Interspecific Phylogenic Relationships within Genus Melilotus Based on Nuclear and Chloroplast DNA

Abstract

Melilotus comprises 19 species, while the phylogenetic relationships between species remain unclear. In the present work, three chloroplast genes, rbcL, matK, trnL-F, and one nuclear region, ITS (internal transcribed spacer) belonging to 48 populations of 18 species of Melilotus were sequenced and phylogenetic trees were constructed to study their interspecific relationships. Based on the phylogenetic tree generated in this study using rbcL analysis, the Melilotus genus is clearly monophyletic in the legume family. Both Bayesian and maximum-parsimony approaches were used to analyze the data. The nrDNA ITS provided more informative characteristics (9.8%) than cpDNA (3.0%). Melilotus contains two closely related groups, clade I and clade II. M. spicatus, M. indicus and M. segetalis have a close relationship. M. infestus, M. siculus and M. sulcatus are closely related. The comparing between molecular phylogeny and flower color classification in Melilotus showed that the flower color is not much informative for phylogenetics of this genus.

Potential DNA barcodes for Melilotus species based on five single loci and their combinations

“…Seeds were selected from 98 accessions ( S1 Table ) representing 18 Melilotus species from Nation Plant Germplasm System (NPGS, USA) [ 32 ] and National Gene Bank of Forage Germplasm (NGBFG, China). Two to three accessions were selected to represent each of the Melilotus species.…”

“…For each sample, total genomic DNA was extracted from whole seedlings using the SDS (sodium dodecyl sulfate) method [ 35 ]. Five pairs of primers, the internal transcribed spacer (ITS) [ 32 ], two chloroplast loci, rbc L [ 36 ] and mat K [ 37 ], and two non-coding regions, trn H- psb A [ 38 ] and trn L-F [ 39 ], were amplified and sequenced. A standard polymerase chain reaction (PCR) in a volume of 25 μL was prepared as follows: 12.25 μL 2×Reaction Mix, 0.25 μL Golden DNA Polymerase, 2 μL each primer (10 μmol/mL), 6.5 μL ddH 2 O and 2 μL template genomic DNA (50 ng/mL).…”

“…In our study, we concerned barcode analysis of Melilotus at the level of species. Regarding the 18 Melilotus species examined in this study, we are quite confident about the materials because we analysed 5 seed morphological traits and 9 agronomic traits for these germplasms [ 31 , 32 ]. Moreover, 40 half-sib (HS) families of M .…”

“…officinalis were obtained to evaluate genotypic variation as well as phenotypic and genotypic correlations [ 33 ]. Simple sequence repeat (SSR) analysis was performed to evaluate the genetic diversity of the 18 Melilotus species [ 34 ], and phylogenetic trees were constructed to study their inter-specific relationships [ 32 ]. No studies to date have measured the resolution of the DNA barcode system using numerous specimens covering almost all Melilotus species, and we established a standard DNA barcode system to assess the discrimination ability of each to propose the most powerful potential barcodes for Melilotus .…”

Genetic Improvement of Key Agronomic Traits in Melilotus albus

“…These are important steps towards identifying an efficient breeding method to maximize genetic gain per cycle of selection. Previous studies on genotypic variation have focused on genetic diversity (Di et al, 2014; Wu et al, 2016) and the phylogenic relationships (Di et al, 2015) within the Melilotus genus by using molecular markers and DNA sequencing. The presence of genotype × environment interactions complicates selection of material for broad adaptation due to variable relative performances across environments (Comstock and Moll, 1963; Cooper and Byth, 1996).…”