School of Biotechnology, Devi Ahilya University, Khandwa Rd., Indore-452001, India

Corresponding author Email: singhgauravbiotech@gmail.com

Article Publishing History

Introduction

Synteny word came from greek where ‘syn’ means together and ‘taenia’ means ribbon. When gene order is conserved between the organism is commonly refered as Synteny. It is of two different type’s viz. macrosynteny when many genes or large chromosomal segments of different organisms are syntenic; and microsynteny when only few genes are conserved in different species (Zhu et al., 2005 and Passoupathy, 2016). Relationship between plants, pathogens and pests have been currently discussed in different models (Andolfo and Ercol-ano, 2015). This models mainly include proteins which are coded by clustered disease-resistance (R) genes in plant genomes (Hulbert et. al., 2001). In earlier study, the R gene-encoded proteins were divided into eight major group on the basis of amino acid motif organization and localization in the cell (Gururani et. al., 2012). Among
these group two main R gene proteins are (CC) NBS-LRR or CNL proteins and TNL proteins. NBS-LRR genes have been grouped into three classes (TNL, CNL and R NLs) (Shao et. al., 2016 Neupane et. al., 2018).

The legumes are extremely diverse and can be distributed into 3 subfamilies viz. Caesalpinioideae, Mimosoideae and Papilionoideae (Doyle and Luckow, 2003). The subfamily, Papilionoideae consists of almost all commercially important legumes like Arachis hypogaea (peanut), Cicer arietinum (chick pea), Glycine max (soybean), Phaseolus vulgaris (common bean), Medicago sativa (alfalfa), Vigna radiata (mungbean), Lens culinaris (lentil) and Pisum sativum (pea). Although these crop legumes have close phylogenetic relationship but differ extremely in their chromosome number, genome size and ploidy level (Table 1). However, previous studies on comparative genetic mapping suggested that the Papilionoideae subfamily members showed broad genome conservation (Weeden et al., 1992; Zhu et al., 2005). Young et al (2003) showed that legumes form a systematic taxonomic group with ubiquitous and prevailing macro- and micro- synteny. There are reports indicating that closely related species contain genomes having various genes with same map positions (Bennetzen, 2000; Devos and Gale, 2000; Paterson et al., 2000; Schmidt, 2000; Gualtieri et al., 2002).

The two most important members of legume family Phaseoleae are soybean and common bean. These two species are commercially valuable legumes, common bean as a nutritional crop for poor population and soybean for its various human and animal uses (Mc Clean et al., 2010). Common bean has been diverged from soybean 19 million years ago. Considering syntenic relationship between two species is crucial to determine the potential for comparative genomic analysis. Whole genome duplication is one of the most salient characteristics of the soybean genome (Cannon and Shoemaker, 2012).Earlier, in plants, synteny analyses were aimed only on the species belonging to the families Poaceae and Brassicaceae. In recent time, such studies are being extended on other plants especially legumes (Gualtieri et al., 2002). It has been shown that sequence based tools help to study the evolution, organization and syntenic relationships of genomes. Macro- and micro- synteny of various species can be compared using linkage maps (Mc Connell et al., 2010). The studies indicated that level of synteny is found high in closely related species, and it decreases with growing phylogenetic distance (Choi et al., 2004). Recently, a linkage map for Apios Americana (a tuberous perennial legume in phaseolae tribe) has been reported and it showed synteny with selected warm-seasoned legumes. It also revealed a translocation event in Glycine max and common bean against Apios and Vigna species (Singh et al., 2018).

The RFLP mapping (Shoemaker et al., 1996) and EST Ks analysis have provided substantial evidences that whole genome duplication (WGD) in soybean has occurred about 13 million years ago (Schmutz et al., 2010 and Schlueter et al., 2004). Boutin et al (1995) have reported a number of syntenic linkage blocks between common bean and soybean by exploiting shared RFLP markers. They used using low density RFLP mapping, they showed that these two species share a high degree of homology in sequence, however, synteny has been found over short blocks of genomes. A clear one to two relationship between common bean and soybean genomes was shown by Lee et al (2001). A few synteny analyses have been reported between soybean and common bean. Mc Clean et al., (2010) studied the synteny between soybean and common bean on the basis of 300 gene base loci. Synteny blocks of averaging 32 cM in common bean and 4.9 Mb in soybean were found for all 11 common bean linkage groups which were mapped to all soybean linkage groups. A total 55 syntenic blocks each having 7 loci were observed between the common bean and soybean. By analysing the location of these blocks, it was revealed that each locus of common bean genome mapped to two loci of soybean genome (Mc Clean et al., 2010).

In 2014, Schmutz and co-workers observed considerable synteny between Phaseolus vulgaris and Glycine max, except in pericentromeric regions, where due to genomic expansion in one or both genomes micro- colinearity was much extended and due to that was lesser dense. Conservation of genome macrostructure has been reported between the soybean and other legumes including Phaseolus vulgaris (Lee et al., 2017). Studies showed the correlation between Phaseolus linkage group Pv01 and Glycine max chromosomes Gm06 and Gm04 (Cannon and Shoemaker 2012). It is also reported that Gm5 and Gm8 chromosomes share synteny to common bean Pv2 (Mc Clean et al., 2010). In another study, nearly 63% of common bean unigenes were shown to have homology to soybean (Kalavacharla et al., 2011). Microsynteny analysis was also reported between common bean and soybean where 6 BAC clones were sequenced and analyzed for microsynteny (Yadegari, 2013). Recently the genomes of G.max and P. vulgaris were analyzed for investigating synteny. Syntenic blocks were found between chromosomes Gm03 and Pv10, Gm10 and Pv07 as well as Gm14 and Pv01. A large number of present research make use of whole genome sequences to study R genes in legumes (Anderson 2016, Christie et. al., 2016 and Neupane et. al., 2018).

Earlier many research were done on NBS-LRR genes in Glycine max and other legumes by using bioinformatics approach (Benson 2014, Shao et. al., 2016 and Neupane et. al., 2018). Nepal and Benson (2015) showed the complete evolutionary relationship of the CNL- R genes in soybean and common bean. In the present study, synteny analysis between chromosome 13 of soybean having NBS-LRR enriched sequences and whole genome of common bean have been carried out since common bean has been reported to have NBS-LRR enriched regions which help in disease resistance. The results from our study will be helpful in understanding evolutionary relationships of NBS-LRR genes with potential implication in crop improvement.

Materials and Methods

The gene synteny alignment, and genomic linkage analyses between the chromosome 13 of G. max and whole genome of P. vulgaris was performed by using ‘SATSUMA version 3.1.0 freely available on the website, http://satsuma.sourceforge.net/. All the data in FASTA and GFF files required for genome sequence and annotation of soybean and common bean were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/ genome/). For synteny analysis, only sequence alignments of more than 200 bp and percent identity of more than 85% were considered and rest were filtered out. The results were visualized by developing custom script in Biopython module of BioPython, version 3.7 freely available on the website, http://biopython.org/DIST/ docs/install/Installation.html.

Results and Discussion

Comparison of orthologous regions of soybean and common bean

The syntenic analysis between soybean and common bean showed many conserved regions between the two as shown in Fig. 1. Twenty one genes mentioned in Table 2, which were screened on the basis of few parameters of our interest as highest, lowest percent similarity, protein kinase, diseases resistance protein, NBS-LRR proteins represent less than 1/4th of the genes we have obtained after synteny mapping. These genes were mentioned with their gene id, locus tag along with the product formed as shown in table 2. Soybean chromosome 13 region having start from 22449055 bp and up to 22449570 bp showed gene homology with the sequence starting from 31929662 to 31930177 of Phaseolus vulgaris. This genomic region in both the species codes for guanine nucleotide binding protein subunit gamma 3-like isoform X1. The guanine nucleotide binding protein is important for signalling pathway. It is to be noted that the Rpg1b genes in soybean mainly codes for NB and LRR proteins. Various NB-LRR protein coding genes which are present on chromosome 13 of G. max are also showing synteny with P. vulgaris. For instance, F-box/ LRR-repeat protein 4-like (LOC100816140) is located at 27041793 bp to 27042286 bp in Phaseolus vulgaris. A mitogen-activated protein kinase2 (LOC100789734) located on chromosome 13 of Glycine max is showing homology with Phaseolus vulgaris.

A G type lectin S-receptors like serine/threonine-protein kinase coding gene located at chromosome 13 in Glycine max is also present in Phaseolus vulgaris genomic region having starts from 30144768 bp to 30146245 bp. It is having several molecular function like ATP binding, Calmodulin binding and protein serine/threonine kinase activity and few biological functions like protein phosphorylation and recognition of pollen. Glycogen phosphorylase 1-like isoform X1 coding gene which is present at locus LOC100779066 on Glycine max chromosome 13 is present in Phaseolus vulgaris. Uncharacterized ATPdependent helicase C23E6.02-like (LOC100792901) and B3 domain-containing protein Os07g0563300-like both were conserved in these legumes namely Glycine max and Phaseolus vulgaris. Highest synteny was found to be 91.794 % similarity between Glycine max and Phaseolus vulgaris for a region which codes for an uncharacterized protein LOC100775655 in both the legume. Similarly, lowest synteny was found to be 85.049 % similarity at loci LOC102661544 of Glycine max.

Figure 1: Syntenic regions between Glycine max and Phaseolus vulgaris. A synteny region has been amplified where synteny length has been indicated using colors. The colors of the links are based on the magnitude of synteny length (Light grey color indicates minimum synteny length whereas red brick color maximum synteny length).

Various syntenic regions were found between the two legumes, which codes for different proteins like MAP Kinase kinase2, Guanine nucleotide binding protein, 1-phosphatidylinositol-3-Phosphate 5-kinase and several Serine threonine kinases. All the proteins have different molecular and biological functions like ATP and ADP binding, Kinase activity, Metal and Calcium Ion binding, signal transduction, defense responses respectively.
Different gene functions and their position in soybean and common bean are described with details in Table 3.

In the present study, a different approach has been used for comparison of soybean genome with common bean. Advantage of synteny analysis is based on the concept that the species which are evolutionary related are diverged from their common ancestor and conserved genome synteny can be efficiently interpreted from a well-studied species to another less characterized genomes. Synteny is found to be higher between the closely related species (Lee et. al., 2017) and this concept is confirmed in this study. The outcome from the analysis identifies the best match to the Glycine max genome. Here focus was done only on chromosome 13 of Glycine max. We selected this chromosome because in Glycine max, diseases resistance coding genes are known to be found mostly on this region (Chr. 13). By the comparison of both the legumes sequences we get NB-LRR protein coding genes, which are responsible for disease resistance in Glycine max and Phaseolus vulgaris.

Identifi cation of conserved synteny has noticeable advantage in understanding the legumes genetics. Large region of synteny occurs between Glycine max and Phaseolus vulgaris (Mc Clean et. al., 2010), our results also showed the gene conservation between both the legumes. Studies of the NBS-LRR gene family in legume plants can provide knowledge on the genomic and molecular mechanism that form the basis of gene regulation and protein functions. In this study, various NBS-LRR proteins (as mentioned in table 3 showing different functions of gene products) were found to be conserved in Glycine max and Phaseolus vulgaris. Now a day’s gene cloning approach is advancing to make disease resistance varieties of legumes as in Medicago sativa cloning of a disease resistance gene was done by utilizing Medicago truncatula gene information through synteny (Yang et. at., 2008). In the same way this synteny analysis result will be useful for evolutionary studies that help in long term planning, breeding and developing disease resistance varieties of legumes.

Table 2: max cultivar_Williams_82 chromosome_13, Glycine_max_v2.0, whole_genome shotgun sequence) with Phaseolus vulgaris.

Table 3: Different functions of gene products of Glycine max and Phaseolus vulgaris.

Acknowledgements

Authors acknowledge the facilities of the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi (DBT) under the Bioinformatics Sub Centre as well as the golden jubilee research fellowship (GJRF) provided by School of Biotechnology, Devi Ahilya University Indore. M.P. India.

Conflict of Interest

The authors confirm that they have no conflict of interest.

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