Genome-wide identification of <Emphasis Type="Italic">SERK</Emphasis> genes in apple and analyses of their role in stress responses and growth

Background

Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases associated with various signaling pathways. These kinases have a relationship with stress signals, and they are also believed to be important for regulating plant growth. However, information about this protein family in apple is limited.

Results

Twelve apple SERK genes distributed across eight chromosomes were identified. These genes clustered into three distinct groups in a phylogenetic analysis. All of the encoded proteins contained typical SERK domains. The chromosomal locations, gene/protein structures, synteny, promoter sequences, protein–protein interactions, and physicochemical characteristics of MdSERK genes were analyzed. Bioinformatics analyses demonstrated that gene duplications have likely contributed to the expansion and evolution of SERK genes in the apple genome. Six homologs of SERK genes were identified between apple and Arabidopsis. Quantitative real-time PCR analyses revealed that the MdSERK genes showed different expression patterns in various tissues. Eight MdSERK genes were responsive to stress signals, such as methyl jasmonate, salicylic acid, abscisic acid, and salt (NaCl). The application of exogenous brassinosteroid and auxin increased the growth and endogenous hormone contents of Malus hupehensis seedlings. The expression levels of seven MdSERK genes were significantly upregulated by brassinosteroid and auxin. In addition, several MdSERK genes showed higher expression levels in standard trees of ‘Nagafu 2’ (CF)/CF than in dwarf trees of CF/‘Malling 9’ (M.9), and in CF than in the spur-type bud mutation “Yanfu 6” (YF).

Conclusion

This study represents the first comprehensive investigation of the apple SERK gene family. These data indicate that apple SERKs may function in adaptation to adverse environmental conditions and may also play roles in controlling apple tree growth.

     

Phylogeny and Molecular Evolution of miR820 and miR396 microRNA Families in <Emphasis Type="Italic">Oryza</Emphasis> AA Genomes

The phylogeny and evolution of the microRNA families, miR820 and miR396, was analysed across the AA genomes of the Oryza species, the close relatives of domesticated rice. A highly dynamic evolution of the miR820 family was revealed. The number of copies of MIR820 genes, their chromosomal location and the mature microRNA sequence varied greatly with a total of 16 novel miR820 variants being identified. The phylogeny of pre-MIR820 sequences revealed that MIR820 genes of recently evolved Oryza AA genomes may have derived from sequence divergence of one or a few ancestral genes found in wild Australian perennial rice populations, Taxon B (jpn2)-MIR820 genes. Genomic scale duplication played an important role in the evolution of some of the miR396 family genes in AA genome Oryza species. miR396 family contained a MIR396 gene cluster (MIR396a and MIR396c) which was conserved across the cereal genomes. Nucleotide diversity analysis at these two MIR396 loci revealed that domesticated rice has retained less than 10% of the total diversity present in wild species. In contrast, the nucleotide sequence of four MIR396 loci remained almost conserved across domesticated and wild rices, indicating that they were under extreme functional constraint and may be involved in regulating some fundamental processes which are important both for wild and domesticated rices. Expression analysis demonstrated that miR820 variants were expressed in O. glaberrima O. barthi and O. longistaminata genome. These findings pose new challenges to explain the possible role of miR820 variants identified.     

Genome-wide identification and expression analysis of the molecular chaperone binding protein <Emphasis Type="Italic">BiP</Emphasis> genes in <Emphasis Type="Italic">Citrus</Emphasis>

Here, we report for the first time the genome-wide identification and expression analysis of the molecular chaperone BiP genes in Citrus. Six genes encoding the conserved protein domain family GPR78/BiP/KAR2 were identified in the genome of Citrus sinensis and C. clementina. Two of them, named here as CsBiP1 and CsBiP2, were classified as true BiPs based on their deduced amino acid sequences. Alignment of the deduced amino acid sequences of CsBiP1 and CsBiP2 with BiP homologs from soybean and Arabidopsis showed that they contain all the conserved functional motifs of BiPs. Analysis of the promoter region of CsBiPs revealed the existence of cis-acting regulatory sequences involved in abiotic, heat-shock, and endoplasmic reticulum (ER) stress responses. Publicly available RNA-seq data indicated that CsBiP1 is abundantly expressed in leaf, flower, fruit, and callus, whereas CsBiP2 expression is rarely detected in any tissues under normal conditions. Comparative quantitative real-time PCR (qPCR) analysis of expression of these genes between C. sinensis grafted on the drought-tolerant “Rangpur” lime (C. limonia) and -sensitive “Flying Dragon” trifoliate orange (Poncirus trifoliata) rootstocks showed that CsBiP1 was upregulated by drought stress on the former but downregulated on the latter, whereas the CsBiP2 mRNA levels were downregulated on drought-stressed “Flying Dragon,” but remained constant on “Rangpur.” CsBiP2 upregulation was only observed in C. sinensis seedlings subjected to osmotic and cold treatments. Taken together, these results indicate the existence of two highly conserved BiP genes in Citrus that are differentially regulated in the different tissues and in response to abiotic stresses.     

High-throughput sequencing of <Emphasis Type="Italic">Prunus ferganensis</Emphasis> indicates that it is a geographical population of <Emphasis Type="Italic">P. persica</Emphasis>

Peach belongs to the genus Prunus, which includes Prunus persica and its relative species, P. mira, P. davidiana, P. kansuensis, and P. ferganensis. Of these, P. ferganensis have been classified as a species, subspecies, or geographical population of P. persica. To explore the genetic difference between P. ferganensis and P. persica, high-throughput sequencing was used in different peach accessions belonging to different species. First, low-depth sequencing data of peach accessions belonging to four categories revealed that similarity between P. ferganensis and P. persica was similar to that between P. persica accessions from different geographical populations. Then, to further detect the genomic variation in P. ferganensis, the P. ferganensis accession “Xinjiang Pan Tao 1” and the P. persica accession “Xia Miao 1” were sequenced with high depth, and sequence reads were assembled. The results showed that the collinearity of “Xinjiang Pan Tao 1” with the reference genome “Lovell” was higher than that of “Xia Miao 1” and “Lovell” peach. Additionally, the number of genetic variants, including single nucleotide polymorphisms (SNPs), structural variations (SVs), and the specific genes annotated from unmapped sequence in “Xia Miao 1” was higher than that in “Xinjiang Pan Tao 1” peach. The data showed that there was a close distance between “Xinjiang Pan Tao 1” (P. ferganensis) and reference genome which belong to P. persica, comparing “Xia Miao 1” (P. persica) and reference ones. The results accompany with phylogenetic tree and structure analysis confirmed that P. ferganensis should be considered as a geographic population of P. persica rather than a subspecies or a distinct species. Furthermore, gene ontology analysis was performed using the gene comprising large-effect variation to understand the phenotypic difference between two accessions. The result revealed that the pathways of gene function affected by SVs but SNPs and insertion-deletions markedly differed between the two peach accessions.     

Towards improvement of marker assisted selection of apple scab resistant cultivars: <Emphasis Type="Italic">Venturia inaequalis</Emphasis> virulence surveys and standardization of molecular marker alleles associated with resistance genes

Molecular breeding for pathogen resistance faces two major problems that delay its widespread adoption, resistance breakdown and difficulties in unambiguously identifying the alleles of the markers associated with specific resistance genes. Since the breakdown of the Rvi6 (Vf) gene in the Northern part of Europe breeders have intensified the search for new resistance sources to be introduced into their breeding programs. Alternative major genes to Rvi6 are available (e.g. Rvi2, Rvi4, Rvi5, Rvi10; Rvi11, Rvi12, Rvi13, and Rvi15, respectively Vh2, Vh4, Vm, Va, Vbj, Vb, Vd, Vr2 according to the old apple scab resistance gene nomenclature) but, with few exceptions (i.e., Rvi4, Rvi5 and, Rvi13), they have so far not been incorporated in commercial varieties. Pyramiding, i.e., combining several of these major resistance genes (R-genes) in individual plants, is one of the most promising strategies currently available to develop apple cultivars with durable apple scab resistance. But, which genes are the best suited to produce such new cultivars? Although the most interesting genes are surely those whose resistance so far has not been broken by the pathogen, genes with resistance that has been overcome coupled with only limited spread of the virulence may also be used in the pyramiding process. However, obtaining information on whether an R-gene is overcome and if so, the extent of the spread of the virulence is difficult and time consuming. Furthermore, often such reports are not up-to-date and the correctness of the data is difficult to verify. To solve these problems, the initiative “Monitoring of Venturia inaequalis virulences” has been proposed. The monitoring is based on a network of orchards of selected differential hosts. Incidence and severity of scab on these genotypes will be collected yearly; and after validation, the data will be published through the homepage of the project (www.vinquest.ch). Here, we present an outline of this initiative. A second major obstacle for broad adoption of marker assisted selection is the lack of tools to align marker analyzes performed in different laboratories to unambiguously identify the alleles linked to specific resistances. The identification of the alleles of the markers in coupling with the resistance genes is often very difficult, if the same genotype used to develop the markers is not simultaneously analyzed. In this paper we present an approach to standardize the size of the alleles in coupling with the resistance genes, using easily accessible cultivars. The proposed procedure has been applied to selected markers for the apple scab resistance genes Rvi2, Rvi4, Rvi5, Rvi6, Rvi11, Rvi12, Rvi13, Rvi14 and Rvi15 (respectively Vh2, Vh4, Vm, Vf, Vbj, Vb, Vd, Rvi14 and Vr2 according to the old nomenclature).     

The isolation of the <Emphasis Type="Italic">IGT</Emphasis> family genes in <Emphasis Type="Italic">Malus × domestica</Emphasis> and their expressions in four idiotype apple cultivars

IGT family genes share the highly conserved motif GφL-(A/T) IGT in domain II and play an essential role in plant form. The tree architecture of apple (Malus ×?domestica Borkh.) affects fruit quality and yield. However, little information is available regarding IGT family genes in apple. Apple cultivars of four ideotypes (columnar, tip bearer, spur, and standard) were selected to characterize IGT family genes. Four IGT family members named MdoTAC1a, MdoTAC1b, MdoLAZY1, and MdoLAZY2 were found in the apple genome, sharing four conserved domains. In addition, MdoLAZY1 and MdoLAZY2 contain a fifth domain (EAR motif) at the C-terminus. There was no difference in the coding sequences of each gene in the four cultivars, but several mutated sites were found in their promoters. The four genes displayed lower expression levels in all tested tissues and organs of the columnar cultivar than in the other three cultivars, while expression levels of MdoTAC1a and MdoTAC1b in shoot tips and vegetative buds were highest in the standard cultivar, followed by spur, tip bearing, and columnar cultivars in decreasing order. These results indicate that IGT gene promoters are of great importance in the development of apple tree architecture and lay a theoretical basis for developing gene-specific markers for marker-assisted selection in breeding programs.     

Divergent Evolution of Symbiotic Bacteria: Rhizobia of the Relic Legume <Emphasis Type="Italic">Vavilovia formosa</Emphasis> Form an Isolated Group within <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.     

microRNA-mediated <Emphasis Type="Italic">R</Emphasis> gene regulation: molecular scabbards for double-edged swords

Plant resistance (R) proteins are immune receptors that recognize pathogen effectors and trigger rapid defense responses, namely effector-triggered immunity. R protein-mediated pathogen resistance is usually race specific. During plant-pathogen coevolution, plant genomes accumulated large numbers of R genes. Even though plant R genes provide important natural resources for breeding disease-resistant crops, their presence in the plant genome comes at a cost. Misregulation of R genes leads to developmental defects, such as stunted growth and reduced fertility. In the past decade, many microRNAs (miRNAs) have been identified to target various R genes in plant genomes. miRNAs reduce R gene levels under normal conditions and allow induction of R gene expression under various stresses. For these reasons, we consider R genes to be double-edged “swords” and miRNAs as molecular “scabbards”. In the present review, we summarize the contributions and potential problems of these “swords” and discuss the features and production of the “scabbards”, as well as the mechanisms used to pull the “sword” from the “scabbard” when needed.