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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. [ABSTRACT FROM AUTHOR]

Plant mitochondria are crucial for species evolution, phylogenetics, classification, and identification as maternal genetic material. However, the presence of numerous repetitive sequences, complex structures, and a low number of genes in the mitochondrial genome has hindered its complete assembly and related research endeavors. In this study, we assembled two mitochondrial genomes of alfalfa varieties of Zhongmu No.1 (299,123 bp) and Zhongmu No.4 (306,983 bp), based on a combination of PacBio, Illumina, and Hi-C sequences. The comparison of genome assemblies revealed that the same number of mitochondrial genes, including thirty-three protein-coding genes, sixteen tRNA genes, and three rRNA genes existed in the two varieties. Additionally, large fragments of repetitive sequences were found underlying frequent mitochondrial recombination events. We observed extensive transfer of mitochondrial fragments into the nuclear genome of Zhongmu No.4. Analysis of the cox1 and rrn18s genes in 35 Medicago accessions revealed the presence of population-level deletions and substitutions in the rrn18s gene. We propose that mitochondrial structural reorganizations may contribute to alfalfa evolution. [ABSTRACT FROM AUTHOR]

Medicago is a genus of legumes (Fabaceae) that resemble common clovers with pinnately trifoliate leaves and spirally coiled seed pods, and Medicago sativa is a famous forage crop throughout the world. In this study, we systematically assembled the complete plastid genomes of 18 Medicago species, representing 35 Medicago accessions, whose genome size ranged from ~119 to 125 kb, and identified one novel inverted repeat (IR) in two accessions of Medicago soleirolii (PI537242 and PI537243), albeit of no IRs in the most accessions. We built a phylogenetic tree based on common protein‐coding sequences of 55 Medicago accessions in 38 species, which were placed into five clades with a divergence since 9.37 million years ago. Global alignment revealed independent genome evolution events, including eight inversions in nine species and four intron losses (ILs) in 10 species, among which four inversions and two ILs have not been reported previously. Within 109–111 unique genes, ndhA, rpl2, and ycf3 were under positive selection in 54 Medicago accessions. Finally, by aligning chloroplast genes against the nuclear genome assembly of M. sativa cultivar "Zhongmu No.1", we found that a large number of chloroplast gene fragments were horizontally transferred to nuclear chromosomes in alfalfa, especially on the chr3:47518422–48722257 coordinates of chromosome 3. Our comprehensive exploration of Medicago chloroplast genomes provided insights for the understanding of Medicago diversity and their genomic evolution events. [ABSTRACT FROM AUTHOR]

The ability of roots to penetrate compacted soils can be beneficial in avoiding drought stress. However, breeding for root penetration is hindered by the lack of an efficient, reliable screening technique. Our objectives in this study were to develop and evaluate the use of a wax-petrolatum layer system as a reference to measure genotypic variation in root penetration ability of 11 rice (Oryza sativa L.) cultivars from dryland and wetland hydrological origins. The wax-petrolatum layers consisted of 60% wax and 40% petrolatum white, with a penetration resistance strength of 1.4 MPa at 27 [degrees] C as measured with a soil penetrometer. The wax-petrolatum layers were placed in pots and served as a partial barrier to root growth. Rice cultivars were sown in a randomized complete block design with three replications and grown in a growth chamber. Root penetration ability was measured as the ratio of the number of roots that penetrated the wax-petrolatum layer to the total number of roots. Consistently, rice cultivars from dryland hydrological origins had greater root penetration ability than did cultivars from wetland origins. Root penetration ability ranged from 6 to 26%. Root thickness ranged from 0.9 to 1.5 mm and was positively correlated (r = 0.81, P < 0.001) with root penetration ability. Data indicated that the use of wax-petrolatum layers was an effective technique for measuring differences in root penetration ability.

Autotetraploid alfalfa is a major hay crop planted all over the world due to its adaptation in different environments and high quality for animal feed. However, the genetic basis of alfalfa quality is not fully understood. In this study, a diverse panel of 200 alfalfa accessions were planted in field trials using augmented experimental design at three locations in 2018 and 2019. Thirty-four quality traits were evaluated by Near Infrared Reflectance Spectroscopy (NIRS). The plants were genotyped using a genotyping by sequencing (GBS) approach and over 46,000 single nucleotide polymorphisms (SNPs) were obtained after variant calling and filtering. Genome-wide association studies (GWAS) identified 28 SNP markers associated with 16 quality traits. Among them, most of the markers were associated with fiber digestibility and protein content. Phenotypic variations were analyzed from three locations and different sets of markers were identified by GWAS when using phenotypic data from different locations, indicating that alfalfa quality traits were also affected by environmental factors. Among different sets of markers identified by location, two markers were associated with nine traits of fiber digestibility. One marker associated with lignin content was identified consistently in multiple environments. Putative candidate genes underlying fiber-related loci were identified and they are involved in the lignin and cell wall biosynthesis. The DNA markers and associated genes identified in this study will be useful for the genetic improvement of forage quality in alfalfa after the validation of the markers. [ABSTRACT FROM AUTHOR]

In this study, we found that one of the TIR-NBS-LRR genes, I MsVR39 i responded positively to VW in I M. sativa i , while negative effect was observed on the other gene I MsVR38 i . Keywords: VW; R-gene; TIR-NBS-LRR; mutant EN VW R-gene TIR-NBS-LRR mutant 619 621 3 04/11/22 20220401 NES 220401 Introduction Verticillium wilt (VW) is a soil-borne fungus disease in a wide range of plant species including alfalfa and caused yield loss up to 50% in less than 3 years due to the rapid spreading of the pathogen in soil (Vandemark I et al i ., 2006). [Extracted from the article]

Wheat stem rust (Puccinia graminis f. sp. tritici Eriks. and E. Henn.) is one of the most destructive diseases world-wide. Races belonging to Ug99 (or TTKSK) continue to cause crop losses in East Africa and threaten global wheat production. Developing and deploying wheat varieties with multiple race-specific genes or complex adult plant resistance is necessary to achieve durability. In the present study, we applied genome-wide association studies (GWAS) for identifying loci associated with the Ug99 stem rust resistance (SR) in a panel of wheat lines developed at the International Maize and Wheat Improvement Center (CIMMYT). Genotyping was carried out using the wheat 9K iSelect single nucleotide polymorphism (SNP) chip. Phenotyping was done in the field in Kenya by infection of Puccinia graminis f. sp. tritici race TTKST, the Sr24-virulent variant of Ug99. Marker-trait association identified 12 SNP markers significantly associated with resistance. Among them, 7 were mapped on five chromosomes. Markers located on chromosomes 4A and 4B overlapped with the location of the Ug99 resistance genes SrND643 and Sr37, respectively. Markers identified on 7DL were collocated with Sr25. Additional significant markers were located in the regions where no Sr gene has been reported. The chromosome location for five of the SNP markers was unknown. A BLASTN search of the NCBI database using the flanking sequences of the SNPs associated with Ug99 resistance revealed that several markers were linked to plant disease resistance analogues, while others were linked to regulatory factors or metabolic enzymes. A KASP (Kompetitive Allele Specific PCR) assay was used for validating six marker loci linked to genes with resistance to Ug99. Of those, four co-segregated with the Sr25-pathotypes while the rest identified unknown resistance genes. With further investigation, these markers can be used for marker-assisted selection in breeding for Ug99 stem rust resistance in wheat. [ABSTRACT FROM AUTHOR]

Verticillium wilt (VW) is a fungal disease that causes severe yield losses in alfalfa. The most effective method to control the disease is through the development and use of resistant varieties. The identification of marker loci linked to VW resistance can facilitate breeding for disease-resistant alfalfa. In the present investigation, we applied an integrated framework of genome-wide association with genotyping-by-sequencing (GBS) to identify VW resistance loci in a panel of elite alfalfa breeding lines. Phenotyping was performed by manual inoculation of the pathogen to healthy seedlings, and scoring for disease resistance was carried out according to the standard test of the North America Alfalfa Improvement Conference (NAAIC). Marker-trait association by linkage disequilibrium identified 10 single nucleotide polymorphism (SNP) markers significantly associated with VW resistance. Alignment of the SNP marker sequences to the M. truncatula genome revealed multiple quantitative trait loci (QTLs). Three, two, one and five markers were located on chromosomes 5, 6, 7 and 8, respectively. Resistance loci found on chromosomes 7 and 8 in the present study co-localized with the QTLs reported previously. A pairwise alignment ( blastn) using the flanking sequences of the resistance loci against the M. truncatula genome identified potential candidate genes with putative disease resistance function. With further investigation, these markers may be implemented into breeding programmes using marker-assisted selection, ultimately leading to improved VW resistance in alfalfa. [ABSTRACT FROM AUTHOR]

Maize kernel development is particularly sensitive to water stress at the early post-pollination phase. Endosperm plays an essential role in support of embryo development and represents the bulk of carbohydrate storage in the kernel. In the present study, we compared transcriptomes of developing maize endosperms between an ABA-insensitive mutant, viviparous1 ( vp1 ), and wild type (wt) under water deficit. A total of 122 and 164 transcripts were significantly affected by water deficit in vp1 and wt, respectively. Transcript profiles indicated that the Vp1 transcription factor contributed to regulation in response to water stress at early stages of maize endosperm development. Genes involved in transcriptional regulation and signal transduction were particularly dependent on presence of a functional Vp1 allele, as 83% the stress-affected genes in these categories were up-regulated by water stress in the wt, but only 34% were up-regulated in the mutant. This suggests that the loss of ABA sensitivity altered signaling networks in response to water deficit such that the mutant was unable to up-regulate the expression of many genes that normally play regulatory roles. Thus, the Vp1 gene plays a role in regulating transcript expression in maize endosperm development in response to water deficit, and this regulation is likely modulated via ABA signaling pathway. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A standard conservation strategy for plant genetic resources integrates in situ (on-farm or wild) and ex situ (gene or field bank) approaches. Gene bank managers collect ex situ accessions that represent a comprehensive snap shot of the genetic diversity of in situ populations at a given time and place. Although simple in theory, achieving complementary in situ and ex situ holdings is challenging. Using Trifolium thompsonii as a model insect-pollinated herbaceous perennial species, we used AFLP markers to compare genetic diversity and structure of ex situ accessions collected at two time periods (1995, 2004) from four locations, with their corresponding in situ populations sampled in 2009. Our goal was to assess the complementarity of the two approaches. We examined how gene flow, selection and genetic drift contributed to population change. Across locations, we found no difference in diversity between ex situ and in situ samples. One population showed a decline in genetic diversity over the 15 years studied. Population genetic differentiation among the four locations was significant, but weak. Association tests suggested infrequent, long distance gene flow. Selection and drift occurred, but differences due to spatial effects were three times as strong as differences attributed to temporal effects, and suggested recollection efforts could occur at intervals greater than fifteen years. An effective collecting strategy for insect pollinated herbaceous perennial species was to sample >150 plants, equalize maternal contribution, and sample along random transects with sufficient space between plants to minimize intrafamilial sampling. Quantifying genetic change between ex situ and in situ accessions allows genetic resource managers to validate ex situ collecting and maintenance protocols, develop appropriate recollection intervals, and provide an early detection mechanism for identifying problematic conditions that can be addressed to prevent further decline in vulnerable in situ populations. [ABSTRACT FROM AUTHOR]

Profilin is an actin-binding protein involved in the dynamic turnover and restructuring of the actin cytoskeleton in all eukaryotic cells. We previously cloned a profilin gene, designated as LePro1 from tomato pollen. To understand its biological role, in the present study, we investigated the temporal and spatial expression of LePro1 during pollen development and found that the transcript was only detected at late stages during microsporogenesis and pollen maturation. Using antisense RNA, we successfully knocked down the expression of LePro1 in tomato plants using stable transformation, and obtained two antisense lines, A2 and A3 showing significant down-regulation of LePro1 in pollen resulting in poor pollen germination and abnormal pollen tube growth. A disorganized F-actin distribution was observed in the antisense pollen. Down-regulation of LePro1 also appeared to affect hydration of pollen deposited on the stigma and arrested pollen tube elongation in the style, thereby affecting fertilization. Our results suggest that LePro1 in conjunction with perhaps other cytoskeletal proteins, plays a regulatory role in the proper organization of F-actin in tomato pollen tubes through promoting actin assembly. Down-regulation of LePro1 leads to interruption of actin assembly and disorganization of the actin cytoskeleton thus arresting pollen tube growth. Based on the present and previous studies, it is likely that a single transcript of profilin gives rise to multiple forms displaying multifunctionality in tomato pollen. [ABSTRACT FROM AUTHOR]

Profilin is an actin-binding protein involved in the dynamic turnover and restructuring of the actin cytoskeleton in all eukaryotic cells. We previously cloned a profilin gene, designated as LePro1 from tomato pollen. To understand its biological role, in the present study, we investigated the temporal and spatial expression of LePro1 during pollen development and found that the transcript was only detected at late stages during microsporogenesis and pollen maturation. Using antisense RNA, we successfully knocked down the expression of LePro1 in tomato plants using stable transformation, and obtained two antisense lines, A2 and A3 showing significant down-regulation of LePro1 in pollen resulting in poor pollen germination and abnormal pollen tube growth. A disorganized F-actin distribution was observed in the antisense pollen. Down-regulation of LePro1 also appeared to affect hydration of pollen deposited on the stigma and arrested pollen tube elongation in the style, thereby affecting fertilization. Our results suggest that LePro1 in conjunction with perhaps other cytoskeletal proteins, plays a regulatory role in the proper organization of F-actin in tomato pollen tubes through promoting actin assembly. Down-regulation of LePro1 leads to interruption of actin assembly and disorganization of the actin cytoskeleton thus arresting pollen tube growth. Based on the present and previous studies, it is likely that a single transcript of profilin gives rise to multiple forms displaying multifunctionality in tomato pollen. [ABSTRACT FROM AUTHOR]

Asparagus cochinchinensis is a traditional Chinese medicine used for treating lung and spleen-related diseases. In this study, we compared the medicinal effects of A. cochinchinensis root and stem extracts on the activity of superoxide dismutase (SOD), the content of malonaldehyde (MDA) and total protein content in the brain, liver and plasma of mice. Polysaccharides and aqueous extracts of the roots significantly increased the spleen index and the SOD activity but reduced the MDA content and slowed down the aging process. In contrast, feeding with the stem extracts significantly reduced the SOD activity and increased the MDA accumulation in the brain and liver of mice, suggesting that the stem extracts may not be appropriate for treating aging-related diseases. [ABSTRACT FROM AUTHOR]

Cigarette smoke contains free radicals that are considered to be a major group of carcinogens. In the present study, we used plant antioxidants to treat cigarette filters for scavenging gas phase free radicals in cigarette smoke. The scavenging activities of four antioxidants against gas phase free radicals were analyzed using an improved spin trap protocol. The scavenging rates ranked as lycopene > grape seed extract > pycnogenol > tomato powder. A scavenging rate of 62% (p < 0.001) was obtained from the lycopene treatment. The use of plant antioxidants as potential scavengers for reducing free-radicals in cigarette smoke is discussed. [ABSTRACT FROM AUTHOR]

Agronomic traits such as biomass yield and abiotic stress tolerance are genetically complex and challenging to improve through conventional breeding approaches. Genomic selection (GS) is an alternative approach in which genome-wide markers are used to determine the genomic estimated breeding value (GEBV) of individuals in a population. In alfalfa (Medicago sativa L.), previous results indicated that low to moderate prediction accuracy values (<70%) were obtained in complex traits, such as yield and abiotic stress resistance. There is a need to increase the prediction value in order to employ GS in breeding programs. In this paper we reviewed different statistic models and their applications in polyploid crops, such as alfalfa and potato. Specifically, we used empirical data affiliated with alfalfa yield under salt stress to investigate approaches that use DNA marker importance values derived from machine learning models, and genome-wide association studies (GWAS) of marker-trait association scores based on different GWASpoly models, in weighted GBLUP analyses. This approach increased prediction accuracies from 50% to more than 80% for alfalfa yield under salt stress. Finally, we expended the weighted GBLUP approach to potato and analyzed 13 phenotypic traits and obtained similar results. This is the first report on alfalfa to use variable importance and GWAS-assisted approaches to increase the prediction accuracy of GS, thus helping to select superior alfalfa lines based on their GEBVs. [ABSTRACT FROM AUTHOR]

Soil salinity is a growing problem in world production agriculture. Continued improvement in crop salt tolerance will require the implementation of innovative breeding strategies such as marker-assisted selection (MAS) and genomic selection (GS). Genetic analyses for yield and vigor traits under salt stress in alfalfa breeding populations with three different phenotypic datasets was assessed. Genotype-by-sequencing (GBS) developed markers with allele dosage and phenotypic data were analyzed by genome-wide association studies (GWAS) and GS using different models. GWAS identified 27 single nucleotide polymorphism (SNP) markers associated with salt tolerance. Mapping SNPs markers against the Medicago truncatula reference genome revealed several putative candidate genes based on their roles in response to salt stress. Additionally, eight GS models were used to estimate breeding values of the training population under salt stress. Highest prediction accuracies and root mean square errors were used to determine the best prediction model. The machine learning methods (support vector machine and random forest) performance best with the prediction accuracy of 0.793 for yield. The marker loci and candidate genes identified, along with optimized GS prediction models, were shown to be useful in improvement of alfalfa with enhanced salt tolerance. DNA markers and the outcome of the GS will be made available to the alfalfa breeding community in efforts to accelerate genetic gains, in the development of biotic stress tolerant and more productive modern-day alfalfa cultivars. [ABSTRACT FROM AUTHOR]