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.     

Mapping Quantitative Trait Loci Associated with Toot Traits Using Sequencing-Based Genotyping Chromosome Segment Substitution Lines Derived from 9311 and Nipponbare in Rice (Oryza sativa L.)

Identification of quantitative trait loci (QTLs) associated with rice root morphology provides useful information for avoiding drought stress and maintaining yield production under the irrigation condition. In this study, a set of chromosome segment substitution lines derived from 9311 as the recipient and Nipponbare as donor, were used to analysis root morphology. By combining the resequencing-based bin-map with a multiple linear regression analysis, QTL identification was conducted on root number (RN), total root length (TRL), root dry weight (RDW), maximum root length (MRL), root thickness (RTH), total absorption area (TAA) and root vitality (RV), using the CSSL population grown under hydroponic conditions. A total of thirty-eight QTLs were identified: six for TRL, six for RDW, eight for the MRL, four for RTH, seven for RN, two for TAA, and five for RV. Phenotypic effect variance explained by these QTLs ranged from 2.23% to 37.08%, and four single QTLs had more than 10% phenotypic explanations on three root traits. We also detected the correlations between grain yield (GY) and root traits, and found that TRL, RTH and MRL had significantly positive correlations with GY. However, TRL, RDW and MRL had significantly positive correlations with biomass yield (BY). Several QTLs identified in our population were co-localized with some loci for grain yield or biomass. This information may be immediately exploited for improving rice water and fertilizer use efficiency for molecular breeding of root system architectures.     

A Comparison of Screening Techniques for Resistance to Verticillium Wilt in Alfalfa (Medicago sativa L.)

Three screening tests for resistance to Verticillium wilt in alfalfa using stem cuttings were compared including: stem infusion with a fungal culture filtrate (SIF), leaf injection with a spore inoculum (LII) and stem infusion with a spore inoculum (SII). The disease severity indice (DSI) determined by the three tests were very similar on average and were significantly correlated for a population of 142 plants regenerated from tissue culture. The DSIs determined by the LII test were also significantly correlated with the DSIs determined by the North American Standard test in a population of 20 Vertus (c.v.) plants. The results suggest that the assays using stem cuttings are effective for determining resistance to Verticillium wilt in alfalfa. Because they are nondestructive and quicker than the North American Standard test, these methods should find application in alfalfa breeding.     

大孔吸附树脂对苜蓿皂苷的吸附和解吸附作用(英文)

采用5种不同极性的树脂(AB-8、S-8、NKA-9、D-101和X-5)来评价对苜蓿皂苷的吸附和解吸附作用,其中中等极性的AB-8树脂对苜蓿皂苷具有最大的吸附量,用55%~65%的乙醇溶液能有效地将吸附的皂苷洗脱下来。当苜蓿粗提物量和AB-8树脂量为1∶1时,树脂的吸附量达到饱和。采用AB-8树脂,用90%乙醇洗脱,苜蓿提取物的最大解吸附量为108.4 mg/g干重树脂。通过大孔树脂吸附和解吸附,将90%乙醇洗脱液浓缩,皂苷含量(53%)是苜蓿粗提物含量(5.68%)的9倍。结果表明,AB-8大孔吸附树脂可用于苜蓿皂苷的大规模制备。     

根癌农杆菌介导的苜蓿体胚转化

以苜蓿体细胞胚胎作为根癌农杆菌介导转化的受体,通过对GUS基因瞬时表达率的分析,研究该转化体系的最佳实验参数。实验结果显示,负压处理10min和共培养5d时表达率最高(可达17．4％)。以这一转化方法分别对带有3种不同启动于的表达载体进行比较,发现由CMV35S启动于驱动的GUS基因的瞬时表达率可达82．7％,Ubil启动于驱动的可达57．8％,而Actl启动于驱动的则未见表达。     

Pathways of Nitrogen Metabolism in Nodules of Alfalfa (Medicago sativa L.)

Exposure of intact alfalfa nodules to ¹⁵N₂ showed that in bacteroids the greatest flow of ¹⁵N was to NH₃. Label was also detected in glutamic acid, aspartic acid, and asparagine (Glu, Asp and Asn), but at far lower levels. In the host plant cytosols, more ¹⁵N was incorporated into Asn than into other compounds. Detached nodules were also used to study the metabolic pathway of N assimilation after exposure to ¹⁵N₂ or vacuum infiltration with (¹⁵NH₄)₂SO₄ in the presence or absence of different inhibitors of nitrogen assimilation: methionine sulfoximine (MSO), azaserine (AZA), or amino-oxyacetate (AOA). Treatment with MSO, an inhibitor of glutamine synthetase (GS), inhibited the flow of the label to glutamine (Gln)-amide, resulting in subsequently decreased label in Asnamide. Aza, which inhibits the formation of Glu from Gln by glutamate synthase (GOGAT), enhanced the labeling of the amide groups of both Gln and Asn, while that of Asn-amino decreased. When AOA was used to block the transamination reaction very little label was found in Asp and Asn-amino. The results are consistent with the role of GS/GOGAT in the cytosol for the assimilation of NH₃ produced by N₂ fixation in the bacteroids of alfalfa nodules. Asn, a major nitrogen transport compound in alfalfa, is mainly synthesized by a Gln-dependent amidation of Asp, according to feeding experiments using the ¹⁵N-labeled amide group of glutamine. Data from ¹⁵NH₄⁺ feeding support some direct amidation of Asp to form Asn.     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.     

Isolation and Identification of Endogenous Cytokinins from Alfalfa (Medicago sativa L)

Endogenous cytokinins in alfalfa were isolated, and identified by mass spectrometry, trans- Ribosylzeatin (RZ) and ribosyldihydrozeatin (DHRZ) were identified from the root, and the combined content (benzyladenine equivalent) was estimated to be approximately 0.5/μg/100 g of fresh weight, eis- and trans-KL were identified from the stems and leaves. The relative content of the m-isomer was approximately five times greater than that of the trans-isomer.     

Identification of Promising Mutants Associated with Egg Production Traits Revealed by Genome-Wide Association Study

Egg number (EN), egg laying rate (LR) and age at first egg (AFE) are important production traits related to egg production in poultry industry. To better understand the knowledge of genetic architecture of dynamic EN during the whole laying cycle and provide the precise positions of associated variants for EN, LR and AFE, laying records from 21 to 72 weeks of age were collected individually for 1,534 F₂ hens produced by reciprocal crosses between White Leghorn and Dongxiang Blue-shelled chicken, and their genotypes were assayed by chicken 600 K Affymetrix high density genotyping arrays. Subsequently, pedigree and SNP-based genetic parameters were estimated and a genome-wide association study (GWAS) was conducted on EN, LR and AFE. The heritability estimates were similar between pedigree and SNP-based estimates varying from 0.17 to 0.36. In the GWA analysis, we identified nine genome-wide significant loci associated with EN of the laying periods from 21 to 26 weeks, 27 to 36 weeks and 37 to 72 weeks. Analysis of GTF2A1 and CLSPN suggested that they influenced the function of ovary and uterus, and may be considered as relevant candidates. The identified SNP rs314448799 for accumulative EN from 21 to 40 weeks on chromosome 5 created phenotypic differences of 6.86 eggs between two homozygous genotypes, which could be potentially applied to the molecular breeding for EN selection. Moreover, our finding showed that LR was a moderate polygenic trait. The suggestive significant region on chromosome 16 for AFE suggested the relationship between sex maturity and immune in the current population. The present study comprehensively evaluates the role of genetic variants in the development of egg laying. The findings will be helpful to investigation of causative genes function and future marker-assisted selection and genomic selection in chickens.     

Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection

In order to quickly and efficiently evaluate the salt tolerance of alfalfa, salinity tests were conducted on Medicago sativa L. var. australis, var. icon, var. loi, and var. gea, under in vitro conditions. Pregerminated seeds of four varieties were subjected to five different NaCl concentrations (0, 50, 100, 150, 200 mM). The influence of saline stress was estimated on the basis of survival percentage, growth parameters, and electrolyte leakage. The seedlings surviving on the medium enriched with salt at the highest concentration were presumed to be tolerant and represented the mother plants for the production of in vitro clones. In the following step, the clones were evaluated in vitro to confirm the salt tolerance. The influence of mild salt stress (75 mM NaCl) on the growth parameters of selected clones was examined. At the end of this trial, the proline accumulation and sodium content in alfalfa shoots were also quantified. The results suggest an increased level of proline promotes salt tolerance. Medicago sativa L. var. icon is highly tolerant in comparison with the other varieties tested. In vitro selection of M. sativa L. varieties on salt-containing media allowed us to obtain clones with increased salinity tolerance.     

Occurrence of Transgenic Feral Alfalfa (Medicago sativa subsp. sativa L.) in Alfalfa Seed Production Areas in the United States

The potential environmental risks of transgene exposure are not clear for alfalfa (Medicago sativa subsp. sativa), a perennial crop that is cross-pollinated by insects. We gathered data on feral alfalfa in major alfalfa seed-production areas in the western United States to (1) evaluate evidence that feral transgenic plants spread transgenes and (2) determine environmental and agricultural production factors influencing the location of feral alfalfa, especially transgenic plants. Road verges in Fresno, California; Canyon, Idaho; and Walla Walla, Washington were surveyed in 2011 and 2012 for feral plants, and samples were tested for the CP4 EPSPS protein that conveys resistance to glyphosate. Of 4580 sites surveyed, feral plants were observed at 404 sites. Twenty-seven percent of these sites had transgenic plants. The frequency of sites having transgenic feral plants varied among our study areas. Transgenic plants were found in 32.7%, 21.4.7% and 8.3% of feral plant sites in Fresno, Canyon and Walla Walla, respectively. Spatial analysis suggested that feral populations started independently and tended to cluster in seed and hay production areas, places where seed tended to drop. Significant but low spatial auto correlation suggested that in some instances, plants colonized nearby locations. Neighboring feral plants were frequently within pollinator foraging range; however, further research is needed to confirm transgene flow. Locations of feral plant clusters were not well predicted by environmental and production variables. However, the likelihood of seed spillage during production and transport had predictive value in explaining the occurrence of transgenic feral populations. Our study confirms that genetically engineered alfalfa has dispersed into the environment, and suggests that minimizing seed spillage and eradicating feral alfalfa along road sides would be effective strategies to minimize transgene dispersal.     

Identification of Genetic Loci Associated with Quality Traits in Almond via Association Mapping

To design an appropriate association study, we need to understand population structure and the structure of linkage disequilibrium within and among populations as well as in different regions of the genome in an organism. In this study, we have used a total of 98 almond accessions, from five continents located and maintained at the Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA; Spain), and 40 microsatellite markers. Population structure analysis performed in ‘Structure’ grouped the accessions into two principal groups; the Mediterranean (Western-Europe) and the non-Mediterranean, with K = 3, being the best fit for our data. There was a strong subpopulation structure with linkage disequilibrium decaying with increasing genetic distance resulting in lower levels of linkage disequilibrium between more distant markers. A significant impact of population structure on linkage disequilibrium in the almond cultivar groups was observed. The mean r² value for all intra-chromosomal loci pairs was 0.040, whereas, the r² for the inter-chromosomal loci pairs was 0.036. For analysis of association between the markers and phenotypic traits, five models comprising both general linear models and mixed linear models were selected to test the marker trait associations. The mixed linear model (MLM) approach using co-ancestry values from population structure and kinship estimates (K model) as covariates identified a maximum of 16 significant associations for chemical traits and 12 for physical traits. This study reports for the first time the use of association mapping for determining marker-locus trait associations in a world-wide almond germplasm collection. It is likely that association mapping will have the most immediate and largest impact on the tier of crops such as almond with the greatest economic value.     

Six Novel Loci Associated with Circulating VEGF Levels Identified by a Meta-analysis of Genome-Wide Association Studies

Vascular endothelial growth factor (VEGF) is an angiogenic and neurotrophic factor, secreted by endothelial cells, known to impact various physiological and disease processes from cancer to cardiovascular disease and to be pharmacologically modifiable. We sought to identify novel loci associated with circulating VEGF levels through a genome-wide association meta-analysis combining data from European-ancestry individuals and using a dense variant map from 1000 genomes imputation panel. Six discovery cohorts including 13,312 samples were analyzed, followed by in-silico and de-novo replication studies including an additional 2,800 individuals. A total of 10 genome-wide significant variants were identified at 7 loci. Four were novel loci (5q14.3, 10q21.3, 16q24.2 and 18q22.3) and the leading variants at these loci were rs114694170 (MEF2C, P = 6.79x10^-13), rs74506613 (JMJD1C, P = 1.17x10^-19), rs4782371 (ZFPM1, P = 1.59x10^-9) and rs2639990 (ZADH2, P = 1.72x10^-8), respectively. We also identified two new independent variants (rs34528081, VEGFA, P = 1.52x10^-18; rs7043199, VLDLR-AS1, P = 5.12x10^-14) at the 3 previously identified loci and strengthened the evidence for the four previously identified SNPs (rs6921438, LOC100132354, P = 7.39x10^-1467; rs1740073, C6orf223, P = 2.34x10^-17; rs6993770, ZFPM2, P = 2.44x10^-60; rs2375981, KCNV2, P = 1.48x10^-100). These variants collectively explained up to 52% of the VEGF phenotypic variance. We explored biological links between genes in the associated loci using Ingenuity Pathway Analysis that emphasized their roles in embryonic development and function. Gene set enrichment analysis identified the ERK5 pathway as enriched in genes containing VEGF associated variants. eQTL analysis showed, in three of the identified regions, variants acting as both cis and trans eQTLs for multiple genes. Most of these genes, as well as some of those in the associated loci, were involved in platelet biogenesis and functionality, suggesting the importance of this process in regulation of VEGF levels. This work also provided new insights into the involvement of genes implicated in various angiogenesis related pathologies in determining circulating VEGF levels. The understanding of the molecular mechanisms by which the identified genes affect circulating VEGF levels could be important in the development of novel VEGF-related therapies for such diseases.     

Photoperiodic Effects on the Emanation of Volatiles from Alfalfa (Medicago sativa L.) Florets

Alfalfa (Medicago sativa L.) plants acclimated to photoperiods of 18 hours light, 6 hour dark in plant growth chambers exhibited a daily cyclic pattern of floret volatile emanation with a maximum emanation of about 6.5 nanograms of hydrocarbons/floret·30 minutes. This maximum was reached about 6 to 8 hours into the light period. After 8 hours of light, emanation of volatiles decreased rapidly to less than 0.1 ng/floret·30 min even though light and temperature remained constant. Under continuous illumination, only a small increase of volatile emanation occurred during the following 24 hours. It appeared that a dark period was necessary to promote floret volatile emanation. Floret volatile emanation was drastically affected for at least 7 days following a photoperiod change. A photoperiod change caused 6-fold concentration oscillations every 2 hours. The results are interpreted on the basis of a very active floral metabolism controlled by photoperiodically induced rhythms.     

Characterization of a Small Sulphur-Rich Storage Albumin in Seeds of Alfalfa (Medicago sativa L.)

A 2S albumin fraction was characterized in seeds of alfalfa{^{Medicago sativa} L.). This low molecular weight (LMW) familyof disulphide-bonded proteins represents a major nitrogen andsulphur storage reserve for the alfalfa seed Characteristicof seed storage proteins, the 2S albumins are abundant in nitrogen-richglutarrune/glutamate/asparagine/aspartate (32%) In addition,this LMW fraction is high in cysteine (9%) and methionine (4%),amino acids which are under-represented in legume seed globulins.These 2S proteins start to accumulate during the early cotyledonstage of development, and are mobilized following germinationPulse-chase labelling experiments show that the 2S proteinsare synthesized as 'preproproteins', similar to 2S proteinsin other seeds. However, alfalfa 2S albumins are immunologicallyunrelated to these proteins. Key words: Seed development, sulphur-containing 2S storage protein, alfalfa (Medicago sativa)