Fan Wu

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

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.

Genotypic Variation in a Breeding Population of Yellow Sweet Clover (Melilotus officinalis)

Yellow sweet clover is a widely spread legume species that has potential to be used as a forage crop in Western China. However, limited information is available on the genetic variation for herbage yield, key morphological traits, and coumarin content. In this study, 40 half sib (HS) families of M. officinalis were evaluated for genotypic variation and phenotypic and genotypic correlation for the traits: LS (leaf to stem ratio), SV (spring vigor), LA (leaf area), PH (plant height), DW (herbage dry weight), SD (stem diameter), SN (stem number), Cou (coumarin content), SY (seed yield), across two locations, Yuzhong and Linze, in Western China. There was significant (P < 0.05) genotypic variation among the HS families for all traits. There was also significant (P < 0.05) genotype-by-environment interaction for the traits DW, PH, SD, SN, and SV. The estimates of HS family mean repeatability across two locations ranged from 0.32 for SN to 0.89 for LA. Pattern analysis generated four HS family groups where group 3 consisted of families with above average expression for DW and below average expression for Cou. The breeding population developed by polycrossing the selected HS families within group 3 will provide a significant breeding pool for M. officinalis cultivar development in China.

Co-transforming bar and CsLEA enhanced tolerance to drought and salt stress in transgenic alfalfa (Medicago sativa L.)

Drought and high salinity are two major abiotic factors that restrict alfalfa productivity. A dehydrin protein, CsLEA, from the desert grass Cleistogenes songorica was transformed into alfalfa (Medicago sativa L.) via Agrobacterium-mediated transformation using the bar gene as a selectable marker, and the drought and salt stress tolerances of the transgenic plants were assessed. Thirty-nine of 119 transformants were positive, as screened by Basta, and further molecularly authenticated using PCR and RT-PCR. Phenotype observations revealed that the transgenic plants grew better than the wild-type (WT) plants after 15d of drought stress and 10d of salt stress: the leaves of WT alfalfa turned yellow, whereas the transgenic alfalfa leaves only wilted; after rewatering, the transgenic plants returned to a normal state, though the WT plants could not be restored. Evaluation of physiologic and biochemical indices during drought and salt stresses showed a relatively lower Na(+) content in the leaves of the transgenic plants, which would reduce toxic ion effects. In addition, the transgenic plants were able to maintain a higher relative water content (RWC), higher shoot biomass, fewer photosystem changes, decreased membrane injury, and a lower level of osmotic stress injury. These results demonstrate that overexpression of the CsLEA gene can enhance the drought and salt tolerance of transgenic alfalfa; in addition, carrying the bar gene in the genome may increase herbicide resistance.

Cross-species transferability of EST-SSR markers developed from the transcriptome of Melilotus and their application to population genetics research

Melilotus is one of the most important legume forages, but the lack of molecular markers has limited the development and utilization of Melilotus germplasm resources. In the present study, 151 M clean reads were generated from various genotypes of Melilotus albus using Illumina sequencing. A total of 19,263 potential EST-SSRs were identified from 104,358 unigene sequences. Moreover, 18,182 primer pairs were successfully designed, and 550 primer pairs were selected using criteria of base repeat type, fragment length and annealing temperature. In addition, 550 primer pairs were screened by using PCR amplification products and used to assess polymorphisms in 15 M. albus accessions. A total of 114 primer pairs were detected as being highly polymorphic, and the average polymorphism information content (PIC) value was 0.79. Furthermore, those 114 polymorphic primer pairs were used to evaluate the transferability to 18 species of the genus Melilotus, and 70 EST-SSR markers were found to be transferable among the 18 Melilotus species. According to the UPGMA dendrogram and STRUCTURE analysis, the 18 Melilotus species were classified into three clusters. This study offers a valuable resource for the genetic diversity and molecular assisted breeding of germplasm resources in the genus Melilotus.

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

Melilotus, an annual or biennial herb, belongs to the tribe Trifolieae (Leguminosae) and consists of 19 species. As an important green manure crop, diverse Melilotus species have different values as feed and medicine. To identify different Melilotus species, we examined the efficiency of five candidate regions as barcodes, including the internal transcribed spacer (ITS) and two chloroplast loci, rbcL and matK, and two non-coding loci, trnH-psbA and trnL-F. In total, 198 individuals from 98 accessions representing 18 Melilotus species were sequenced for these five potential barcodes. Based on inter-specific divergence, we analysed sequences and confirmed that each candidate barcode was able to identify some of the 18 species. The resolution of a single barcode and its combinations ranged from 33.33% to 88.89%. Analysis of pairwise distances showed that matK+rbcL+trnL-F+trnH-psbA+ITS (MRTPI) had the greatest value and rbcL the least. Barcode gap values and similarity value analyses confirmed these trends. The results indicated that an ITS region, successfully identifying 13 of 18 species, was the most appropriate single barcode and that the combination of all five potential barcodes identified 16 of the 18 species. We conclude that MRTPI is the most effective tool for Melilotus species identification. Taking full advantage of the barcode system, a clear taxonomic relationship can be applied to identify Melilotus species and enhance their practical production.

Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content

Coumarin and its derivatives are widely used as fragrances in industrial products and have medical value. The goal of the present study was to discover genes and pathways related to coumarin biosynthesis in Melilotus albus using transcriptome analysis. The genes of five M. albus near-isogenic lines (NILs) that had different coumarin content and β-glucosidase activity according to the investigation of pedigree were quantified and then analysed by RNA-Seq. Using transcriptome analysis, differentially expressed genes (DEGs) were identified in two pairwise comparisons that differed in coumarin content as well as in two pairwise comparisons that differed in β-glucosidase activity. Gene expression pattern analysis suggested similar transcriptional trends in the genotypes with the same coumarin levels. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database of DEGs was used to identify functional pathways associated with coumarin biosynthesis. We identified 111 unigenes, with several DEGs among them possibly being related to coumarin synthesis pathways. Unigenes encoding a hexokinase, an abscisic acid receptor, a phenylalanine ammonia-lyase (PAL) and two peroxidases particularly showed correspondence with the coumarin content of different genotypes. These results will contribute to a better understanding of the coumarin biosynthesis in M. albus.

Co-transforming bar and CsALDH Genes Enhanced Resistance to Herbicide and Drought and Salt Stress in Transgenic Alfalfa (Medicago sativa L.)

Drought and high salinity are two major abiotic factors that restrict the productivity of alfalfa. By application of the Agrobacterium-mediated transformation method, an oxidative responsive gene, CsALDH12A1, from the desert grass Cleistogenes songorica together with the bar gene associated with herbicide resistance, were co-transformed into alfalfa (Medicago sativa L.). From the all 90 transformants, 16 were positive as screened by spraying 1 mL L-1 10% Basta solution and molecularly diagnosis using PCR. Real-time PCR analysis indicated that drought and salt stress induced high CsALDH expression in the leaves of the transgenic plants. The CsALDH expression levels under drought (15 d) and salt stress (200 mM NaCl) were 6.11 and 6.87 times higher than in the control plants, respectively. In comparison to the WT plants, no abnormal phenotypes were observed among the transgenic plants, which showed significant enhancement of tolerance to 15 d of drought and 10 d of salinity treatment. Evaluation of the physiological and biochemical indices during drought and salt stress of the transgenic plants revealed relatively lower Na+ content and higher K+ content in the leaves relative to the WT plants, a reduction of toxic on effects and maintenance of osmotic adjustment. In addition, the transgenic plants could maintain a higher relative water content level, higher shoot biomass, fewer changes in the photosystem, decreased membrane injury, and a lower level of osmotic stress. These results indicate that the co-expression of the introduced bar and CsALDH genes enhanced the herbicide, drought and salt tolerance of alfalfa and therefore can potentially be used as a novel genetic resource for the future breeding programs to develop new cultivars.

Analysis of miRNAs and their target genes in five Melilotus albus NILs with different coumarin content

MicroRNAs (miRNAs) exhibit diverse and important roles in regulation of various biological processes at the post-transcriptional level in plants. In this study, Melilotus albus miRNA and their target genes were elucidated from five M. albus near-isogenic lines which differ in coumarin content to construct small RNA libraries through high-throughput sequencing. A total of 417 known miRNAs and 76 novel miRNAs were identified in M. albus. In addition, 4155 different target genes for 114 known miRNA families and 14 target genes for 2 novel miRNAs were identified in M. albus. Moreover, mtr-miR5248 and mtr-miR7701-5p target c35498_g3 and gma-miR396a-3p target c37211_g1 involved in coumarin biosynthesis were identified by using the differential expression of the miRNAs and their target genes correlation analysis. The abundance of miRNAs and potential target genes were validated by qRT-PCR analysis. We also found that there were both positive and negative expression changing patterns between miRNAs and their related target genes. Our first and preliminary study of miRNAs will contribute to our understanding of the functions and molecular regulatory mechanisms of miRNAs and their target genes, and provide information on regulating the complex coumarin pathway in M. albus for future research.

Analysis of microRNA reveals cleistogamous and chasmogamous floret divergence in dimorphic plant

Cleistogenes songorica, a grass species that exhibits two spatially different type of inflorescence, chastogamy (CH), flowers localized at the top, and cleistogamy (CL) flowers embedded in leaf sheath. This study aimed at dissecting reasons underlying these distinct floral development patterns at morphological and microRNA level. Phenotyping for CH and CL was conducted and four small RNA libraries were constructed from the CH and CL flowers for high-throughput sequencing to identify the differentiated miRNAs. As results, spikelet, stigma, anther, lemma and lodicule length of CH flowers were found larger than that of CL, and so was seed setting. Also, 17 flower-related differential expression miRNAs were identified which were associated with floral organ development and morphogenesis, and the flower development. Further results showed that miR159a.1-CL3996.Contig2 pair was related to anther development, miR156a-5p-CL1954.Contig2 was linked to response to high light intensity, miR408-3p/miR408d-Unigene429 was related to pollination and Unigene429 positively regulated flower development. To our knowledge, this is the first study on differential miRNA accumulation between CH and CL flowers and our study serves as a foundation to the future elucidation of regulatory mechanisms of miRNAs in the divergent development of CL and CH flowers in a single plant.

Genetic variation and diversity in 199 Melilotus accessions based on a combination of 5 DNA sequences

Melilotus is an important genus of legume plants and an herbage with excellent nitrogen fixation; it can tolerate extreme environmental conditions and possesses important medicinal value. However, there is limited genetic information about the genus; thus, we analysed four chloroplast loci (rbcL, matK, psbA-trnH and trnL-F) and one nuclear region (ITS) to determine the genetic diversity of 199 accessions from 18 Melilotus species. The rbcL and matK sequences were highly conserved, whereas the trnL-F and ITS sequences contained variable and parsimony-informative sites. In our analyses of the single and combined regions, we calculated the pairwise distance, haplotype and nucleotide diversity and gaps and then constructed phylogenetic trees to assess the genetic diversity, and our results revealed significant variations among the different accessions. The genetic distance values were between zero and nine, and based on the combined regions, the highest frequency value was approximately four. Melilotus showed high haplotype and nucleotide diversity, particularly in the ITS sequences, with values of 0.86 and 0.0087, respectively. The single ITS sequence, psbA-trnH, and the combined matK+rbcL+trnL-F (MRT) and matK+rbcL+psbA-trnH+trnL-F+ITS (MRPTI) regions showed interspecific variation in the gap analysis. Phylogenetic trees calculated using ITS, psbA-trnH and MRPTI sequences indicated distinct genetic relationship in 18 species, and these species could be divided into two groups. By determining the genetic diversity of plants, we can evaluate the genetic relationships among species and accessions, providing a basis for preserving and utilizing the genetic resources of Melilotus.