Identification of BAHD genes in seven Rosaceae species
The BAHD superfamily characteristic domain (Pfam: PF02458) and the BAHD HMM configuration file (PF02458) were used as candidate genes to identify the BAHD members by the Hidden Markov model. The online site SMART (http://smart.embl-heidelberg.de/) was used to analysis protein sequences of candidate genes, and to determine the domain PF02458 of BAHD. We obtained 822 BAHD family candidate genes based on above analysis. Furthermore, multiple sequence alignment was conducted to verify the presence of two characteristic conserved domains (HXXXD and DFGWG) of BAHD family genes [6]. Six genes were removed from Chinese white pear due to the absence of two domains, while 17 in European pear, seven in woodland strawberry, eight in apple, 20 in sweet cherry, three in peach, and six in black raspberry. Finally, 114 BAHD genes were identified in Pyrusbretschneideri (the Chinese white pear), while 135 in Pyrus communis (European pear), 141 in Malus domestican (apple), 89 in Fragaria vesca (woodland strawberry), 125 in Prunus aviumsweet cherry), 82 in Prunus persicapeach), 69 in Rubus occidentalisblack raspberry) (Table 1). Detailed information of gene features of BAHD genes can be retrieved (Additional file 2: Table S1).
Table 1 Genome information and BAHD genes number identified in Rosaceae species
Common name
|
Scientific name
|
Chromosome number
|
Release version
|
Genome gene number
|
Identified BAHD genes
|
Gene name prefix
|
|
Chinese white pear
|
Pyrus bretschneideri
|
34
|
NJAU, v1.1
|
42,341
|
114(120)
|
Pbr
|
|
Apple
|
Malus domestica
|
34
|
JGI, v1.1
|
63,541
|
141(149)
|
Mdo
|
|
Strawberry
|
Fragaria vesca
|
14
|
GDR, v4.0
|
32,831
|
89(96)
|
Fve
|
|
European pear
|
Pyrus communis
|
34
|
GDR, v1.1
|
43,419
|
135(152)
|
pcp
|
|
Sweet cherry
|
Prunus avium
|
16
|
GDR, v1.0
|
43,679
|
125(145)
|
Pav
|
|
Peach
|
Prunus persica
|
16
|
JGI, v1.1
|
27,864
|
82(85)
|
Ppa
|
|
Black raspberry
|
Rubus occidentalis
|
14
|
GDR, v3.0
|
33,286
|
69(75)
|
Roc
|
|
The database address were listed below: NJAU (http://peargenome.njau.edu.cn/); GDR (http://www.rosaceae.org/); JGI (http://www.jgi.doe.gov/); The numbers in parentheses show the count of genes before filtering for unanchored and missing conserved domain genes.
Phylogenetic analysis and conserved motifs analysis of BAHD genes in Chinese white pear
BAHD amino acid sequences from Chinese white pear, European pear, Arabidopsis and Populus were used to construct a phylogenetic tree. The neighbor-joining phylogeny of BAHD from European pear was also constructed. (Additional file 1: Figure S1). The Chinese white pear BAHD family protein genes were classified as five clades (I, II, III-a, IV, V) according to the classification results from model plants (Fig.1). Clade I consist of two subclades (Clade I-a and Clade I-b). This result is consistent with that reported by Yu et al (2009). The Arabidopsis genes belong to Clade I-a have been found to be involved in modifying aromatic- and aliphatic alcohols in Arabidopsis and Populus Yu et al (2009) [25, 26], mirroring the function of BAHD genes in Chinese white pear. The members of Clade I-b are related to lignin monomeric intermediates biosynthesis [27, 28], such as tobacco and Arabidopsis shikimate hydroxycinnamoyltransferases [29]. Clade II consist of two subclades (Clade II-a and Clade II-b). The function of Arabidopsis genes belong to Clade II-a is unknown. Clade II-b contains 2 Arabidopsis genes, AT3G29590.1 (At5MAT) and AT1G03940.1 (At3AT1),, which are associated with anthocyanin biosynthesis [30, 31]. Clade III-a contains less members, including four genes from Chinese white pear, three genes from Arabidopsis, and one gene from poplar. Clade V contains nine members, including one well-studied Arabidopsis genes AT4G24510.1 (CER2) involved in regulating the cuticular wax biosynthesis [32]. Clade IV contains many members which are involved in catalyzing the acetylation of aromatic alcohols and acetylating small- or medium-chain alcohols (Yu et al). Above all, only three and four members were clustered in Clade III-a and Clade V. However, about 28.1% (32 of 114) BAHD genes were involved in Clade I and 36.0% (41 of 114) were involved in Clade IV, respectively. BAHD genes closely related to pear volatile esters content, such as alcohol acyltransferase (AAT),, may be belong to Clade I and Clade IV. We detected 20 conserved motifs of the Chinese white pear using the online software MEME (Fig.2). All BAHD family members have motif1 or motif3 and about 65.8% (75 of 114) members contain both of them. Based on gene structure analysis, we found that motif1 and motif3 correspond to domains HXXXD and DFGWG, respectively. And motif sequences were CGGFAIGLSMSHKVADGSSLSTFINSWAE and FYEADFGWGKP, respectively. Members of the I-a, I-b, II-a, II-b, III-a, and V subfamilies do not contain motif17 except for Pbr005916.1; members of the I-a, I-b, II-a, II-b, III-a, and V subfamilies do not contain motif19 except for Pbr010925.1; Except for Pbr005746.1, Pbr014025.1, Pbr035166.1 and Pbr036245.1, members of IV subfamilies do not contain motif10. Motif14 and motif16 were only detected in the Clade II-b. The type and distribution of the conservative motifs of the same subfamily are similar, further supporting the evolutionary tree classification (Fig.2). Related information of conservative motifs is shown in Additional file 3: Table S2.
Gene duplication events identified in pear BAHD superfamily and BAHD collinearity analysis in the seven Rosaceae species
Different patterns of gene replication have jointly promoted the evolution of the BAHD family, including Whole-genome duplication (WGD) or segmental duplication, tandem duplication (TD), proximal duplication (PD), transposed duplication (TRD), dispersed duplication (DSD) [33, 34]. We use DupGen_finder software [35] to detect duplicated gene pairs among BAHD family genes for seven Rosaceae genomes respectively. All the member of BAHD gene family was assigned to relative types: WGD, PD, TD, TRD or DSD. The number of WGD duplications in Chinese white pear and apple were 29 and 59, but only three in strawberry and peach, and four in black raspberry and sweet cherry, and nine in European pear. The number of dispersed duplications in Chinese white pear and sweet cherry were 113 and 142, and 91 of BAHDs in strawberry and 81 of BAHDs in apple; the number is higher than those in European Pear (29) and peach (76) and black raspberry (70). Genomic rearrangements and gene loss may lead to the large proportion of dispersed duplicates in these species, moreover, the RNA- and DNA-based transposable gene duplication can also result in this [33]. WGD and dispersed duplication made a crucial impact in the evolution of the BAHD superfamily in Chinese white pear, apple and European pear (Additional file 4: Table S3). In peach and strawberry tandem and dispersed duplication is the main force; proximal and dispersed duplication played major roles in black raspberry and sweet cherry. In pear, about 57.1% (113 of 198) BAHD genes were involved in dispersed gene duplication, while 66.9% (91of 136) in woodland strawberry, 44.0% (81 of 184) in apple, 60.3% (76 of 126) in peach, 67.3% (70 of 104) in black raspberry, 66.4% (142 of 214) in sweet cherry and 52.7% (29 of 55) in European Pear, respectively (Fig.3). The result indicated that the dispersed duplication is ubiquitous in all investigated species.
In addition, we identified intra-genomic synteny blocks for each species [33]. As shown in Fig.4a, BAHD genes of Chinese white pear were distributed on 17 chromosomes with a random distribution; there is only one gene on chromosome 13. Similarly to that in Chinese white pear, the BAHD genes were detected randomly distributed in the other species. We found a total of 61 syntenic gene pairs from six Rosaceae species. Among them, 21 syntenic pairs were identified in Chinese white pear (Fig.4a), 29 pairs in apple (Fig.4b), compared only three in strawberry (Fig.4f), peach (Fig.4d), black raspberry (Fig.4e) and sweet cherry (Fig.4c) (Additional file 5: Table S4).
Ka, Ks, and Ka/Ks analysis for BAHD family genes
The stage of evolution for the WGD is usually estimated using Ks (synonymous substitutions per site) [36–38]. In addition to the original WGD (Ks~1.5–1.8, ~140 Mya) (denoted asγpaleohexaploidization event) that shared by core eudicots [39], a more recent WGD was detected in pear, and dated to 30–45 Mya (Ks~0.15 to 0.3) [24]. As shown in Supplementary file S5, Ks values of WGD-derived gene pairs in Chinese white pear ranged from 0.006 to 3.909, the range of Ks values for gene pairs derived from TD, PD, TRD and DSD are 0.001~4.247,0.07~3.670,0.029~4.381 and 0.005~5.066, respectively. Similar results were found in apple. In Chinese white pear, there are nine WGD-derived genes pairs with Ks values ranged from 0.15 to 0.30, demonstrating that they may be derived from the current WGD (30–45 MYA) [24]. Some other duplicated gene pairs possessed higher Ks values (1.992~3.909), implying that they have probably originated from more ancient duplication events. We also found that the Ks values of the WGD-derived gene pairs in black raspberry is 1.356~2.965, in European pear is 0.153~4.362, in peach is 1.416~4.357, and in sweet cherry is 1.469~4.210. The higher Ks values of WGD-derived gene pairs in peach, black raspberry and sweet cherry suggest that they were duplicated and retained from more ancient genome duplication events, supporting the absence of more recent WGD events in these species.
Deleterious mutations can be removed by negative selection (purifying selection). Conversely, new favorable mutations could be accumulated by positive selection (Darwinian selection) and spread through the Population [40]. To detect the selection pressure acting on BAHD genes, we analyzed the nonsynonymous substitutions value (Ka) and nonsynonymous/synonymous substitution ratio (Ka/Ks) in the seven Rosaceae species (Additional file 6: Table S5). The direction and magnitude of selection pressure were inferred based on Ka/Ks ratio ( Ka/Ks > 1: positive selection; Ka/Ks = 1: neutral evolution; and Ka/Ks < 1: purifying selection) [41]. The results showed that Ka/Ks values of all BAHD gene pairs of strawberry (Fig.5d), peach (Fig.5c) and European pear (Fig.5b) were less than one, indicating that these genes evolved through dramastic purifying selection (Fig.5). Similar results in the other four Rosaceae species (the Chinese pear (Fig.5f), sweet cherry (Fig.5a), black raspberry (Fig.5g) and apple (Fig.5e)), except for a few gene pairs with Ka/Ks values greater than one. It also can be seen from the box plot that these data distributions are more concentrated, especially in Chinese white pear, sweet cherry and apple.
Expression pattern of BAHD genes in Chinese white pear
Based on transcriptome data (Additional file 7: Table S6) from different pear tissues, we found that most genes in Chinese white pear expressed higher in root (Fig.6) and we discovered that 37 of the BAHD genes were expressed in all four stages of fruit development; Pbr014238.1 only expressed in the four periods of pollen tube; Pbr020016.1, Pbr027303.1, Pbr029551.1, Pbr014028.1 and Pbr006821.1 were highly expressed in the late stage of fruit development (Fruit_S4); Most members of the BAHD superfamily showed no expression during the four periods of the pollen tube.
Gene expression analyses with qRT-PCR
Reference to transcriptome expression profiles and the ester content analysis, we selected five potential Chinese white pear genes (Pbr020016.1, Pbr019034.1, Pbr014028.1, Pbr006821.1 and Pbr029551.1) that show strong correlation with content change of ester during fruit development. We used qRT-PCR to examine aforementioned candidate genes. In our study, we found that the individual genes’ expression patterns were highly correlated with the ester content changes during pear fruit development (Fig.7). The expression pattern of Pbr020016.1 (Fig.7a) rised first and then decreased and attained the maximum at the last stage. Pbr006821.1 (Fig.7e), Pbr014028.1 (Fig.7b), Pbr019034.1 (Fig.7c) and Pbr029551.1 (Fig.7d) showed similar expression pattern, they all reached the lowest value in the second period and then began to increase. Pbr006821.1 had a slow increase in regulation from S2 (75DAF) to S4 (145DAF). Conversely, Pbr014028.1, Pbr019034.1 and Pbr029551.1 had a sharp increase from S2 to S4. The expression peak of Pbr006821.1 emerged in the first stage; and the overall trend of Pbr020016.1, Pbr029551.1 and Pbr019034.1 is increased, so we speculate that they may play a role during fruit ripening.