RLPs were closely situated in the chromosomes, forming regions rich in RLKs and RLPs (Figure 1, Figure 2, Figure 3 and Figure 4).
In total, 16 RLK clusters were observed in the B. juncea genome, where 10 of them resided in the A genome, more than in the B genome with six gene clusters (Figure 1 and Figure 2; Table S1). The RLK clusters were almost evenly distributed in the chromosomes of B. juncea, except for on six chromosomes (A04, A07, A08, B01, B06 and B07) (Table S1). However, there were only seven RLP gene clusters, which were evenly distributed in seven chromosomes of the B. juncea genome, with three in the A genome, less than in the B genome which had four clusters (Figure 3 and Figure 4; Table S2). All of the RLP clusters were homogeneous, since LysM-RLP genes were not involved in generating gene clusters (Figure 3 and Figure 4; Table S2). By contrast, one LysM-RLK gene, located on chromosome A06, participated in the formation of RLK clusters, resulting in the presence of a heterogeneous cluster (Figure 1 and Figure 2; Table S1).
Most of the large RLK clusters were situated in the B genome chromosomes of B. juncea, where the largest clusters containing six RLKs were found on chromosomes B04 and B05, followed by clusters of five genes on B02 and B08 (Figure 2; Table S1). Additionally, the average gene number of a RLK cluster in the A genome was three genes, smaller than in the B genome with five genes (Table S1). Contrastingly, the highest gene number in a RLP cluster, having five genes, was observed on chromosome B04 (Figure 3 and Figure 4; Table S2). Most of the RLP clusters, which were located on chromosomes A03, A07, B03 and B06, contained three genes (Figure 3 and Figure 4; Table S2).
The size of a cluster was determined by the sequence length between the two RLKs or RLPs situated at both ends of the gene cluster. The size of RLK clusters, with an average of 118.82 kb, ranged from 29.45 kb on A03 to 293.95 kb on chromosome B03 (Figure 1 and Figure 2). Similarly, RLP cluster size also ranged from 33.38 kb to 186.32 kbp on chromosomes A03 and B06, respectively, compared with the average size of 83.80 kb (Figure 3 and Figure 4). The size of RLK and RLP clusters was not related to the gene number in the cluster (Figure 1, Figure 2, Figure 3 and Figure 4).

3.4. Analysis of Duplications and Paralogues of RLK and RLP Genes in B. juncea

In this study, duplicated genes were detected using BLASTp by comparing all predicted proteins against each other. The RLK and RLP gene families were analysed separately. Duplicated genes, which occupy two different positions in the same genome, were defined as paralogous copies [51,52]. As B. juncea is an allotetraploid species, [32,33], two types of genomic duplications were observed; intra- and inter-genomic duplications (Figure 5; Table 2, Tables S1 and S2). The former is where duplication occurs within the A or B sub-genome of B. juncea. By contrast, the duplication events that occur between the A and B sub-genomes are considered as inter-genomic duplications (Figure 5).
A total of 413 RLKs (90.17% of the mapped RLKs), consisting of seven LysM-RLKs and 406 LRR-RLKs, were found to be involved in duplication events. Accordingly, 413 paralogous copies were formed in the B. juncea genome (Table S1). A total of 52.83% of the assigned RLPs were defined as duplications, with 112 genes including two (1.79%) LysM-RLPs and 110 (98.21%) LRR-RLPs. There were 112 RLP paralogous copies in the B. juncea genome (Table S2). In B. juncea, most of the assigned RLK genes (90.17%) had more than one copy, while approximately half of the mapped RLPs (47.17%) were a single copy.
The outer circle illustrates the position of RLK/RLP clusters. The LysM-RLK/RLP (black) and LRR-RLK/RLP (red) type genes were mapped on 18 chromosomes (A01–A10, B01–B08) in the inner circle. The coloured lines in the centre represent the duplication events between chromosomes: inter-genomic duplications of LRR-RLKs/RLPs (yellow) and LysM-RLKs/RLPs (black); intra-genomic duplication events of LRR-RLKs/RLPs (blue) and LysM-RLKs/RLPs (red).
Segmental duplications made minor contributions to RLK and RLP duplications. However, tandem duplications play a major role in RLP duplications compared to RLK duplications. In the duplicated RLK genes, 14.04% (58 genes) were derived from tandem duplications, while only 3.87% (16 genes) were from segmental duplications (Table S1). For the RLPs, 46.43% (52 genes) of the duplicated genes resulted from tandem duplication, while only 3.57% (4 genes) were from segmental duplications (Table S2).
The duplicated RLKs were mostly evenly distributed in the A and B sub-genomes in B. juncea, with 209 and 204 genes, respectively, consistent with the genomic distribution of duplicated RLPs, with 52 and 60 genes, respectively (Tables S1 and S2). However, these duplicated RLKs and RLPs were unevenly distributed over the 18 chromosomes of B. juncea (Figure 5; Tables S1 and S2). Additionally, the highest number of duplicated RLKs was found on chromosome B02, containing 34 LRR-RLKs, followed by B05 with 32 LRR-RLKs (Table S1). All of the LRR-RLKs residing on chromosomes A05 (21) and B07 (16) were involved in duplication events (Table S1). Chromosomes B03 and B04 had the highest number of duplicated RLPs, each containing 11 genes, while the least number (one) was found on chromosomes A02, A05 and A10 (Table S2).
In total, 82% (51 genes) of the clustered RLKs were found to be duplicated, as well as 88% (22 out of 25 genes) of clustered RLPs (Tables S1 and S2). Six out of 16 RLK clusters were formed by tandem duplication, while almost all of the RLP clusters (six out of seven) were defined as tandem clusters (Figure 1, Figure 2 and Figure 3; Tables S1 and S2). For instance, the gene cluster on chromosome A02 contained three LRR-RLK genes, of which two were duplicates. Another example was the cluster on chromosome B08, constructed with three tandem duplicates.
A total of 471 and 110 duplication events were detected in the RLK and RLP families, respectively (Table 2). For both the RLK and RLP families, almost all of the duplication events occurred in the LRR sub-family, with 466 in LRR-RLKs and 109 in LRR-RLPs, in sharp contrast with five in LysM-RLKs and one in LysM-RLPs (Table 2).
Two types of duplication events, inter and intra duplications, were observed in both B. juncea RLK and RLP families (Table 2). For B. juncea RLKs, the number of inter-genomic duplications was much higher than intra-genomic, with 308 and 163, respectively. For the RLP family, the number of these two types of duplications was smaller, with 52 intra-genomic duplications and 58 inter-genomic duplications (Table 2). Furthermore, the intra-genomic duplications of RLKs were evenly distributed in B. juncea A and B genomes, with 83 and 80, respectively, while the RLP duplication events in the A genome (20) were less than in the B genome of 32 (Table 2). Almost all of the LysM-RLK duplications (four out of five) were defined as inter-genomic duplications, and in LysM-RLP, there was only one duplication event found between the A and B sub-genomes (Table 2). These two kinds of duplications for RLKs could be observed in all 18 chromosomes of B. juncea (Figure 5; Table S1). For RLPs, the B sub-genome contained both inter- and intra- genomic duplication events, while in the A sub-genome, the chromosomes A02 and A06 did not have intra-genomic duplication and A05 and A10 did not have the inter-genomic type (Table S2). The duplicated events could be observed across the B. juncea genome (Figure 5).

3.5. Phylogenetic Analysis of RLK and RLP Genes in B. juncea

The evolutionary relationship between predicted proteins was studied by the construction of phylogenetic trees, using the neighbour-joining (NJ) method with 1000 bootstrap replicates (Figures S1 and S2). Here, the RLKs and RLPs were analysed separately.
A total of 493 RLKs were divided into seven major groups, which were further classified into subgroups and clades (Figure S1). However, the size of the groups significantly varied, ranging from four in Group VI to 145 in Group I, with an average of 70 genes in each group. Additionally, all of the LysM-RLKs displayed a close relationship with some LRR-RLKs in Group I. In the phylogenetic tree, the predicted paralogous RLKs were grouped together with high bootstrap value. The majority of subgroups or clades consisted of paralogous genes located on the same chromosome or homeologous chromosomes, which were derived from duplications events. For example, LysM-RLK genes (BjuA002911 on chromosome A08, BjuA021332 on A06, and BjuB004835 on B04), defined as paralogous genes, were grouped together in Group I.
In total, 228 RLPs were classified into three primary groups through phylogenetic analysis (Figure S2). Similar to LysM-RLKs, LysM-RLPs were also closely related to some LRR-RLPs in Group III (Figure S2). Differently from RLKs, many branches were formed with a single RLP gene, suggesting the distant evolutionary relationship and the functional diversity among RLP genes (Figure S2).

3.6. Comparison and Conservation Analysis of RLK and RLP Genes between B. juncea and Its Diploid Progenitor Species B. rapa and B. nigra

B. juncea, an allotetraploid species (AABB), was generated through interspecific hybridisation between two ancestral diploid genomes of B. rapa (AA) and B. nigra (BB) [32,33]. Genomic comparison of RLKs and RLPs is important to understand the genetic diversity and evolution between these Brassica species.
In total, 493 RLKs and 228 RLPs were identified in B. juncea, of which 236 RLKs and 95 RLPs were located in the A genome, and the remaining genes resided in the B genome (Tables S1 and S2). In B. rapa, genome-wide identification indicated that 0.651% (300 genes) of the total predicted genes were detected as RLK genes, consisting of three LysM-RLKs and 297 LRR-RLKs, which is higher than in the B. juncea A genome (Table 1 and Table 3). Conversely, 65 RLPs (0.141%) in B. rapa, which included two LysM-RLPs and 63 LRR-RLPs, were less than those found in the B. juncea A genome with 95 genes (Table 1 and Table 3). A total of 317 RLK genes (0.636% of the total predicted genes) were identified from the B. nigra scaffold sequence, with five LysM-RLKs and 312 LRR-RLKs more than in the B genome of B. juncea. Similarly, the number of RLPs (0.353%) in B. nigra was also higher than in B. juncea, with 176 and 117 genes, respectively (Table 1, Table 4 and Table 5).
In these three Brassica species, the proportions of RLK genes were similar (around 0.613% to 0.651%), while those of RLPs varied from 0.141% to 0.353% (Table 2). Additionally, LysM and LRR subgroups could be observed in each RLK and RLP family, while the percentage of the LysM subfamily was found to be extremely low, below 4%, in both RLK and RLP families (Table 3, Table 4 and Table 5).
The chromosomal distribution and clustering of RLK and RLP genes were not analysed in B. nigra, as a good quality assembled genome was not available. Genes of the RLK family in the A genome of B. juncea shared a similar distribution pattern with B. rapa in gene number and distribution (Figure 6; Table 3 and Table 4). For instance, chromosome A06 in B. juncea and B. rapa contained the highest number of RLKs, and the least number was observed in A04 and A08. Additionally, the chromosomal distribution pattern of the LRR-RLK subfamily was consistent with the RLK family in the A genomes of B. juncea and B. rapa (Figure 6; Table 3 and Table 4). However, the distribution of LysM-RLK genes was slightly different between these two Brassica species, as the presence of LysM-RLK on chromosome A05 of B. juncea was not observed in B. rapa (Figure 6; Table 3 and Table 4).
The chromosomal distribution of RLPs differed between the A genome of B. juncea and B. rapa (Figure 6; Table 3 and Table 4). For instance, the highest number (17 genes) of RLPs were found on chromosomes A03 and A09 in the A genome of B. juncea, while it was observed on A01 and A06 of B. rapa, with 10 genes. Furthermore, a different distribution pattern could be observed in both the LRR-RLP or LysM-RLP subfamilies between the A genome of these two Brassica species (Figure 6; Table 3 and Table 4). For example, the LysM-RLP gene, lost in B. juncea A08 was present in B. rapa.
In B. rapa, 20.34% (60 genes) of the mapped RLKs were found to be organised in 13 gene clusters, which was higher than 10 clusters grouped by 33 genes (13.98%) in the B. juncea A genome (Table 3 and Table S1). Furthermore, the cluster distribution of RLKs and RLPs differed between B. juncea and B. rapa (Figure 7 and Figure 8; Table 3, Tables S1 and S2). For instance, the RLP clusters in the B. juncea A genome were evenly located on chromosomes A03, A07 and A08, while they were equally distributed on A05 and A06 of B. rapa.
The identification of orthologous genes was performed between B. juncea and its diploid progenitors by comparing the protein sequences in each RLK and RLP family, respectively. In B. juncea, a total of 383 RLKs (83.62%) and 89 RLPs (41.98%) were considered as orthologous genes, of which 194 RLKs and 24 RLPs were mapped in the A genome, and the remaining genes were distributed in the B genome (Table 4 and Table 6). The orthology analysis also indicated that there were 214 RLKs (72.54%) and 27 RLPs (41.54%) conserved in B. rapa and 227 RLKs and 72 RLPs maintained in B. nigra, with 71.61% and 40.91%, respectively (Table 3, Table 4, Table 5 and Table 6). In these Brassica species, the proportion of conserved RLK genes was higher than RLP genes.
Genomic distribution of orthologous genes indicated that the proportion of conserved RLKs in each sub-genome of B. juncea (82.20% and 85.14%) was higher than B. rapa (72.54%) and B. nigra (71.61%), respectively (Table 3, Table 4, Table 5 and Table 6). Similarly, the percentage of conserved RLPs was also higher in the B genome of B. juncea (55.56%) than its progenitor B. nigra (40.91%), while obviously lower in the A genome of B. juncea than B. rapa, with 25.26% and 41.54%, respectively (Table 3, Table 4, Table 5 and Table 6).
In B. juncea, 86 RLKs and 38 RLPs were found to be lost in the A genome, and 90 RLKs and 104 RLPs were absent in the B genome, compared to its diploid progenitors (Table 6). Additionally, some RLKs and RLPs were only observed in B. juncea, including 42 RLKs and 71 RLPs in the A genome and 33 RLKs and 52 RLPs in the B genome (Table 6).
The synteny relationship of RLK and RLP genes in the A genome was assessed between B. juncea and its diploid progenitor B. rapa on the basis of an orthologous analysis and their physical positions. This analysis, which was separately performed in the RLK and RLP families, indicated that the distribution of RLKs and RLPs displayed obvious synteny and a collinear relationship between the A genomes of B. juncea and B. rapa (Figure 7 and Figure 8). In the B. juncea A genome, a total of 192 RLKs (81.36%) were found to be in synteny and collinear with B. rapa (Table 4). However, only 28.42% (27 genes) of the total RLP genes in the B. juncea A genome were observed to be in synteny and collinear with B. rapa (Table 4).

4. Discussion

4.1. Genome Wide Identification of RLK and RLP Genes in B. juncea

The number of LRR-RLKs in B. juncea is notably higher than the number in B. rapa with 303 genes [52], A. thaliana containing 213 genes [54], O. sativa having 309 [55] and P. trichocarpa having 379 [56]. This is probably due to the large allotetraploid genome of B. juncea (AABB) [33]. Similarly, a higher number of LRR-RLKs has also been identified in polyploid T. aestivum and G. max, with 531 and 467 genes, respectively [48,57,58,59], and allotetraploid B. napus cv. Darmor V4 (596 genes) [60]. Accordingly, the LRR-RLP number in B. juncea is also higher than in the diploids B. rapa and B. nigra analysed in this study and the plants previously reported, including A. thaliana (57) [12], O. sativa (90) [21] and P. trichocarpa (82) [20].
Here, the content of LysM of RLKs and RLPs is extremely low in the studied species. Similarly, the number of LysM-RLKs and LysM-RLPs is also very low in previous reports across species from the Brassicaceae family [60]. To date, the highest number (17) of LysM-RLKs have been separately observed in L. japonicas and M. truncatula [61,62], followed by G. max and P. trichocarpa with 12 and 11 genes, respectively [63], while less than 10 genes have been observed in A. thaliana (5 genes) and O. sativa (6 genes) [62,63]. For LysM-RLPs, there are three genes found in A. thaliana, [64,65], four in G. max, two in M. truncatula, seven in P. trichocarpa and six in O. sativa [65].
The distribution of RLKs and RLPs on the majority of chromosomes was uneven. The distribution pattern of LRR-RLK in B. rapa, detected in this study, is consistent with the previous report [52]. The uneven distribution of LRR-RLKs is not novel in plants’ genomes [18], as has been demonstrated in other plant species recently, including S. lycopersicum [46], T. aestivum [48], two Glycine species (G. max and G. latifolia) [57,66] and two Citrus species (C. clementina and C. sinensis) [67]. While the genome-wide study of LRR-RLPs is limited in several plant species, only O. sativa and Arabidopsis had their distribution analysed, where they exhibit a similar pattern [21] compared with our study. Additionally, the unequal distribution of genes across sub-genomes was also observed in other Brassica gene families, such as the NLR family in B. juncea [41], B. napus [45] and B. rapa [68] and the chitinase family in B. rapa [69]. Moreover, in this study, many RLKs and RLPs were grouped within the gene clusters. The formation of resistance-gene clusters is thought to facilitate the evolution of resistance genes through sequence exchanges via unequal crossing over, recombinatorial mispairing, or generating high haplotypic diversity [70,71].

4.2. RLK and RLP Gene Duplication in B. juncea

Gene duplication, as a result of whole-genome duplication, segmental duplication, tandem duplication and transposition events, is believed to contribute significantly to evolutionary innovation and gene family expansion, which allow the plants to be “buffered” against selective pressure in natural environments [72,73].
In this study, approximately 90% of the assigned RLKs have been found to be duplicated, suggesting that gene duplications contribute significantly to enlarge the RLK family in B. juncea. Although the duplicated proportion of RLPs is lower than RLKs, the 53% duplication rate indicates that gene duplication also plays major roles in the expansion of B, juncea RLPs. Only around 4% of duplications in both the RLK and RLP families is considered as segmental duplications, suggesting that segmental duplications contribute minimally to the expansion of RLKs and RLPs. However, tandem duplications are likely to be mainly responsible for RLP duplications, due to around 50% duplications being defined as tandem duplications, yet they play minor roles in RLK duplications. Tandem duplication has been reported to be mainly responsible for the expansion of the LRR-RLK subfamily in O. sativa [55,74] and five Rosaceae species [75], as well as two additional O. sativa gene families, RLP and NLR [21,76]. However, this kind of duplication plays a minor role to enlarge the LRR-RLK sub-family in A. thaliana, G. max and S. lycopersicum, with around 15%, 20.3% and 12% of LRR-RLKs being tandemly duplicated, respectively [46,57,74]. In P. trichocarpa and G. max, the explosion of LRR-RLKs is significantly caused by segmental duplications, with approximately 82% and 73.3%, respectively [56,57], compared with about 11% in O. sativa and around 26% in A. thaliana [74]. Additionally, these two major mechanisms proved to play primary roles in the expansion of LRR-RLK subfamily in B. rapa and RLK/Pelle family in A. thaliana [22,52].
In the Brassica species, all of the genomes have undergone a lineage-specific whole-genome triplication. The number of genes increased after many rounds of duplication [77,78,79,80]. Thus, we speculate that the expansion of RLK and RLP genes is probably mainly caused by whole-genome duplication. After speciation, B. juncea underwent substantial genome reshuffling and chromosomal rearrangements [33,81,82,83], which probably resulted in the random distribution of duplicated RLKs and RLPs across the B. juncea genome.
Here, 37.50% RLK clusters and 85.71% RLP clusters in B. juncea were generated by tandemly duplicated genes, indicating that tandem duplication events should be responsible for the origin of the genes in each cluster, which also have been proved in some species, including monocots A. trichopoda and O. sativa [55,84], eudicots P. trichocarpa and G. max [56,57]. Some inter-genomic duplications are likely to be from homoeologous regions and the duplication is due to polyploidization.

4.3. Phylogenetic Analysis

In this study, the phylogenetic trees were generated using full-length protein sequences of RLKs and RLPs. Full protein sequences of LRR-RLKs, which can provide excellent evolutionary inference, have been used for phylogenetic analysis with the neighbour-joining (NJ) method in many plants [48,52]. In both the RLK and RLP families, the predicted paralogous sequences, which are derived by gene duplications, are closely grouped together on the same or a close clade. Similarly, this has been observed in A. thaliana, B. rapa, O. sativa, P. trichocarpa and T. aestivum, due to paralogous sequences generated by duplication events and having similar domain architecture [21,48,52]. This number of paralogous sequences indicates that gene duplication plays an important role in the expansion of this family. The divergence of the sequences may be consistent with divergence in function of the genes in these families, as they are known to play roles in a large number of processes and not just disease resistance. Linking the genes involved in disease to specific clades may aid in the identification of candidate genes.
In phylogenetic analysis, protein sequences of the RLP family have formed many distinct clades constructed by branches with a single gene, compared to RLKs. This indicates that RLPs have undergone faster evolution and maintained relatively more diversity in sequence than RLKs, due to greater selective pressure on the RLPs. This phenomenon is also shown in the NLR family, of which the CNL family is more highly diverged than the TNL family in the Cucurbitaceae, Fabaceae, Brassicaceae, Poaceae and Solanaceae species [45,76,85,86].

4.4. Comparative Analysis of RLK and RLP Genes in B. juncea to Its Related Diploid Progenitor Species B. rapa and B. nigra

The comparison between B. juncea and its progenitors (B. rapa and B. nigra) reveals RLKs and RLPs are reduced in B. juncea, probably due to gene loss during the polyploidization events. This was also observed in the B. juncea NLR family [41]. The gene reduction of NLR family has also been reported in B. napus, compared with its progenitors B. rapa and B. oleracea [45]. Additionally, LRR-RLK gene reduction has been observed in other species. For example, Triticum aestivum (wheat), an allohexaploid species with AABBDD genome, was found to contain 531 LRR-RLKs, distributed in the A, B and D sub-genomes, with 166, 195 and 170 genes, respectively, while the gene number of A and D sub-genomes is lower than its progenitors Triticum urartu (AA, 217 genes) and Aegilops tauschii (DD, 248 genes) [48,58,87,88,89].
The proportions of LRR-RLK genes in B. juncea, B. rapa and B. nigra are consistent with estimates of LRR-RLK for other angiosperm species, ranging from 0.67% to 1.39% [90]. Therefore, the gene number of LRR-RLKs seems not to be correlated with genome size, which can also be observed in LysM-RLKs and RLPs in this study. Similarly, the gene number of NLRs seems to be nonlinearly related with the total predicted gene number, and is unproportional to genome size in B. napus, B. rapa and B. oleracea [45,91]. It has been reported that a total of 303 LRR-RLK genes were identified in B. rapa [52], 10 genes more than that found in the present study. Thus, the difference of gene number is likely caused by using different detection criteria and different reference versions of B. rapa genome, here we used B. rapa v2.5 updated by Cai, Wang, Liu, Wu, Liang, Cui, Cheng and Wang [42].
The RLPs and RLKs in the A genome of B. juncea are consistent with B. rapa in their distribution and synteny. In particular, the gene families are highly conserved in size and orientation, similar to the B. napus NLR family [45]. The gene conservation and syntenic relationship has been found previously in many plants, such as G. max, A. thaliana and B. nigra, Solanaceae species, T. urartu and T. aestivum [92,93,94,95,96,97].
Furthermore, significant genomic co-linearity has been reported between B. napus and its diploid progenitors B. rapa and B. oleracea [98,99]. Therefore, we speculate that RLKs and RLPs in the B. juncea B genome are also syntenic to B. nigra, although a high quality genome assembly of B. nigra is not yet available for comparison. However, the absence of synteny in some gene clusters and the presence of non-orthologous genes, which have been observed in the A genome of B. juncea in contrast with B. rapa, suggest occurrences of gene deletion, translocation and divergence in the B. juncea A genome after speciation. This was also reported in the NLR gene family between B. napus and its progenitors [45].
Orthology analysis indicates that over 82% RLKs are found to be conserved and maintained in B. juncea, compared to B. rapa and B. nigra. For RLP genes, the conserved proportion is consistent between the B genome of B. juncea and B. nigra, whereas the RLP conserved proportion is much lower in the B. juncea A genome than its progenitor B. rapa. This indicates that RLPs in the A genome are more diverse than the in the B genome. The presence of orthologous genes suggests these genes have been retained after genome polyploidization. In plants, disease resistance genes, such as NLR, RLK and RLP families are highly duplicated and have undergone diversifying selection compared to other developmental genes [21,100,101]. Thus, these orthologous copies, which are found to be located in syntenic region between B. juncea and its progenitors, probably maintain their original function in the growth and development of B. juncea. Non-orthologous genes in B. juncea are likely to be specific or gained novel disease resistance.
The gene number of RLKs and RLPs varies within species due to gene losses and/or expansion through duplication and/or divergence [48,67,75,84,90,101]. Moreover, gene loss and gain was reported in B. juncea previously, compared to its related diploids [33]. In this study, the differences in gene number of conserved RLKs and RLPs between B. juncea and its diploid progenitors indicates the gene loss and gain in B. juncea. Reduction in RLKs and RLPs, which is primarily caused by gene loss during polyploidizations, was observed in B. juncea, compared to B. rapa and B. nigra. Simultaneously, gene duplication and recombination of RLKs and RLPs does not generate novel disease resistance genes specific to B. juncea.
In polyploids, the duplicated disease resistance genes were found to be preferentially lost after genome duplication, and most of the gene losses were probably driven by a deletion mechanism [102]. In B. juncea, the gene loss of RLKs and RLPs are likely caused by species-specific gene deletion after natural hybridisation of B. juncea from B. rapa and B. nigra. A similar kind of deletion was also reported in T. aestivum gene families: LRR-RLK, lectin receptor kinases and Glyceraldehyde-3-phosphate dehydrogenase [48,49,103], and the NLR family in B. napus [45].
In summary, a notable deletion of RLKs and RLPs was not observed in B. juncea, compared to its diploid progenitors, B. rapa and B. nigra. Evolutionary selection may play a minor role in maintaining the properties of B. juncea RLKs, compared with B. rapa and B. nigra, while being responsible for the diversity of B. juncea RLP genes.

5. Conclusions

In this study, the sub-families of B. juncea LRR-RLK and LRR-RLP are found to be larger than those in most reported plants. Furthermore, most of the RLK and RLP genes are not grouped within the clusters (singletons). Moreover, the occurrence of numerous paralogous genes of RLKs and RLPs indicates that gene duplications significantly contribute to the expansion of RLK and RLP families during the evolution process. Additionally, tandem duplications play major roles in RLP duplications, while segmental duplications make limited contributions to RLK and RLP duplications.
The comparative genome analysis indicates that gene loss and gain of RLKs and RLPs are observed in B. juncea, compared to its diploid progenitors, B. rapa and B. nigra. Furthermore, RLK and RLP genes show syntenic relationships with B. rapa in the A genome. The presence of orthologous genes suggests the remaining of conserved RLKs and RLPs after B. juncea speciation. However, RLPs are likely to have a faster evolution than RLKs.
The identification and characterisation of these genes will provide a useful resource for Brassica researchers undertaking the identification of resistance genes for Brassica crop improvement or for evolutionary studies.

Supplementary Materials

The following are available online at https://www.mdpi.com/2079-7737/10/1/17/s1, Figure S1: Phylogenetic analysis of RLK proteins of B. juncea, Figure S2: Phylogenetic analysis of RLP proteins of B. juncea, Table S1: The distribution and organisation of candidate RLK genes in B. juncea, Table S2: The distribution and organisation of candidate RLP genes in B. juncea.

Author Contributions

Conceptualization, H.Y., D.E. and J.B.; methodology, H.Y. and P.E.B.; validation, H.Y. formal analysis, H.Y., S.T. and P.EB.; investigation, H.Y. and P.E.B.; writing—original draft preparation, H.Y.; writing—review and editing, D.E., J.B. and S.T.; supervision, J.B. and D.E.; funding acquisition, J.B. and D.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by ARC, grant number FT130100604, DP1601004497, LP140100537 and LP160100030.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in supplementary Table S1.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Physical location of receptor-like kinase (RLK) genes (LysM-RLK and LRR-RLK) on the chromosomes of the A genome of B. juncea. Solid circles above the chromosome (grey bars) represent RLK genes: LysM-RLK (red) and LRR-RLK (blue). Rectangles represent gene clusters: homogeneous cluster (red) and heterogeneous cluster (green). Numbers below the rectangles indicate the size of gene cluster in kb. Unfilled black ellipses indicate regions rich in RLK genes. Black brackets indicate tandemly-duplicated genes within a gene cluster. Chromosome lengths are displayed in Mbp (megabase pairs) on the scale at top.
Figure 1. Physical location of receptor-like kinase (RLK) genes (LysM-RLK and LRR-RLK) on the chromosomes of the A genome of B. juncea. Solid circles above the chromosome (grey bars) represent RLK genes: LysM-RLK (red) and LRR-RLK (blue). Rectangles represent gene clusters: homogeneous cluster (red) and heterogeneous cluster (green). Numbers below the rectangles indicate the size of gene cluster in kb. Unfilled black ellipses indicate regions rich in RLK genes. Black brackets indicate tandemly-duplicated genes within a gene cluster. Chromosome lengths are displayed in Mbp (megabase pairs) on the scale at top.
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Figure 2. Physical location of RLK genes (LysM-RLK and LRR-RLK) on the chromosomes of the B genome of B. juncea. Solid circles above the chromosome (grey bars) represent RLK genes: LysM-RLK (red) and LRR-RLK (blue). Red rectangles represent gene clusters. Numbers below the rectangles indicate the size of gene cluster in kb. Unfilled black ellipses indicate regions rich in RLK genes. Black brackets indicate tandemly-duplicated genes within a gene cluster. Chromosome lengths are displayed in Mbp (megabase pairs) on the scale at top.
Figure 2. Physical location of RLK genes (LysM-RLK and LRR-RLK) on the chromosomes of the B genome of B. juncea. Solid circles above the chromosome (grey bars) represent RLK genes: LysM-RLK (red) and LRR-RLK (blue). Red rectangles represent gene clusters. Numbers below the rectangles indicate the size of gene cluster in kb. Unfilled black ellipses indicate regions rich in RLK genes. Black brackets indicate tandemly-duplicated genes within a gene cluster. Chromosome lengths are displayed in Mbp (megabase pairs) on the scale at top.
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Figure 3. Physical location of receptor-like protein (RLP) genes (LysM-RLP and LRR-RLP) on the chromosomes of the A genome of B. juncea. Solid circles above the chromosome (grey bars) represent RLP genes: LysM-RLP (red) and LRR-RLP (blue). Red rectangles represent the gene cluster. Numbers below the rectangles indicate the size of gene cluster in kb. Black brackets indicate tandemly-duplicated genes within a gene cluster. Chromosome lengths are displayed in Mbp on the scale at top.
Figure 3. Physical location of receptor-like protein (RLP) genes (LysM-RLP and LRR-RLP) on the chromosomes of the A genome of B. juncea. Solid circles above the chromosome (grey bars) represent RLP genes: LysM-RLP (red) and LRR-RLP (blue). Red rectangles represent the gene cluster. Numbers below the rectangles indicate the size of gene cluster in kb. Black brackets indicate tandemly-duplicated genes within a gene cluster. Chromosome lengths are displayed in Mbp on the scale at top.
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Figure 4. Physical location of RLP genes (LysM-RLP and LRR-RLP) on the chromosomes of the B genome of B. juncea. Solid circles above the chromosome (grey bars) represent RLP genes: LysM-RLP (red) and LRR-RLP (blue). Red rectangles represent the gene cluster. Numbers below the rectangles indicate the size of gene cluster in kb. Unfilled black ellipses indicate regions rich in RLP genes. Black brackets indicate tandemly-duplicated genes within a gene cluster. Chromosome lengths are displayed in Mbp (megabase pairs) on the scale at top. In B. juncea, the mapped RLKs for each chromosome ranged from 2.62% (12 genes on chromosome A08) to 7.86% (36 genes on B02) (Table S1). However, 10.85% (23 genes) of the assigned RLPs were found to be located on chromosome B04, followed by 19 genes (8.96%) on B03, and the least number were observed on A10 with three genes (1.42%) (Table S2). In addition, the gene number of both RLKs and RLPs was found to be higher on four chromosomes (B03 to B05, and B08), compared with the other chromosomes (Tables S1 and S2).
Figure 4. Physical location of RLP genes (LysM-RLP and LRR-RLP) on the chromosomes of the B genome of B. juncea. Solid circles above the chromosome (grey bars) represent RLP genes: LysM-RLP (red) and LRR-RLP (blue). Red rectangles represent the gene cluster. Numbers below the rectangles indicate the size of gene cluster in kb. Unfilled black ellipses indicate regions rich in RLP genes. Black brackets indicate tandemly-duplicated genes within a gene cluster. Chromosome lengths are displayed in Mbp (megabase pairs) on the scale at top. In B. juncea, the mapped RLKs for each chromosome ranged from 2.62% (12 genes on chromosome A08) to 7.86% (36 genes on B02) (Table S1). However, 10.85% (23 genes) of the assigned RLPs were found to be located on chromosome B04, followed by 19 genes (8.96%) on B03, and the least number were observed on A10 with three genes (1.42%) (Table S2). In addition, the gene number of both RLKs and RLPs was found to be higher on four chromosomes (B03 to B05, and B08), compared with the other chromosomes (Tables S1 and S2).
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Figure 5. Chromosomal distribution, organisation and duplication of 458 candidate RLK genes (A) and 212 candidate RLP genes (B) in the B. juncea genome.
Figure 5. Chromosomal distribution, organisation and duplication of 458 candidate RLK genes (A) and 212 candidate RLP genes (B) in the B. juncea genome.
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Figure 6. Distribution of candidate RLK (A) and RLP (B) genes in the A genome of B. juncea and B. rapa.
Figure 6. Distribution of candidate RLK (A) and RLP (B) genes in the A genome of B. juncea and B. rapa.
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Figure 7. Comparative organisation of RLK genes between Aj genome of B. juncea and Ar genome of B. rapa (chromosomes A01 to A10). Solid circles above the chromosome (grey bars) represented RLK genes: LysM-RLK (red) and LRR-RLK (blue). Rectangles represented gene clusters: homogeneous cluster (black) and heterogeneous cluster (red). Pink straight lines between genes displayed the genes with syntenic relationship between B. juncea and B. rapa. Chromosome lengths were displayed in Mbp (megabase pairs) on the scale at top.
Figure 7. Comparative organisation of RLK genes between Aj genome of B. juncea and Ar genome of B. rapa (chromosomes A01 to A10). Solid circles above the chromosome (grey bars) represented RLK genes: LysM-RLK (red) and LRR-RLK (blue). Rectangles represented gene clusters: homogeneous cluster (black) and heterogeneous cluster (red). Pink straight lines between genes displayed the genes with syntenic relationship between B. juncea and B. rapa. Chromosome lengths were displayed in Mbp (megabase pairs) on the scale at top.
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Figure 8. Comparative organisation of RLP genes between Aj genome of B. juncea and Ar genome of B. rapa (chromosomes A01 to A10). Solid circles above the chromosome (grey bars) represented RLK genes: LysM-RLP (red) and LRR-RLP (blue). Black rectangles represented homogeneous cluster. Pink straight lines between genes displayed the genes with syntenic relationship between B. juncea and B. rapa. Chromosome lengths were displayed in Mbp (megabase pairs) on the scale at top.
Figure 8. Comparative organisation of RLP genes between Aj genome of B. juncea and Ar genome of B. rapa (chromosomes A01 to A10). Solid circles above the chromosome (grey bars) represented RLK genes: LysM-RLP (red) and LRR-RLP (blue). Black rectangles represented homogeneous cluster. Pink straight lines between genes displayed the genes with syntenic relationship between B. juncea and B. rapa. Chromosome lengths were displayed in Mbp (megabase pairs) on the scale at top.
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Table
Table 1. The total number of predicted receptor-like kinase (RLK) and receptor-like protein (RLP) genes identified in the Brassica genomes.
Table 1. The total number of predicted receptor-like kinase (RLK) and receptor-like protein (RLP) genes identified in the Brassica genomes.
SpeciesB. junceaB. rapaB. nigra
Gene content80,43046,09849,826
RLKs493 (0.613%)300 (0.651%)317 (0.636%)
LRR-RLKs484 (0.602%)297 (0.644%)312 (0.626%)
LysM-RLKs9 (0.011%)3 (0.007%)5 (0.010%)
RLPs228 (0.283%)65 (0.141%)176 (0.353%)
LRR-RLPs226 (0.281%)63 (0.137%)175 (0.351%)
LysM-RLPs2 (0.002%)2 (0.004%)1 (0.002%)
Table
Table 2. The duplication events defined in the B. juncea genome.
Table 2. The duplication events defined in the B. juncea genome.
Duplication TypeRLKRLP
TotalIntra Sub-FamilyInter
Sub-Family		TotalIntra Sub-FamilyInter
Sub-Family
LysM-LRR-LysM-LRR-
Intra A genomic831820200200
Intra B genomic800800320320
Total of intra genome16311620520520
Inter genome30843040581570
Total4715466011011090
Table
Table 3. The total number of candidate RLK and RLP genes identified in B. rapa. The total number of genes in each chromosome, as well as the conserved genes obtained from the comparison with B. juncea.
Table 3. The total number of candidate RLK and RLP genes identified in B. rapa. The total number of genes in each chromosome, as well as the conserved genes obtained from the comparison with B. juncea.
ChromosomeRLKRLP
TotalLysM-RLKLRR-RLKGene ClusterConservedTotalLysM-RLPLRR-RLPGene ClusterConserved
No. of ClusterNo. of Gene in ClusterTotalLysM-RLKLRR-RLKNo. of ClusterNo. of Gene in ClusterTotalLysM-RLPLRR-RLP
A genome B. rapaA013603615290291001000505
A0228028291701770700202
A0338038003003090900202
A0416115151411310100101
A0525025171901960613404
A064914831536135101913211
A07370373122102140400202
A0819118001511471600413
A0926026001601690900404
A1021021271701720200101
Total mapped genes2953,
1.02% 1		292,
98.98% 1		1360,
20.34% 1		214,
72.54% 1		3,
100.00% 2		211,
72.26% 3		652,
3.08% 1		63,
96.92% 1		26,
9.23% 1		27,
41.54% 1		2,
100.00% 2		25,
39.68% 3
Unassigned505-----000-----
Total3003,
1.00% 4		297,
99.00% 4		13----652,
3.08% 4		63,
96.92% 4		2----
1 The proportion in all mapped RLKs or RLPs. 2 The proportion in mapped LysM-RLKs or LysM-RLPs. 3 The proportion in mapped LRR-RLKs or LRR-RLPs. 4 The proportion in all candidate RLKs or RLPs.
Table
Table 4. The total number of candidate RLK and RLP genes identified in B. juncea. The total number of genes in each chromosome, as well as the conserved genes obtained from the comparison with B. nigra and B. rapa. The total number of syntenic genes in each chromosome of B. juncea A genome, in contrast with B. rapa.
Table 4. The total number of candidate RLK and RLP genes identified in B. juncea. The total number of genes in each chromosome, as well as the conserved genes obtained from the comparison with B. nigra and B. rapa. The total number of syntenic genes in each chromosome of B. juncea A genome, in contrast with B. rapa.
ChromosomeRLKRLP
TotalLysM-LRR-ConservedSyntenic GenesTotalLysMLRR-ConservedSyntenic Genes
Total LysM-LRR-Total LysM-LRR-
A genome B. junceaA01300302402422120126065
A022502517017145052022
A03290292602628170171011
A041621413112139091011
A052212118018196061013
A063113027126327162112
A07260262202221110112022
A081211112111118083035
A09230231701715170175055
A102202218018173031011
Total in A genome236,
51.53% 1		5,
2.12% 2		231,
97.88% 2		194,
82.20% 2		3,
60.00% 3		191,
82.68% 4		192,
81.36% 2		95,
44.81% 1		1,
1.05% 2		94,
98.95% 2		24,
25.26% 2		1,
100.00% 3		23,
24.47% 4		27,
28.42% 2
B genome B. junceaB012722524222-606606-
B023603630030-10010303-
B033003027027-1911813112-
B043012923122-2302311011-
B053303328028-1801811011-
B062002017017-11011606-
B071601616016-16016909-
B083003024024-14014606-
Total in B genome222,
48.47% 1		3,
1.35% 2		219, 98.65% 2189, 85.14% 23,
100.00% 3		186,
84.93% 4		-117,
55.19% 1		1,
0.85% 2		116,
99.15% 2		65,
55.56% 2		1,
100.00% 3		64,
55.17% 4		-
Total mapped genes4588,
1.75% 1		450,
98.25% 1		383,
83.62% 1		6,
75.00% 3		377,
83.78% 4		-2122,
0.94% 1		210,
99.06% 1		89,
41.98% 1		2,
100.00% 3		87,
41.43% 4		-
1 The proportion in all mapped RLKs or RLPs. 2 The proportion in all mapped RLKs or RLPs in each sub-genome of B. juncea. 3 The proportion in mapped LysM-RLKs or LysM-RLPs across the entire genome and each sub-genome of B. juncea. 4 The proportion in mapped LRR-RLKs or LRR-RLPs across the entire genome and each sub-genome of B. juncea.
Table
Table 5. The total number of candidate RLK and RLP genes identified in B. nigra scaffolds. The conserved genes obtained from the comparison with B. juncea and B. nigra.
Table 5. The total number of candidate RLK and RLP genes identified in B. nigra scaffolds. The conserved genes obtained from the comparison with B. juncea and B. nigra.
RLKTotal317
LysM-RLK5 (1.58% 1)
LRR-RLK312 (98.42% 1)
ConservedTotal227 (71.61% 1)
LysM-RLK4
LRR-RLK223
RLPTotal176
LysM-RLP1 (0.57% 2)
LRR-RLP175 (99.43% 2)
ConservedTotal72 (40.91% 2)
LysM-RLP1
LRR-RLP71
1 The proportion in all RLKs. 2 The proportion in all RLPs.
Table
Table 6. Comparative analysis of the number of RLK and RLP genes in B. juncea, B. nigra and B. rapa.
Table 6. Comparative analysis of the number of RLK and RLP genes in B. juncea, B. nigra and B. rapa.
Gene FamilyGenomeB. juncea
A Genome		B. rapa
A Genome		B. juncea
B Genome 		B. nigra
B Genome
RLK familyTotal (LysM, LRR)236 (5, 231)300 (3, 297)222 (3, 219)317 (5, 315)
Total conserved194 (82.20%)214 (71.33%)189 (85.14%)227 (71.61%)
Conserved LysM-RLK3 (60.00%)3 (100.00%)3 (100.00%)4 (80.00%)
Conserved LRR-RLK191 (82.68%)211 (71.04%)186 (84.93%)223 (70.79%)
Not conserved42 (17.80%)86 (28.67%)33 (14.86%)90 (28.39%)
Lost086090
Gained420330
RLP familyTotal (LysM, LRR)95 (1, 94)65 (2, 63)117 (1, 116)176 (1, 175)
Total conserved24 (25.26%)27 (41.54%)65 (55.56%)72 (40.91%)
Conserved LysM-RLP1 (100.00%)2 (100.00%)1 (100.00%)1 (100.00%)
Conserved LRR-RLP23 (24.47%)25 (39.68%)64 (55.17%)71 (40.57%)
Not conserved71 (74.74%)38 (58.46%)52 (44.44%)104 (59.09%)
Lost0380104
Gained710520
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Yang, H.; Bayer, P.E.; Tirnaz, S.; Edwards, D.; Batley, J. Genome-Wide Identification and Evolution of Receptor-Like Kinases (RLKs) and Receptor like Proteins (RLPs) in Brassica juncea. Biology 2021, 10, 17. https://doi.org/10.3390/biology10010017

AMA Style

Yang H, Bayer PE, Tirnaz S, Edwards D, Batley J. Genome-Wide Identification and Evolution of Receptor-Like Kinases (RLKs) and Receptor like Proteins (RLPs) in Brassica juncea. Biology. 2021; 10(1):17. https://doi.org/10.3390/biology10010017

Chicago/Turabian Style

Yang, Hua, Philipp E. Bayer, Soodeh Tirnaz, David Edwards, and Jacqueline Batley. 2021. "Genome-Wide Identification and Evolution of Receptor-Like Kinases (RLKs) and Receptor like Proteins (RLPs) in Brassica juncea" Biology 10, no. 1: 17. https://doi.org/10.3390/biology10010017

APA Style

Yang, H., Bayer, P. E., Tirnaz, S., Edwards, D., & Batley, J. (2021). Genome-Wide Identification and Evolution of Receptor-Like Kinases (RLKs) and Receptor like Proteins (RLPs) in Brassica juncea. Biology, 10(1), 17. https://doi.org/10.3390/biology10010017

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