Genome-Wide Identification, Phylogenetic and Expression Analysis of Expansin Gene Family in Medicago sativa L.

Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar "XinjiangDaye") reference genome, we identified 168 Expansin members (MsEXPs). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I-EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis-acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress.

Photosynthesis, fluorescence, and nutrition of Zhonglan No. 2, a new alfalfa cultivar.

BACKGROUND: The years after planting play an important role in the above-ground biomass and nutritive value of alfalfa. Zhonglan No. 2 (Medicago sativa L. cv. Zhonglan No. 2) is a new breeding alfalfa cultivar characterized by high drought tolerance and high yield. To determine the optimum time for utilization of Zhonglan No. 2, we examined growth traits, chlorophyll content, photosynthetic and fluorescence parameters, and composition and nutritive values at the late vegetative and early flowering stages of the first stubble in the second, third, fourth, sixth, and eleventh years after planting. RESULTS: In general, the height and leaf area decreased with increasing number of years after planting. At the late vegetative stage, the fourth-year alfalfa exhibited higher stomatal conductance (Gs) and intercellular CO2 concentration (Ci), and better water use efficiency, and at the early flowering stage, the fourth-year alfalfa had the highest (P < 0.05) leaf net photosynthetic rate (Pn) and carboxylation efficiency (CE). Total digestible nutrients did not differ among years, but, in the early flowering stage, crude protein content decreased with years (P < 0.05). Malondialdehyde (MDA) content and total antioxidant capacity did not differ among years after planting, suggesting aging did not impose oxidative stress on this alfalfa cultivar. CONCLUSIONS: Based on height, chlorophyll content, crude protein (CP) content, and photosynthetic and fluorescence parameters, the fourth year after planting, at the early flowering stage, was the best for using Zhonglan No. 2. © 2021 Society of Chemical Industry.

Cultivar Discrimination of Single Alfalfa (Medicago sativa L.) Seed via Multispectral Imaging Combined with Multivariate Analysis.

Rapid and accurate discrimination of alfalfa cultivars is crucial for producers, consumers, and market regulators. However, the conventional routine of alfalfa cultivars discrimination is time-consuming and labor-intensive. In this study, the potential of a new method was evaluated that used multispectral imaging combined with object-wise multivariate image analysis to distinguish alfalfa cultivars with a single seed. Three multivariate analysis methods including principal component analysis (PCA), linear discrimination analysis (LDA), and support vector machines (SVM) were applied to distinguish seeds of 12 alfalfa cultivars based on their morphological and spectral traits. The results showed that the combination of morphological features and spectral data could provide an exceedingly concise process to classify alfalfa seeds of different cultivars with multivariate analysis, while it failed to make the classification with only seed morphological features. Seed classification accuracy of the testing sets was 91.53% for LDA, and 93.47% for SVM. Thus, multispectral imaging combined with multivariate analysis could provide a simple, robust and nondestructive method to distinguish alfalfa seed cultivars.

Genome-Wide Identification and Expression Profiling of the ERF Gene Family in Medicago sativa L. Under Various Abiotic Stresses.

The AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR) transcription factor represents one of the largest plant-specific transcriptional regulators in plants. ERF plays important roles in the regulation of various developmental processes and acts as a mediator in plant external stress responses. However, the research of the ERF gene family is still limited in alfalfa (Medicago sativa L.), one of the most important forage legume species in the world. In the present study, a total of 159 ERF genes were identified, and the phylogenetic reconstruction, classification, conserved motifs, signal peptide prediction, and expression patterns under salt, drought, and low-temperature stresses of these ERF genes were comprehensively analyzed. The ERF genes family in alfalfa could be classified into 10 groups and predicted to be strongly homologous. Based on the structure and functions relationships, the III and IV subfamilies were more likely to play functions in abiotic stresses and 18 MsERF genes were selected for further quantitative real-time PCR validation in different stresses treatment. The results showed that all these MsERF genes were upregulated under three stresses except MsERF008. This study identified the possibility of abiotic tolerance candidate genes playing various roles in stress resistance at the whole-genome level, which would provide primary understanding for exploring ERF-mediated tolerance in alfalfa.

Isolation and Functional Characterization of MsFTa, a FLOWERING LOCUS T Homolog from Alfalfa (Medicago sativa).

The production of hay and seeds of alfalfa, an important legume forage for the diary industry worldwide, is highly related to flowering time, which has been widely reported to be integrated by FLOWERING LOCUS T (FT). However, the function of FT(s) in alfalfa is largely unknown. Here, we identified MsFTa, an FT ortholog in alfalfa, and characterized its role in flowering regulation. MsFTa shares the conserved exon/intron structure of FTs, and MsFTa is 98% identical to MtFTa1 in Medicago trucatula. MsFTa was diurnally regulated with a peak before the dark period, and was preferentially expressed in leaves and floral buds. Transient expression of MsFTa-GFP fusion protein demonstrated its localization in the nucleus and cytoplasm. When ectopically expressed, MsFTa rescued the late-flowering phenotype of ft mutants from Arabidopsis and M. trucatula. MsFTa over-expression plants of both Arabidopsis and M. truncatula flowered significantly earlier than the non-transgenic controls under long day conditions, indicating that exogenous MsFTa strongly accelerated flowering. Hence, MsFTa functions positively in flowering promotion, suggesting that MsFTa may encode a florigen that acts as a key regulator in the flowering pathway. This study provides an effective candidate gene for optimizing alfalfa flowering time by genetically manipulating the expression of MsFTa.

Study on differentially expressed genes related to defoliation traits in two alfalfa varieties based on RNA-Seq.

BACKGROUND: Alfalfa (Medicago sativa) is a widely cultivated, essential commercial forage crop. The protein content in its leaves is the critical factor in determining the quality of alfalfa. Thus far, the understanding of the molecular mechanism of alfalfa defoliation traits remains unclear. The transcriptome database created by RNA-Seq is used to identify critical genes related to defoliation traits. RESULTS: In this study, we sequenced the transcriptomes of the Zhungeer variety (with easy leaf abscission) and WL319HQ variety (without easy leaf abscission). Among the identified 66,734 unigenes, 706 differentially expressed genes (DEGs) upregulated, and 392 unigenes downregulated in the Zhungeer vs WL319HQ leaf. KEGG pathway annotations showed that 8,414 unigenes were annotated to 87 pathways and contained 281 DEGs. Six DEGs belonging to the "Carotenoid biosynthesis", "Plant hormone signal transduction" and "Circadian rhythm-plant" pathways involved in defoliation traits were identified and validated by RT-qPCR analyses. CONCLUSIONS: This study used RNA-Seq to discover genes associated with defoliation traits between two alfalfa varieties. Our transcriptome data dramatically enriches alfalfa functional genomic studies. In addition, these data provide theoretical guidance for field production practice and genetic breeding, as well as references for future study of defoliation traits in alfalfa.

Karyotypic evolution of the Medicago complex: sativa-caerulea-falcata inferred from comparative cytogenetic analysis.

BACKGROUND: Polyploidy plays an important role in the adaptation and speciation of plants. The alteration of karyotype is a significant event during polyploidy formation. The Medicago sativa complex includes both diploid (2n = 2× = 16) and tetraploid (2n = 2× = 32) subspecies. The tetraploid M. ssp. sativa was regarded as having a simple autopolyploid origin from diploid ssp. caerulea, whereas the autopolyploid origin of tetraploid ssp. falcata from diploid form ssp. falcata is still in doubt. In this study, detailed comparative cytogenetic analysis between diploid to tetraploid species, as well as genomic affinity across different species in the M. sativa complex, were conducted based on comparative mapping of 11 repeated DNA sequences and two rDNA sequences by a fluorescence in situ hybridization (FISH) technique. RESULTS: FISH patterns of the repeats in diploid subspecies caerulea were highly similar to those in tetraploid subspecies sativa. Distinctly different FISH patterns were first observed in diploid ssp. falcata, with only centromeric hybridizations using centromeric and multiple region repeats and a few subtelomeric hybridizations using subtelomeric repeats. Tetraploid subspecies falcata was unexpectedly found to possess a highly variable karyotype, which agreed with neither diploid ssp. falcata nor ssp. sativa. Reconstruction of chromosome-doubling process of diploid ssp. caerulea showed that chromosome changes have occurred during polyploidization process. CONCLUSIONS: The comparative cytogenetic results provide reliable evidence that diploid subspecies caerulea is the direct progenitor of tetraploid subspecies sativa. And autotetraploid ssp. sativa has been suggested to undergo a partial diploidization by the progressive accumulation of chromosome structural rearrangements during evolution. However, the tetraploid subspecies falcata is far from a simple autopolyploid from diploid subspecies falcata although no obvious morphological change was observed between these two subspecies.

Five species, many genotypes, broad phenotypic diversity: When agronomy meets functional ecology.

PREMISE OF THE STUDY: Current ecological theory can provide insight into the causes and impacts of plant domestication. However, just how domestication has impacted intraspecific genetic variability (ITV) is unknown. We used 50 ecotypes and 35 cultivars from five grassland species to explore how selection drives functional trait coordination and genetic differentiation. METHODS: We quantified the extent of genetic diversity among different sets of functional traits and determined how much genetic diversity has been generated within populations of natural ecotypes and selected cultivars. KEY RESULTS: In general, the cultivars were larger (e.g., greater height, faster growth rates) and had larger and thinner leaves (greater SLA). We found large (average 63%) and trait-dependent (ranging from 14% for LNC to 95.8% for growth rate) genetic variability. The relative extent of genetic variability was greater for whole-plant than for organ-level traits. This pattern was consistent within ecotypes and within cultivars. However, ecotypes presented greater ITV variability. CONCLUSIONS: The results indicated that genetic diversity is large in domesticated species with contrasting levels of heritability among functional traits and that selection for high yield has led to indirect selection of some associated leaf traits. These findings open the way to define which target traits should be the focus in selection programs, especially in the context of community-level selection.

Assessment of Cultivar Distinctness in Alfalfa: A Comparison of Genotyping-by-Sequencing, Simple-Sequence Repeat Marker, and Morphophysiological Observations.

Cultivar registration agencies typically require morphophysiological trait-based distinctness of candidate cultivars. This requirement is difficult to achieve for cultivars of major perennial forages because of their genetic structure and ever-increasing number of registered material, leading to possible rejection of agronomically valuable cultivars. This study aimed to explore the value of molecular markers applied to replicated bulked plants (three bulks of 100 independent plants each per cultivar) to assess alfalfa ( L. subsp. ) cultivar distinctness. We compared genotyping-by-sequencing information based on 2902 polymorphic single-nucleotide polymorphism (SNP) markers (>30 reads per DNA sample) with morphophysiological information based on 11 traits and with simple-sequence repeat (SSR) marker information from 41 polymorphic markers for their ability to distinguish 11 alfalfa landraces representative of the germplasm from northern Italy. Three molecular criteria, one based on cultivar differences for individual SSR bands and two based on overall SNP marker variation assessed either by statistically significant cultivar differences on principal component axes or discriminant analysis, distinctly outperformed the morphophysiological criterion. Combining the morphophysiological criterion with either molecular marker method increased discrimination among cultivars, since morphophysiological diversity was unrelated to SSR marker-based diversity ( = 0.04) and poorly related to SNP marker-based diversity ( = 0.23, < 0.15). The criterion based on statistically significant SNP allele frequency differences was less discriminating than morphophysiological variation. Marker-based distinctness, which can be assessed at low cost and without interactions with testing conditions, could validly substitute for (or complement) morphophysiological distinctness in alfalfa cultivar registration schemes. It also has interest in sui generis registration systems aimed at marketing alfalfa landraces.

Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa.

BACKGROUND: Medicago sativa (alfalfa) is a low-input forage and potential bioenergy crop, and improving its yield and quality has always been a focus of the alfalfa breeding industry. Transgenic alfalfa plants overexpressing a precursor of alfalfa microRNA156 (MsmiR156) were recently generated by our group. These plants (miR156OE) showed enhanced biomass yield, reduced internodal length, increased shoot branching and trichome density, and a delay in flowering time. Transcripts of three SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes (MsSPL6, MsSPL12, and MsSPL13) were found to be targeted for cleavage by MsmiR156 in alfalfa. RESULTS: To further illustrate the molecular mechanisms underlying the effects of miR156 in alfalfa, two miR156OE genotypes (A11a and A17) were subjected to Next Generation RNA Sequencing with Illumina HiSeq. More than 1.11 billion clean reads were obtained from our available sequenced samples. A total of 160,472 transcripts were generated using Trinity de novo assembly and 4,985 significantly differentially expressed genes were detected in miR156OE plants A11a and A17 using the Medicago truncatula genome as reference. A total of 17 genes (including upregulated, downregulated, and unchanged) were selected for quantitative real-time PCR (qRT-PCR) validation, which showed that gene expression levels were largely consistent between qRT-PCR and RNA-Seq data. In addition to the established SPL genes MsSPL6, MsSPL12 and MsSPL13, four new SPLs; MsSPL2, MsSPL3, MsSPL4 and MsSPL9 were also down-regulated significantly in both miR156OE plants. These seven SPL genes belong to genes phylogeny clades VI, IV, VIII, V and VII, which have been reported to be targeted by miR156 in Arabidopsis thaliana. The gene ontology terms characterized electron transporter, starch synthase activity, sucrose transport, sucrose-phosphate synthase activity, chitin binding, sexual reproduction, flavonoid biosynthesis and lignin catabolism correlate well to the phenotypes of miR156OE alfalfa plants. CONCLUSIONS: This is the first report of changes in global gene expression in response to miR156 overexpression in alfalfa. The discovered miR156-targeted SPL genes belonging to different clades indicate miR156 plays fundamental and multifunctional roles in regulating alfalfa plant development.

Selection of reliable reference genes for quantitative real-time RT-PCR in alfalfa.

Real-time quantitative RT-PCR (qRT-PCR) is the most commonly used method for accurately detecting gene expression patterns. As part of qRT-PCR analysis, normalization of the data requires internal control gene(s) that display uniform expression under different biological conditions. However, no invariable internal control gene exists, and therefore more than one reference gene is needed to normalize RT-PCR results. In this study, we assessed the expression of eight candidate internal control genes, namely 18S ribosomal RNA (18S rRNA), elongation factor-1alpha, ß-Actin, E2 ubiquitin-conjugating enzyme, ß-Tubulin (TUB), ACTIN2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Msc27 of unknown function, in a diverse set of 16 alfalfa (Medicago sativa) samples representing different tissues and abiotic stress challenges, using geNorm and BestKeeper software. The results revealed that the eight candidate genes are inconsistently expressed under different experimental conditions. Msc27 and 18S rRNA are suitable reference genes for comparing different tissue types. Under different abscisic acid and NaCl conditions, three reference genes are necessary. Finally, GAPDH, TUB and ß-Actin are unsuitable for normalization of qRT-PCR data under these given conditions in alfalfa. The relative expression level of MsWRKY33 was analyzed using selected reference genes. These results provide an experimental guideline for future research on gene expression in alfalfa using qRT-PCR.

Identification of Loci Associated with Drought Resistance Traits in Heterozygous Autotetraploid Alfalfa (Medicago sativa L.) Using Genome-Wide Association Studies with Genotyping by Sequencing.

Drought resistance is an important breeding target for enhancing alfalfa productivity in arid and semi-arid regions. Identification of genes involved in drought tolerance will facilitate breeding for improving drought resistance and water use efficiency in alfalfa. Our objective was to use a diversity panel of alfalfa accessions comprised of 198 cultivars and landraces to identify genes involved in drought tolerance. The panel was selected from the USDA-ARS National Plant Germplasm System alfalfa collection and genotyped using genotyping by sequencing. A greenhouse procedure was used for phenotyping two important traits associated with drought tolerance: drought resistance index (DRI) and relative leaf water content (RWC). Marker-trait association identified nineteen and fifteen loci associated with DRI and RWC, respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. Markers associated with DRI are located on all chromosomes while markers associated with RWC are located on chromosomes 1, 2, 3, 4, 5, 6 and 7. Co-localizations of significant markers between DRI and RWC were found on chromosomes 3, 5 and 7. Most loci associated with DRI in this work overlap with the reported QTLs associated with biomass under drought in alfalfa. Additional significant markers were targeted to several contigs with unknown chromosomal locations. BLAST search using their flanking sequences revealed homology to several annotated genes with functions in stress tolerance. With further validation, these markers may be used for marker-assisted breeding new alfalfa varieties with drought resistance and enhanced water use efficiency.

Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing.

Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids.

Screening for salt-responsive proteins in two contrasting alfalfa cultivars using a comparative proteome approach.

A comparative proteomic approach was carried out between two contrasting alfalfa cultivars, nonomu (NM-801; salt tolerant) and vernal (VN; salt intolerant) in terms of salt tolerance. Seedlings were subjected to salt stress (50 and 100 mM NaCl) for three days. Several physiological parameters (leaf water, chlorophyll, root Na(+), K(+), and Ca(2+)) and root proteome profile were analyzed. Comparison of physiological status revealed that NM-801 is more tolerant to salt than VN. Eighty three differentially expressed proteins were found on 2-DE maps, of which 50 were identified by MALDI-TOF or MALDI-TOF/TOF mass spectrometry. These proteins were involved in ion homeostasis, protein turnover and signaling, protein folding, cell wall components, carbohydrate and energy metabolism, reactive oxygen species regulation and detoxification, and purine and fatty acid metabolism. The comparative proteome analysis showed that 33 salt-responsive proteins were significantly changed in both cultivars, while 17 (14 in VN and 3 in NM-801) were cultivar-specific. Peroxidase, protein disulfide-isomerase, NAD synthetase, and isoflavone reductase were up-regulated significantly only in NM-801 in all salt concentrations. In addition, we identified novel proteins including NAD synthetase and biotin carboxylase-3 that were not reported previously as salt-responsive. Taken together, these results provide new insights of salt stress tolerance in alfalfa.

Genomic characterization of the LEED..PEEDs, a gene family unique to the medicago lineage.

The LEED..PEED (LP) gene family in Medicago truncatula (A17) is composed of 13 genes coding small putatively secreted peptides with one to two conserved domains of negatively charged residues. This family is not present in the genomes of Glycine max, Lotus japonicus, or the IRLC species Cicer arietinum. LP genes were also not detected in a Trifolium pratense draft genome or Pisum sativum nodule transcriptome, which were sequenced de novo in this study, suggesting that the LP gene family arose within the past 25 million years. M. truncatula accession HM056 has 13 LP genes with high similarity to those in A17, whereas M. truncatula ssp. tricycla (R108) and M. sativa have 11 and 10 LP gene copies, respectively. In M. truncatula A17, 12 LP genes are located on chromosome 7 within a 93-kb window, whereas one LP gene copy is located on chromosome 4. A phylogenetic analysis of the gene family is consistent with most gene duplications occurring prior to Medicago speciation events, mainly through local tandem duplications and one distant duplication across chromosomes. Synteny comparisons between R108 and A17 confirm that gene order is conserved between the two subspecies, although a further duplication occurred solely in A17. In M. truncatula A17, all 13 LPs are exclusively transcribed in nodules and absent from other plant tissues, including roots, leaves, flowers, seeds, seed shells, and pods. The recent expansion of LP genes in Medicago spp. and their timing and location of expression suggest a novel function in nodulation, possibly as an aftermath of the evolution of bacteroid terminal differentiation or potentially associated with rhizobial-host specificity.

In silico identification of transcription factors in Medicago sativa using available transcriptomic resources.

Transcription factors (TFs) are proteins that govern organismal development and response to the environment by regulating gene expression. Information on the amount and diversity of TFs within individual plant species is critical for understanding of their biological roles and evolutionary history across the plant kingdom. Currently, only scattered information on separate TFs is available for alfalfa, the most extensively cultivated forage legume in the world. In the meantime, several large transcriptomic resources that can be used to identify and characterize alfalfa TF genes are freely accessible online. In this study, we have performed an in silico analysis of transcriptome data generated in our laboratory and publicly acquirable from other sources to reveal and systematize alfalfa transcription factors. Transcriptome-wide mining enabled prediction of 983 TFs along with their sequence features and putative phylogenies of the largest families. All data were assembled into a simple open-access database named AlfalfaTFDB ( http://plantpathology.ba.ars.usda.gov/alfalfatfdb.html ). Transcriptomic analysis used in this work represents an effective approach for the identification of TF genes in plants with incomplete genomes, such as alfalfa. Integrated TF repertoires of Medicago sativa will provide an important tool for studying regulation of gene expression in other complex non-model species of agricultural significance.

Prevalence of single nucleotide polymorphism among 27 diverse alfalfa genotypes as assessed by transcriptome sequencing.

BACKGROUND: Alfalfa, a perennial, outcrossing species, is a widely planted forage legume producing highly nutritious biomass. Currently, improvement of cultivated alfalfa mainly relies on recurrent phenotypic selection. Marker assisted breeding strategies can enhance alfalfa improvement efforts, particularly if many genome-wide markers are available. Transcriptome sequencing enables efficient high-throughput discovery of single nucleotide polymorphism (SNP) markers for a complex polyploid species. RESULT: The transcriptomes of 27 alfalfa genotypes, including elite breeding genotypes, parents of mapping populations, and unimproved wild genotypes, were sequenced using an Illumina Genome Analyzer IIx. De novo assembly of quality-filtered 72-bp reads generated 25,183 contigs with a total length of 26.8 Mbp and an average length of 1,065 bp, with an average read depth of 55.9-fold for each genotype. Overall, 21,954 (87.2%) of the 25,183 contigs represented 14,878 unique protein accessions. Gene ontology (GO) analysis suggested that a broad diversity of genes was represented in the resulting sequences. The realignment of individual reads to the contigs enabled the detection of 872,384 SNPs and 31,760 InDels. High resolution melting (HRM) analysis was used to validate 91% of 192 putative SNPs identified by sequencing. Both allelic variants at about 95% of SNP sites identified among five wild, unimproved genotypes are still present in cultivated alfalfa, and all four US breeding programs also contain a high proportion of these SNPs. Thus, little evidence exists among this dataset for loss of significant DNA sequence diversity from either domestication or breeding of alfalfa. Structure analysis indicated that individuals from the subspecies falcata, the diploid subspecies caerulea, and the tetraploid subspecies sativa (cultivated tetraploid alfalfa) were clearly separated. CONCLUSION: We used transcriptome sequencing to discover large numbers of SNPs segregating in elite breeding populations of alfalfa. Little loss of SNP diversity was evident between unimproved and elite alfalfa germplasm. The EST and SNP markers generated from this study are publicly available at the Legume Information System ( http://medsa.comparative-legumes.org/) and can contribute to future alfalfa research and breeding applications.

Technical improvement of the TBP (tubulin-based polymorphism) method for plant species detection, based on capillary electrophoresis.

Nowadays, feed and food safety and traceability are of primary importance. Hence, a correct labeling of the different products is highly desirable in general, but mandatory for those people who are suffering from eating disorders and food allergies. Among the technologies that have been developed for feed and food analysis, the patented tubulin-based polymorphism (TBP) method emerges as an easy, versatile, and inexpensive diagnostic tool. Initially used to fingerprint different plant species and varieties, TBP was then successfully applied to trace species in mixtures of plant origin such as commercial feeds. TBP is a DNA-based molecular marker, that makes use of PCR for the selective amplification of plant ß-tubulin introns. Amplified fragments are then separated by PAGE and visualized by silver staining. We have now developed an improved version of TBP. Based on capillary electrophoresis and fluorescence detection, it makes the method automatic, more sensible, reproducible, and faster. Compared to the classic TBP, this new version allows to obtain a better data resolution and an easier interpretation of the results, clearing the way to large-scale feed/food diagnostics.

[Molecular identification of astragali radix and its adulterants by ITS sequences].

OBJECTIVE: To explore a new method for identification Astragali Radix from its adulterants by using ITS sequence. METHOD: Thirteen samples of the different Astragali Radix materials and 6 samples of the adulterants of the roots of Hedysarum polybotrys, Medicago sativa and Althaea rosea were collected. ITS sequence was amplified by PCR and sequenced unidirectionally. The interspecific K-2-P distances of Astragali Radix and its adulterants were calculated, and NJ tree and UPGMA tree were constructed by MEGA 4. RESULT: ITS sequences were obtained from 19 samples respectively, there were Astragali Radix 646-650 bp, H. polybotrys 664 bp, Medicago sativa 659 bp, Althaea rosea 728 bp, which were registered in the GenBank. Phylogeny trees reconstruction using NJ and UPGMA analysis based on ITS nucleotide sequences can effectively distinguish Astragali Radix from adulterants. CONCLUSION: ITS sequence can be used to identify Astragali Radix from its adulterants successfully and is an efficient molecular marker for authentication of Astragali Radix and its adulterants.

Estimation of the genetic diversity in tetraploid alfalfa populations based on RAPD markers for breeding purposes.

Alfalfa is an autotetraploid, allogamous and heterozygous forage legume, whose varieties are synthetic populations. Due to the complex nature of the species, information about genetic diversity of germplasm used in any alfalfa breeding program is most beneficial. The genetic diversity of five alfalfa varieties, involved in progeny tests at Institute of Field and Vegetable Crops, was characterized based on RAPD markers. A total of 60 primers were screened, out of which 17 were selected for the analysis of genetic diversity. A total of 156 polymorphic bands were generated, with 10.6 bands per primer. Number and percentage of polymorphic loci, effective number of alleles, expected heterozygosity and Shannon's information index were used to estimate genetic variation. Variety Zuzana had the highest values for all tested parameters, exhibiting the highest level of variation, whereas variety RSI 20 exhibited the lowest. Analysis of molecular variance (AMOVA) showed that 88.39% of the total genetic variation was attributed to intra-varietal variance. The cluster analysis for individual samples and varieties revealed differences in their population structures: variety Zuzana showed a very high level of genetic variation, Banat and Ghareh were divided in subpopulations, while Pecy and RSI 20 were relatively uniform. Ways of exploiting the investigated germplasm in the breeding programs are suggested in this paper, depending on their population structure and diversity. The RAPD analysis shows potential to be applied in analysis of parental populations in semi-hybrid alfalfa breeding program in both, development of new homogenous germplasm, and identification of promising, complementary germplasm.