Alfalfa Breeding Programs Research Articles

Selección de cepas bacterianas con capacidad antifúngica contra fitopatógenos de alfalfa para constituir un consorcio bacteriano

Alfalfa breeding programs require environmentally friendly alternatives to improve plant growth and health. Plant Growth Promoting Rhizobacteria (PGPR) and endophytic bacteria offer a biological solution as they can inhibitphytopathogenic fungi by producing antifungal compounds or enzymes. In Argentina, alfalfa is primarily affected by Colletotrichum trifolii (Ct) and Phytophthora megasperma (Pm). The objectives of this study were to select bacterial isolates with antifungal inhibition against Ct and Pm and identify a biocontroller isolate compatible with the rhizobia INTA5 has and with low-N2O emissions, to constitute an eco-friendly bacterial consortium. Ten isolates demonstrated antifungal inhibition, with mean Inhibition Percentage (IP) values between 41.74-91.28 for Ct and 56.23-77.59 for Pm compared to the control. Among these isolates, B2, B4, SC6, and MN1 showed the highest inhibition performance and were selected to evaluate their compatibility with INTA5. Only B4 exhibited compatibility, which was further evaluated against Ct in an experiment with alfalfa seedlings under controlled conditions. The alfalfaseedlings treated with B4 increased the plant survival number against Ct. The availability of a bacterial consortium that promotes biocontrol and biological nitrogen fixation represents the preferred strategy for alfalfa breeding programs.

Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality.

Alfalfa (Medicago sativa L.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

Genetic Mapping of Tolerance to Bacterial Stem Blight Caused by Pseudomonas syringae pv. syringae in Alfalfa (Medicago sativa L.).

The bacterial stem blight of alfalfa (Medicago sativa L.), first reported in the United States in 1904, has emerged recently as a serious disease problem in the western states. The causal agent, Pseudomonas syringae pv. syringae, promotes frost damage and disease that can reduce first harvest yields by 50%. Resistant cultivars and an understanding of host-pathogen interactions are lacking in this pathosystem. With the goal of identifying DNA markers associated with disease resistance, we developed biparental F1 mapping populations using plants from the cultivar ZG9830. Leaflets of plants in the mapping populations were inoculated with a bacterial suspension using a needleless syringe and scored for disease symptoms. Bacterial populations were measured by culture plating and using a quantitative PCR assay. Surprisingly, leaflets with few to no symptoms had bacterial loads similar to leaflets with severe disease symptoms, indicating that plants without symptoms were tolerant to the bacterium. Genotyping-by-sequencing identified 11 significant SNP markers associated with the tolerance phenotype. This is the first study to identify DNA markers associated with tolerance to P. syringae. These results provide insight into host responses and provide markers that can be used in alfalfa breeding programs to develop improved cultivars to manage the bacterial stem blight of alfalfa.

GENETIC DIVERSITY OF THE BC2 POPULATION OF ALFALFA MULTIFOLIATE LEAVES BASED ON MORPHOLOGICAL TRAITS USING CORRELATION, PRINCIPAL COMPONENT, AND CLUSTERING ANALYSIS

The current research aimed to assess the 179 BC2 population of alfalfa (Medicago sativa L. cv. Huaiyin) and PL34HQ multifoliate leaves and their parents to select genotypes based on morphological traits for future alfalfa breeding programs. A backcross breeding was carried out between the recurrent male parent and alfalfa PL34HQ as the non-recurrent female parent of alfalfa. In the pod setting stage, morphological traits were measured in the BC2 populations. Data were analyzed for descriptive statistics, correlation, principal component (PCA), and agglomerative hierarchical cluster analyses. The result of descriptive statistics revealed high variation in the means, minimum, and maximum values for most of the morphological traits. Therefore, high variability was observed in plant height (PH) 26-98 cm, number of branches per plant (NBP) 6-74, stem diameter (SD) 1.46-5.46 mm, second branch length SBL 0-62 cm, number of multifoliate leaves per plant (NMFLP) 0-95.7%, number of multifoliate leaves on the main branch (NMFLMB) 0-90.6%, number of nodes (NN) 12-24, fresh shoot weight (FSW) 3.10-359.4 g, and dry shoot weight (DSW) 0.40-108.6 g. The results showed that the comprehensive traits of the BC2 population tended to the recurrent parent, about 32.40% of the total population. However, many plants tended to the non-recurrent parent, about 67.40% of the total population, with a high multifoliate rate. Correlation analysis revealed a significant positive association of plant height with stem diameter, fresh shoot weight, dry shoot weight, second branch length, number of nodes, and branch number. Principal component analysis (PCA) discovered the first three components with an eigenvalue greater than one, contributing 78.98% of total variability for different traits. The PC1 showed favorable factor loading for all morphological traits. The contribution of dry shoot weight, fresh shoot weight, stem diameter, and plant height was highest in principal component 1. The average hierarchical cluster analysis method grouped all the genotypes into four main clusters. Alfalfa multifoliate leaf genotype clustering based on morphological traits would help identify potential genotypes for future breeding research and programs. Keywords: - Alfalfa multifoliate leaves, Principal component analysis, Correlation, Cluster analysis.

Selection of alfalfa genotypes for dry matter yield and persistence with repeated measures

The biggest challenge in the alfalfa breeding program is to obtain cultivars with high persistence, high productivity, and adaptability. Therefore, studies about selection methods are necessary for the success of alfalfa breeding programs. This study aimed to evaluate dry matter yield and persistence in alfalfa for selecting genotypes, using appropriate statistical models for experiments with repeated measures. The experiment was conducted at Embrapa Southeast Livestock, in São Carlos, state of São Paulo, Brazil in a randomized blocks design, in plots subdivided in time, with three replicates. Eight genotypes were evaluated, and the agronomic trait evaluated was dry matter yield. The experiments in split-plots were used with two and three errors and generalized linear models with the following correlation structures: composite symmetry (CS), heterogeneous composite symmetry (HCS), auto regressive (AR), heterogeneous auto regressive (HAR), and variance components (VC). The best model was selected according to the lowest value of the Akaike Information Criterion (AIC), and three methodologies were used to identify the genotype with greater productivity and persistence: Average test for multiple comparisons, adaptability, and stability by multi-information, and similarity between genotype and ideotype. The interaction between genotypes and cuts was significant, demonstrating the existence of the different behavior of the alfalfa genotypes over the cuts. Different methodologies allowed to measure the average yield of the alfalfa genotype and the persistence over the cuts. PSB 4 genotype demonstrated promissory behavior in terms of productivity and persistence throughout the production cycle of alfalfa.

Good Statistical Practices in Agronomy Using Categorical Data Analysis, with Alfalfa Examples Having Poisson and Binomial Underlying Distributions

Categorical data derived from qualitative classifications or countable quantitative data are common in biological scientific work and crop breeding. Categorical data analyses are important for drawing correct inferences from experiments. However, categorical data can introduce unique issues in data analysis. This paper discusses common problems arising from categorical variable analysis and modeling, demonstrates the issues or risks of misapplying analysis, and suggests approaches to address data analysis challenges using two data sets from alfalfa breeding programs. For each data set, we present several analysis methods, e.g., simple t-test, analysis of variance (ANOVA), split plot analysis, generalized linear model (glm), generalized linear mixed model (glmm) using R with R markdown, and with the standard statistical analysis software SAS/JMP. The goal is to demonstrate good analysis practices for categorical data by comparing the potential ‘bad’ analyses with better ones, avoiding too much reliance on reaching a significant p-value of 0.05, and navigating the morass of ever-increasing numbers of potential R functions. The three main aspects of this research focus on choosing the right data distribution to use, using the correct error terms for hypothesis test p-values including the right type of sum of the squares (Type I, II, and III), and proper statistical models for categorical data analysis. Our results show the importance of good statistical analysis practice to help agronomists, breeders, and other researchers apply appropriate statistical approaches to draw more accurate conclusions from their data.

A Review of Unreduced Gametes and Neopolyploids in Alfalfa: How to Fill the Gap between Well-Established Meiotic Mutants and Next-Generation Genomic Resources.

The gene flow mediated by unreduced gametes between diploid and tetraploid plants of the Medicago sativa-coerulea-falcata complex is pivotal for alfalfa breeding. Sexually tetraploidized hybrids could represent the best way to exploit progressive heterosis simultaneously derived from gene diversity, heterozygosity, and polyploidy. Moreover, unreduced gametes combined with parthenogenesis (i.e., apomixis) would enable the cloning of plants through seeds, providing a unique opportunity for the selection of superior genotypes with permanently fixed heterosis. This reproductive strategy has never been detected in the genus Medicago, but features of apomixis, such as restitutional apomeiosis and haploid parthenogenesis, have been reported. By means of an original case study, we demonstrated that sexually tetraploidized plants maintain apomeiosis, but this trait is developmentally independent from parthenogenesis. Alfalfa meiotic mutants producing unreduced egg cells revealed a null or very low capacity for parthenogenesis. The overall achievements reached so far are reviewed and discussed along with the efforts and strategies made for exploiting reproductive mutants that express apomictic elements in alfalfa breeding programs. Although several studies have investigated the cytological mechanisms responsible for 2n gamete formation and the inheritance of this trait, only a very small number of molecular markers and candidate genes putatively linked to unreduced gamete formation have been identified. Furthermore, this scenario has remained almost unchanged over the last two decades. Here, we propose a reverse genetics approach, by exploiting the genomic and transcriptomic resources available in alfalfa. Through a comparison with 9 proteins belonging to Arabidopsis thaliana known for their involvement in 2n gamete production, we identified 47 orthologous genes and evaluated their expression in several tissues, paving the way for novel candidate gene characterization studies. An overall view on strategies suitable to fill the gap between well-established meiotic mutants and next-generation genomic resources is presented and discussed.

Validation of UAV-based alfalfa biomass predictability using photogrammetry with fully automatic plot segmentation

Alfalfa is the most widely cultivated forage legume, with approximately 30 million hectares planted worldwide. Genetic improvements in alfalfa have been highly successful in developing cultivars with exceptional winter hardiness and disease resistance traits. However, genetic improvements have been limited for complex economically important traits such as biomass. One of the major bottlenecks is the labor-intensive phenotyping burden for biomass selection. In this study, we employed two alfalfa fields to pave a path to overcome the challenge by using UAV images with fully automatic field plot segmentation for high-throughput phenotyping. The first field was used to develop the prediction model and the second field to validate the predictions. The first and second fields had 808 and 1025 plots, respectively. The first field had three harvests with biomass measured in May, July, and September of 2019. The second had one harvest with biomass measured in September of 2019. These two fields were imaged one day before harvesting with a DJI Phantom 4 pro UAV carrying an additional Sentera multispectral camera. Alfalfa plot images were extracted by GRID software to quantify vegetative area based on the Normalized Difference Vegetation Index. The prediction model developed from the first field explained 50–70% (R Square) of biomass variation in the second field by incorporating four features from UAV images: vegetative area, plant height, Normalized Green–Red Difference Index, and Normalized Difference Red Edge Index. This result suggests that UAV-based, high-throughput phenotyping could be used to improve the efficiency of the biomass selection process in alfalfa breeding programs.

Resistance to the alfalfa weevil in the Iranian collection of <i>Medicago sativa</i>

The Iranian collection of Medicago sativa (alfalfa, 502 accessions) was evaluated under field conditions to identify variation in resistance to the alfalfa weevil (Hypera postica Gyllenhal) between accessions over 4 years. Twenty-one accessions were identified as resistant (score 3) to semi-resistant (score 5) based on K-means cluster analysis. These materials were grouped into 10 groups by means of Duncan's Multiple Range Test. Susceptibility to alfalfa weevil was significantly correlated with some of the agro-morphological traits such as plant height (r = 0.314, P<0.01), regrowth rate (r = 0.435, P<0.01) and fall dormancy score (r = 0.284, P<0.01). In general, the resistant germplasm originated from elevated regions at high latitudes, where a co-evolution between the plant and the pest may have taken place. Multiple regression analysis showed a positive linear relationship between susceptibility to the alfalfa weevil and traits such as growth habit, plant height, length and width of the central leaflet and regrowth rate. The resulting bi-plot based on factor analysis ranked accessions for susceptibility to the pest. The identified germplasm should prove useful in alfalfa breeding programs to develop accessions resistant to the alfalfa weevil.

Cellular and Transcriptional Responses of Resistant and Susceptible Cultivars of Alfalfa to the Root Lesion Nematode, Pratylenchus penetrans.

The root lesion nematode (RLN), Pratylenchus penetrans, is a migratory species that attacks a broad range of crops, including alfalfa. High levels of infection can reduce alfalfa forage yields and lead to decreased cold tolerance. Currently, there are no commercially certified varieties with RLN resistance. Little information on molecular interactions between alfalfa and P. penetrans, that would shed light on mechanisms of alfalfa resistance to RLN, is available. To advance our understanding of the host–pathogen interactions and to gain biological insights into the genetics and genomics of host resistance to RLN, we performed a comprehensive assessment of resistant and susceptible interactions of alfalfa with P. penetrans that included root penetration studies, ultrastructural observations, and global gene expression profiling of host plants and the nematode. Several gene-candidates associated with alfalfa resistance to P. penetrans and nematode parasitism genes encoding nematode effector proteins were identified for potential use in alfalfa breeding programs or development of new nematicides. We propose that preformed or constitutive defenses, such as significant accumulation of tannin-like deposits in root cells of the resistant cultivar, could be a key to nematode resistance, at least for the specific case of alfalfa-P. penetrans interaction.

67 2018 Alfalfa in the South Workshop Series: A review of regional Extension efforts

Abstract Alfalfa research in the Southeast has steadily increased over the past ten years. Researchers have a renewed interest in this perennial legume as new varieties have been released that are better suited to the challenging climate and use in the Southeast. Extensive effort has been put forth in establishing programs promoting the use of alfalfa as a viable high-quality forage option in the south. In 2018, the “Alfalfa in the South” workshop series was designed to provide in-depth extension education, circulate new research findings, and engage in discussions with producers as related to the use of alfalfa in the southeast. The series consisted of 4 one-day workshops, organized by University of Georgia Extension in conjunction with America’s Alfalfa, and included Extension specialists, faculty, and graduate students from both the University of Georgia and Auburn University. Topics were broken into five classroom learning modules: 1) site selection and establishment, 2) improving animal performance with alfalfa, 3) managing alfalfa for persistence, 4) alfalfa breeding programs, and 5) graduate student research updates. Each workshop concluded with an on-farm demonstration where attendees engaged in discussion with producers utilizing alfalfa in their operation. Postworkshop evaluation results determined that, on a scale of 1 to 5, where “1” was “no change” and “5” indicated “greatly improved,” producers rated their change in interest using alfalfa as a 4.73 and their confidence in using alfalfa management skills as 4.36. Additionally, the producer discussion and field tour section rated the highest, 4.67 (1 being “poor” and 5 being “excellent”); however, all topics rated 4.12 or higher, indicating that attendees found this workshop to be very good to excellent. Of the 60 participants, 68% indicated that they would make some major changes and 32% plan to try to make minor management changes following the workshops.

Genomic Prediction for 25 Agronomic and Quality Traits in Alfalfa (Medicago sativa).

Agronomic and quality traits in alfalfa are very important to forage industry. Genomic prediction (GP) based on genotyping-by-sequencing (GBS) data could shorten the breeding cycles and accelerate the genetic gains of these complex traits, if they display moderate to high prediction accuracies. The aim of this study was to investigate the predictive potentials of these traits in alfalfa. A total of 322 genotypes from 75 alfalfa accessions were used for GP of the agronomic and quality traits, which were related to yield and nutrition value, respectively, using BayesA, BayesB, and BayesCπ methods. Ten-fold cross validation was used to evaluate the accuracy of GP represented by the correlation between genomic estimated breeding value (GEBV) and estimated breeding value (EBV). The accuracies ranged from 0.0021 to 0.6485 for different traits. For each trait, three GP methods displayed similar prediction accuracies. Among 15 quality traits, mineral element Ca had a moderate and the highest prediction accuracy (0.34). NDF digestibility after 48 h (NDFD 48 h) and 30 h (NDFD 30 h) and mineral element Mg had prediction accuracies varying from 0.20 to 0.25. Other traits, for example, fat and crude protein, showed low prediction accuracies (0.05 to 0.19). Among 10 agronomic traits, however, some displayed relatively high prediction accuracies. Plant height (PH) in fall (FH) had the highest prediction accuracy (0.65), followed by flowering date (FD) and plant regrowth (PR) with accuracies at 0.52 and 0.51, respectively. Leaf to stem ratio (LS), plant branch (PB), and biomass yield (BY) reached to moderate prediction accuracies ranging from 0.25 to 0.32. Our results revealed that a few agronomic traits, such as FH, FD, and PR, had relatively high prediction accuracies, therefore it is feasible to apply genomic selection (GS) for these traits in alfalfa breeding programs. Because of the limitations of population size and density of SNP markers, several traits displayed low accuracies which could be improved by a bigger reference population, higher density of SNP markers, and more powerful statistic tools.

Direct, indirect and simultaneous selection as strategies for alfalfa breeding on forage yield and nutritive value

ABSTRACT Alfalfa breeding aimed at trait improvement for livestock feed takes longer periods of time, if compared to many other crops. Therefore, better selection methods are necessary for the success of alfalfa breeding programs. Although knowing about selection methods is quite important, there is a notable lack of information, as regards successful solutions. This study aimed to use direct, indirect and simultaneous selection methods for selecting alfalfa cultivars, based on yield traits and nutritive value. The evaluated traits were subdivided into two groups: forage yield and nutritive value. Selection gains were estimated by direct, indirect and simultaneous selection for each group, considering the selection of the 25 % best cultivars. Direct and indirect selections among genotype averages are not efficient to provide the desirable responses to the whole set of traits. The results for simultaneous selection, using the Tai index, provided a more balanced gain distribution to the set of traits in all cuts. The simultaneous selection allowed the identification of the 5681 and Verdor cultivars in the first cut, as well as ProINTA Patricia in the second cut, as superior in the two groups of evaluated traits.

Towards intra-population improvement of alfalfa yield and persistence

Alfalfa (Medicago sativa L.) is the most important forage crop in the world, regarding growing area and economic value, because of its high yield level, good persistence, high protein content and energy value. In alfalfa, concept of intra-population breeding means direct selection for yield, through recurrent phenotypic selection using some form of progeny testing. Significant differences between genotypes were found for green forage and dry matter yields. The observed variation for both yield traits was much higher at population level than for the selected plants that will form new improved population. In the next steps of breeding process selected plants will be intercrossed in isolation to develop a new, improved population. Alfalfa breeding programs should focus on yield per se, with the use of old and new knowledge on the genetic control of yield and with adaptation of existing or developing new breeding procedure.

RESTORATION OF LATVIAN ALFALFA (Medicago sativa) GENETIC RESOURCES PERSPECTIVE FOR BREEDING

We have carried out restoring of some Latvian alfalfa genetic resources from seeds of accessions that did not germinate in soil at all. Two of them were repatriated from the N. Vavilov All-Russian Institute of Plant Industry (multiplied more than 40 years ago) and three from the Research Institute of Agriculture (Latvia) (multiplied 15-20 years ago). For germination of old seeds we used early elaborated in vitro culture conditions. Germination rate ranged 2-60%, depending from the genotype and seeds storage conditions. Plantlets with well developed roots and 2-3 leaves were planted in the substrate in small pots and grown in a greenhouse about a month, then replanted in the soil in field conditions and grown till the maturity. Restored accessions are available now for evaluation and involving in the alfalfa breeding programs.

Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L.) using high-resolution melting.

Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs.

The development of 204 novel EST-SSRs and their use for genetic diversity analyses in cultivated alfalfa

Cultivated alfalfa (Medicago sativa L.) is one of the most important forage legume in the world. Here, we report the development of 204 novel polymorphic expressed sequence tag simple sequence repeat (EST-SSRs) markers from transcript sequences via global Illumina sequencing for cultivated alfalfa. Among the synthesized 750 pairs of primers, 204 EST-SSR primer pairs showed polymorphisms among 10 alfalfa accessions (including five individual plants in each accession) generating a total of 1222 alleles, with the number of alleles per locus, the observed heterozygosity (Ho), the corrected heterozygosity (He) and the Shannon-Wiener diversity index (H'c) averaging at 5.99, 0.73, 0.71 and 1.14, respectively. Of the 204 novel EST-SSRs in alfalfa, 120 can be in silico mapped onto the eight Medicago truncatula chromosomes (Mt3.5.2). Considering the high polymorphism, these EST-SSRs can be applied to assess genetic diversity, population structure, relatedness, evolution, linkage mapping and cultivar protection of cultivated alfalfa to facilitate alfalfa breeding programs.

First Report of Race 2 of Colletotrichum trifolii Causing Anthracnose on Alfalfa (Medicago sativa) in Wisconsin.

Anthracnose of alfalfa (Medicago sativa), caused by Colletotrichum trifolii, is widespread in the United States. In addition to loss of forage due to death of stems, the pathogen causes crown rot, reducing stand life and winter survival (2), making it one of the most serious diseases of alfalfa. Three physiological races have been described (2). Race 1 is reported to be the dominant race that is present wherever alfalfa is grown, while race 2 was reported in a limited area in the Mid-Atlantic states, and race 4 was found in Ohio (1). Conspicuous, straw-colored dead stems with a "shepherd's crook" wilt and large, sunken, diamond-shaped lesions with a dark border were observed in experimental plots and breeding nurseries of experimental lines in Clinton and West Salem, WI, in August 2011 and in West Salem, WI, in mid-August 2012. Acervuli with black setae and orange spore masses were observed in lesions placed in moist chambers for 2 days at room temperature with ambient room lighting. Conidia were germinated on 1% water agar and then single hyphae were transferred to potato dextrose agar (PDA) plates. DNA was extracted from pure cultures of strains DA-1 (Clinton, WI) and FGI-3 (West Salem, WI), the rDNA ITS1-5.8S-ITS2 region was amplified with primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), the products sequenced directly, and the sequences compared to the ITS region of known race 1 and race 2 strains of C. trifolii. The sequences from DA-1 and FGI-3 were identical to the ITS sequence of C. trifolii 2sp2 (race 1; KF444778) and C. trifolii SB-2 (race 2; KF444779), but distinct from the ITS sequence of C. destructivum (JQ005764) and C. dematium (JX567507), which can cause anthracnose on alfalfa (1). Conidia from DA-1 and FGI-3 were harvested from 7-day-old cultures grown on PDA plates, diluted to 2 × 106 conidia/ml, and sprayed to runoff on 10-day-old growth chamber grown plants of three differential cultivars: Saranac (susceptible to races 1 and 2), Arc (resistant to race 1, susceptible to race 2), and Saranac AR (resistant to races 1 and 2). Plants were maintained at 100% relative humidity for 48 h and then grown in a growth chamber at 24°C with a 16-h photoperiod. Symptoms were rated at 14 days after inoculation. In the three repetitions of the experiment using 75 plants of each cultivar in each experiment, less than 10% of the Saranac and Arc plants survived, while survival of Saranac AR was 31 to 44%. The approximate expected survival of differential cultivars inoculated with race 1 is 1% for Saranac, 65 to 70% for Arc, and 45% for Saranac AR (2). Aggressiveness of race 2 strains on Saranac AR is variable, ranging from 12 to 68% plant survival (3). The susceptibility of Arc when inoculated with DA-1 and FGI-3 is consistent with the reaction to race 2 strains, indicating that both strains are race 2. The isolation of race 2 strains in major alfalfa growing regions in Wisconsin indicates that this physiological race is currently more widespread than previously observed. Although most modern alfalfa cultivars have resistance to race 1, few cultivars with resistance to race 2 are available. The occurrence of C. trifolii race 2 in the Midwest United States should be considered in alfalfa breeding programs when developing multi-pest resistant alfalfa cultivars.

Analysis of the Alfalfa Root Transcriptome in Response to Salinity Stress

Salinity is one of the major abiotic factors affecting alfalfa productivity. Identifying genes that control this complex trait will provide critical insights for alfalfa breeding programs. To date, no studies have been published on a deep sequencing-based profiling of the alfalfa transcriptome in response to salinity stress. Observations gathered through research on reference genomes may not always be applicable to alfalfa. In this work, Illumina RNA-sequencing was performed in two alfalfa genotypes contrasting in salt tolerance, in order to estimate a broad spectrum of genes affected by salt stress. A total of 367,619,586 short reads were generated from cDNA libraries originated from roots of both lines. More than 60,000 tentative consensus sequences (TCs) were obtained and, among them, 74.5% had a significant similarity to proteins in the NCBI database. Mining of simple sequence repeats (SSRs) from all TCs revealed 6,496 SSRs belonging to 3,183 annotated unigenes. Bioinformatics analysis showed that the expression of 1,165 genes, including 86 transcription factors (TFs), was significantly altered under salt stress. About 40% of differentially expressed genes were assigned to known gene ontology (GO) categories using Arabidopsis GO. A random check of differentially expressed genes by quantitative real-time PCR confirmed the bioinformatic analysis of the RNA-seq data. A number of salt-responsive genes in both tested genotypes were identified and assigned to functional classes, and gene candidates with roles in the adaptation to salinity were proposed. Alfalfa-specific data on salt-responsive genes obtained in this work will be useful in understanding the molecular mechanisms of salinity tolerance in alfalfa.

10.5937/ratpov50-5059 = Heterosis in alfalfa breeding

The progress of alfalfa breeding has been slow, most notably due to its complex genetic structure (autotetraploidy) and tetrasomic inheritance. Alfalfa breeding programs are based on recurrent phenotypic selection with or without progeny testing. The genetic control of major agronomic traits is determined by both additive gene action (accumulation of frequency of desirable alleles represented by significant general combining ability (GCA) effects, and nonadditive gene action, complementary gene interactions represented by significant specific combining ability (SCA) effects. This type of gene action expression in alfalfa also determines the way in which breeding is carried out and brings about changes in the methods used. It has also given rise to the idea of the semi-hybrid breeding of this crop which involves breeding alfalfas within the population, identification of heterotic germplasm, and the seed production of semi or free hybrid seed. The studies of the relationship between the molecular variability of alfalfa populations and heterosis in their hybrids could contribute to a more precise selection of parental populations to be used for crossing in semi-hybrid alfalfa breeding procedure, aiming to reduce number of necessary crossings and therefore make future alfalfa breeding programs more efficient. Future tasks of alfalfa breeders should be to discover how to translate heterosis from single plants in hybrids planted in dense stand to generate 'yield for free' (capture heterosis) in alfalfa semi hybrids.