Genetic Diversity of Aspergillus flavus on Maize in Guatemala.

Aflatoxin contamination of maize is a leading threat to health in Guatemala. This contamination is the result of infection from Aspergillus flavus and has been effectively reduced in other countries through application of nonaflatoxigenic, indigenous strains of A. flavus. We collected 82 maize samples from throughout Guatemala in two years and isolated 272 A. flavus from these samples, including 126 unique genotypes. We provide here a phenotypic and simple sequence repeat (SSR)-based genotypic description of these isolates, as well as an analysis of the diversity of this population. High levels of genetic diversity were observed with the nonaflatoxigenic isolates in this study, but this information contributes to the development of indigenous aflatoxin biocontrol products.

cgSSR marker-based genome-wide association study identified genomic regions for panicle characters and yield in rice (Oryza sativa L.).

BACKGROUND: To improve production efficiency, positive alleles corresponding to yield-related attributes must be accumulated in a single elite background. We designed and used cgSSR markers, which are superior to random SSR markers in genome-wide association study, to identify genomic regions that contribute to panicle characters and grain yield in this study. RESULTS: As evidenced by the high polymorphic information content value and gene diversity coefficient, the new cgSSR markers were determined to be highly informative. These cgSSR markers were employed to generate genotype data for an association panel evaluated for four panicle characters and grain yield over three seasons. For five traits, 17 significant marker-trait associations on six chromosomes were discovered. The percentage of phenotypic variance that could be explained ranged from 4% to 13%. Unrelated gene-derived markers had a strong association with target traits as well. CONCLUSION: Trait-associated cgSSR markers derived from corresponding or related genes ensure their utility in direct allele selection, while other linked markers aid in allele selection indirectly by altering the phenotype of interest. Through a marker-assisted breeding approach, these marker-trait associations can be leveraged to accumulate favourable alleles for yield enhancement in rice. © 2022 Society of Chemical Industry.

Genetic Diversity of Phymatotrichopsis omnivora Based on Mating Type and Microsatellite Markers Reveals Heterothallic Mating System.

Phymatotrichopsis omnivora is a member of Pezizomycetes and causes root rot disease on a broad range of dicotyledonous plants. Using recently generated draft genome sequence data from four P. omnivora isolates, we developed simple sequence repeat (SSR) markers and identified both mating type genes (MAT1-1-1 and MAT1-2-1) in this fungus. To understand the genetic diversity of P. omnivora isolates (n = 43) and spore mats (n = 29) collected from four locations (Oklahoma, Texas, Arizona, and Mexico) and four host crops (cotton, alfalfa, peach, and soybean), we applied 24 SSR markers and showed that of the 72 P. omnivora isolates and spore mats tested, 41 were distinct genotypes. Furthermore, the developed SSR markers did not show cross-transferability to other close relatives of P. omnivora in the class Pezizomycetes. A multiplex PCR detecting both mating type idiomorphs and a reference gene (TUB2) was developed to screen P. omnivora isolates. Based on the dataset we tested, P. omnivora is a heterothallic fungus with both mating types present in the United States in a ratio close to 1:1. We tested P. omnivora spore mats obtained from spatially distinct disease rings that developed in a center-pivot alfalfa field and showed that both mating types can be present not only in the same field but also within a single spore mat. This study shows that P. omnivora has the genetic toolkit for generating sexually diverse progeny, providing impetus for future studies that focus on identifying sexual morphs in nature.

Genetic Diversity of a Natural Population of Akebia trifoliata (Thunb.) Koidz and Extraction of a Core Collection Using Simple Sequence Repeat Markers.

Understand genetic diversity and genetic structure of germplasm is premise of germplasm conservation and utilization. And core collection can reduce the cost and difficulty of germplasm conservation. Akebia trifoliata (Thunb.) Koidz is an important medicinal, fruit and oil crop, particularly in China. In this study, 28 simple sequence repeat (SSR) markers were used to assess the genetic diversity and genetic structure of 955 A. trifoliata germplasms, determine their molecular identity and extract a core collection. The genetic diversity of the 955 germplasms was moderately polymorphic. The average number of alleles (Na), observed heterozygosity (H O ), expected heterozygosity (H E ), Shannon's information index (I∗), and polymorphic information content (PIC) were 3.71, 0.24, 0.46, 0.81, and 0.41, respectively. Four subpopulations were identified, indicating a weak genetic structure. A 955 germplasms could be completely distinguished by the characters of s28, s25, s74, s89, s68, s30, s13, s100, s72, s77, and s3. And each germplasm's molecular identity was made up of eleven characters. The core collection was composed of 164 germplasms (17.2% of 955 total germplasms in the population) and diversity parameters differed significantly from those of a random core collection. These results have implications for germplasm conservation. At the same time, based on the results, the 955 germplasm could be better used and managed.

Potentials and Challenges of Genomics for Breeding Cannabis Cultivars.

Cannabis (Cannabis sativa L.) is an influential yet controversial agricultural plant with a very long and prominent history of recreational, medicinal, and industrial usages. Given the importance of this species, we deepened some of the main challenges-along with potential solutions-behind the breeding of new cannabis cultivars. One of the main issues that should be fixed before starting new breeding programs is the uncertain taxonomic classification of the two main taxa (e.g., indica and sativa) of the Cannabis genus. We tried therefore to examine this topic from a molecular perspective through the use of DNA barcoding. Our findings seem to support a unique species system (C. sativa) based on two subspecies: C. sativa subsp. sativa and C. sativa subsp. indica. The second key issue in a breeding program is related to the dioecy behavior of this species and to the comprehension of those molecular mechanisms underlying flower development, the main cannabis product. Given the role of MADS box genes in flower identity, we analyzed and reorganized all the genomic and transcriptomic data available for homeotic genes, trying to decipher the applicability of the ABCDE model in Cannabis. Finally, reviewing the limits of the conventional breeding methods traditionally applied for developing new varieties, we proposed a new breeding scheme for the constitution of F1 hybrids, without ignoring the indisputable contribution offered by genomics. In this sense, in parallel, we resumed the main advances in the genomic field of this species and, ascertained the lack of a robust set of SNP markers, provided a discriminant and polymorphic panel of SSR markers as a valuable tool for future marker assisted breeding programs.

Genetic Structure of the Norwegian Parastagonospora nodorum Population.

The necrotrophic fungal pathogen Parastagonospora nodorum causes Septoria nodorum blotch (SNB), which is one of the dominating leaf blotch diseases of wheat in Norway. A total of 165 P. nodorum isolates were collected from three wheat growing regions in Norway from 2015 to 2017. These isolates, as well as nine isolates from other countries, were analyzed for genetic variation using 20 simple sequence repeat (SSR) markers. Genetic analysis of the isolate collection indicated that the P. nodorum pathogen population infecting Norwegian spring and winter wheat underwent regular sexual reproduction and exhibited a high level of genetic diversity, with no genetic subdivisions between sampled locations, years or host cultivars. A high frequency of the presence of necrotrophic effector (NE) gene SnToxA was found in Norwegian P. nodorum isolates compared to other parts of Europe, and we hypothesize that the SnToxA gene is the major virulence factor among the three known P. nodorum NE genes (SnToxA, SnTox1, and SnTox3) in the Norwegian pathogen population. While the importance of SNB has declined in much of Europe, Norway has remained as a P. nodorum hotspot, likely due at least in part to local adaptation of the pathogen population to ToxA sensitive Norwegian spring wheat cultivars.

Development of introgression lines of AA genome Oryza species, O. glaberrima, O. rufipogon, and O. nivara, in the genetic background of O. sativa L. cv. Taichung 65.

To evaluate and utilize potentially valuable quantitative trait loci or genes of wild relatives in the genetic background of domesticated crop species, chromosome segment substitution lines (CSSLs) are a valuable tool. CSSLs can be constructed through the exchange of chromosome segments of AA genome species of the genus Oryza with cultivated rice, Oryza sativa L. Here we report the development of three sets of CSSLs carrying segments of AA genome species closely related to Oryza sativa-O. glaberrima (IRGC 103777 from Mali), O. rufipogon (W1962 from China), and O. nivara (IRGC 105715 from Cambodia)-in the genetic background of ssp. japonica cultivar Taichung 65 through the use of 101 to 121 simple-sequence-repeat markers in whole-genome genotyping and marker-assisted selection. The materials are available via the National Bioresource Project (Rice) Oryzabase Web page.

ISSR Analysis of Genetic Diversity and Structure of Plum Varieties Cultivated in Southern China.

Plums (Prunus spp.) are important deciduous fruit crops in the world. China is a major producer of P. salicina Lindl., but the genetic relationship of Chinese plums in key production regions remain unclear. In this study, 14 University of British Columbia (UBC) inter simple sequence repeats (ISSR) primers were used to analyze 33 plum varieties cultivated in Fujian Province to determine their genetic diversity and population structure. A total of 146 bands were generated, of which 130 were polymorphic. Mean percentage of polymorphic bands was 89.04%, Shannon's information index value was 0.38, and the Nei's genetic index value was 0.24. Using unrooted trees (Neighbor-Joining method), 33 varieties were classified into four groups. Split graph separated them into two major groups, each with two subgroups. The two phylogenetic trees indicate that environmental or natural selection pressure is an important factor influencing their genetic relationship. Analysis of population structure revealed that they have frequent genetic exchanges among closed subpopulations; thus, genetic variation mainly occurs within the population. Additionally, based on the phylogenetic analysis and unique morphological characteristics of fruits, we propose that the Chinese landrace Nai could contribute significantly to development of the famous variety Wickson.

Simple sequence repeat markers associated/linked with agronomic traits, as core primers, are eminently suitable for DNA fingerprinting in Upland cotton.

Analyzing the genetic differences among crop germplasm resources scientifically and accurately is very important for the selection of core accessions, the identification of new cultivars, and the determination of seed purity. However, phenotypic selection per se is not sufficient to identify genetically distinct accessions. In this study, 26 out of 83 simple sequence repeat markers associated/linked with cotton important agronomic traits derived from our previous and other published research, corresponding to the 26 chromosomes of Upland cotton (Gossypium hirsutum L.), were selected as core primers for DNA fingerprinting construction. The 26 markers showed clear band patterns, good repeatability and high polymorphism. The average alleles, gene diversity index and polymorphism information content were 3.12, 0.4312 and 0.3830, respectively. Using TM-1, a genetic standard line for Upland cotton, as the control, DNA fingerprinting pattern and DNA barcodes were obtained based on the core primers. There was a significant positive correlation between genetic distance matrix determined using 26 core primers and that determined using more primers (335) derived from previous research, further suggesting that the core primers were eminently suitable for DNA fingerprinting in Upland cotton. This study provides a molecular basis for assessing identification, authenticity and seed purity of cotton cultivars.

Corrigendum: Transcriptomic Identification of Drought-Related Genes and SSR Markers in Sudan Grass Based on RNA-Seq.

[This corrects the article on p. 687 in vol. 8, PMID: 28523007.].

Transcriptomic Identification of Drought-Related Genes and SSR Markers in Sudan Grass Based on RNA-Seq.

Sudan grass (Sorghum sudanense) is an annual warm-season gramineous forage grass that is widely used as pasture, hay, and silage. However, drought stress severely impacts its yield, and there is limited information about the mechanisms of drought tolerance in Sudan grass. In this study, we used next-generation sequencing to identify differentially expressed genes (DEGs) in the Sudan grass variety Wulate No.1, and we developed simple sequence repeat (SSR) markers associated with drought stress. From 852,543,826 raw reads, nearly 816,854,366 clean reads were identified and used for analysis. A total of 80,686 unigenes were obtained via de novo assembly of the clean reads including 45,065 unigenes (55.9%) that were identified as coding sequences (CDSs). According to Gene Ontology analysis, 31,444 unigenes were annotated, 11,778 unigenes were identified to 25 categories in the clusters of orthologous groups of proteins (KOG) classification, and 11,223 unigenes were assigned to 280 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Additionally, there were 2,329 DEGs under a short-term of 25% polyethylene glycol (PEG) treatment, while 5,101 DEGs were identified under the long-term of 25% PEG treatment. DEGs were enriched in pathways of carbon fixation in photosynthetic organisms and plant hormone signal transduction which played a leading role in short-term of drought stress. However, DEGs were mainly enriched in pathway of plant hormone signal transduction that played an important role under long-term of drought stress. To increase accuracy, we excluded all the DEGs of all controls, specifically, five DEGs that were associated with high PEG concentrations were found through RNA-Seq. All five genes were up-regulated under drought stress, but the functions of the genes remain unclear. In addition, we identified 17,548 SSRs obtained from 80,686 unigenes. The newly identified drought tolerance DEGs will contribute to transgenic breeding efforts, while SSRs developed from high-throughput transcriptome data will facilitate marker-assisted selection for all traits in Sudan grass.

Development of microsatellite markers using Illumina MiSeq sequencing to characterize Ephedra gerardiana (Ephedraceae).

PREMISE OF THE STUDY: Ephedragerardiana (Ephedraceae), occurring in the Himalayan ranges, is an important plant species used in Tibetan medicine. Due to the lack of molecular markers to characterize genetic diversity, knowledge for conservation and uses of E. gerardiana resources is limited; we therefore developed microsatellite markers for use in this species. METHODS AND RESULTS: Using Illumina MiSeq sequencing technology, we developed 29 polymorphic microsatellite loci suitable for E. gerardiana, of which 15 loci also showed polymorphisms in two related Ephedra species, E. saxatilis and E. monosperma. The average number of effective alleles per locus ranged from two to six. The observed and expected heterozygosity ranged from 0.23 to 0.83 and 0.44 to 0.86, respectively, in E. gerardiana populations. CONCLUSIONS: The developed 29 microsatellite markers are effective for the study of genetic structure and genetic diversity of E. gerardiana, and 15 of these markers are suitable for related Ephedra species.

An Assessment of Genetic Diversity and Drought Tolerance in Argan Tree (Argania spinosa) Populations: Potential for the Development of Improved Drought Tolerance.

The argan tree (Argania spinosa) occurs in a restricted area of Southwestern Morocco characterized by low water availability and high evapotranspirative demand. Despite the adaptation of the argan tree to drought stress, the extent of the argan forest has declined markedly due to increased aridity, land use changes and the expansion of olive cultivation. The oil of the argan seed is used for cooking and as the basis for numerous cosmetics. The identification of argan tree varieties with enhanced drought tolerance may minimize the economic losses associated with the decline of the argan forest and constrain the spread of desertification. In this study we collected argan ecotypes from four contrasting habitats and grew them under identical controlled environment conditions to investigate their response to drought. Leaf gas exchange analysis indicated that the argan ecotypes showed a high degree of adaptation to drought stress, maintaining photosynthetic activity at low levels of foliar water content and co-ordinating photosynthesis, stomatal behavior and metabolism. The stomata of the argan trees were highly sensitive to increased leaf to air vapor pressure deficit, representing an adaptation to growth in an arid environment where potential evapotranspiration is high. However, despite originating in contrasting environments, the four argan ecotypes exhibited similar gas exchange characteristics under both fully irrigated and water deficit conditions. Population genetic analyses using microsatellite markers indicated a high degree of relatedness between the four ecotypes; indicative of both artificial selection and the transport of ecotypes between different provinces throughout centuries of management of the argan forest. The majority of genetic variation across the four populations (71%) was observed between individuals, suggesting that improvement of argan is possible. Phenotypic screening of physiological responses to drought may prove effective in identifying individuals and then developing varieties with enhanced drought tolerance to enable the maintenance of argan production as climate change results in more frequent and severe drought events in Northern Africa.

Towards Defining Heterotic Gene Pools in Pearl Millet [Pennisetum glaucum (L.) R. Br.].

Pearl millet is a climate resilient crop and one of the most widely grown millets worldwide. Heterotic hybrid development is one of the principal breeding objectives in pearl millet. In a maiden attempt to identify heterotic groups for grain yield, a total of 343 hybrid parental [maintainer (B-) and restorer (R-)] lines were genotyped with 88 polymorphic SSR markers. The SSRs generated a total of 532 alleles with a mean value of 6.05 alleles per locus, mean gene diversity of 0.55, and an average PIC of 0.50. Out of 532 alleles, 443 (83.27%) alleles were contributed by B-lines with a mean of 5.03 alleles per locus. R-lines contributed 476 alleles (89.47%) with a mean of 5.41, while 441 (82.89%) alleles were shared commonly between B- and R-lines. The gene diversity was higher among R-lines (0.55) compared to B-lines (0.49). The unweighted neighbor-joining tree based on simple matching dissimilarity matrix obtained from SSR data clearly differentiated B- lines into 10 sub-clusters (B1 through B10), and R- lines into 11 sub-clusters (R1 through R11). A total of 99 hybrids (generated by crossing representative 9 B- and 11 R- lines) along with checks were evaluated in the hybrid trial. The 20 parents were evaluated in the line trial. Both the trials were evaluated in three environments. Based on per se performance, high sca effects and standard heterosis, F1s generated from crosses between representatives of groups B10R5, B3R5, B3R6, B4UD, B5R11, B2R4, and B9R9 had high specific combining ability for grain yield compared to rest of the crosses. These groups may represent putative heterotic gene pools in pearl millet.

Sophora microphylla (Fabaceae) microsatellite markers and their utility across the genus.

PREMISE OF THE STUDY: Genus-specific microsatellite markers were developed for Sophora for population genetic and systematic studies of the group in New Zealand, and potentially elsewhere in the geographic range. • METHODS AND RESULTS: From sequencing a total genomic DNA library (using Roche 454), we identified and developed 29 polymorphic microsatellite markers for S. microphylla and S. chathamica. We tested 12 of these markers on 14 S. chathamica individuals and four S. microphylla populations. All loci amplified in both species and species-specific alleles occurred at seven loci. In S. microphylla populations, the observed and expected heterozygosities ranged from 0.000-0.960 and 0.000-0.908, respectively, with alleles per locus ranging from seven to 23. • CONCLUSIONS: The developed markers will be valuable in studies of phylogenetics, population structure, mating system, and selection of provenances for restoration projects.

Analysis of in situ diversity and population structure in Ethiopian cultivated Sorghum bicolor (L.) landraces using phenotypic traits and SSR markers.

Genetic diversity is a fundamental input for every plant breeding program, genetic resources conservation, and evolutionary studies. In situ diversity and population genetic structure of eight cultivated sorghum landrace populations were investigated in the center of origin, Ethiopia using seven phenotypic traits and 12 highly polymorphic sorghum SSR markers. In farmers' fields, DNA samples were collected using Whatman® plant saver card and quantitative phenotypic traits were measured from 160 individual plant samples belonging to the eight populations representing three diverse geographical regions. High diversity was observed among the various populations for the measured phenotypic traits. The 12 SSR loci produced a total of 123 alleles of which 78 (63.41%) were rare (frequency ≤0.05) with an average of 10.25 alleles per polymorphic locus. The polymorphism information content (PIC) was in the range 0.39-0.85 showing the good discriminatory power of the SSR loci used. Average observed heterozygosity and gene diversity across all populations and loci ranged 0.04-0.33 and 0.41-0.87, respectively. Neighbor-joining and STRUCTURE analyses grouped the 160 samples from the eight populations differently. AMOVA showed 54.44% of the variation to be within populations, 32.76% among populations within regions, and 12.8% among the regions of origin. There was high divergence in the total populations (FST = 0.40) indicating low level of gene flow (Nm = 0.38), but high gene flow was also observed in some adjacent populations. The populations from Wello displayed close relationship with remote Gibe and Metekel populations indicating that the variation followed human migration patterns. Implications of the results for sorghum improvement and germplasm conservation are discussed.

Analysis of microsatellites in the vulnerable orchid Gastrodia flavilabella: the development of microsatellite markers, and cross-species amplification in Gastrodia.

BACKGROUND: Gastrodia flabilabella is a mycoheterotrophic orchid that obtains carbohydrates and nutrients from its symbiotic mycorrhizal fungi. The species is an endemic and vulnerable species enlisted in the "A Preliminary Red List of Taiwanese Vascular Plants" according to the IUCN Red List Categories and Criteria Version 3.1. G. flabilabella dwells the underground of broadleaf and coniferous forest with richness litter. Based on herbarium records, this species is distributed in central Taiwan. Twenty eight microsatellite loci were developed in G. flabilabella and were tested for cross-species amplification in additional taxa of G. confusoides, G. elata, and G. javanica. We estimated the genetic variation that is valuable for conservation management and the development of the molecular identification system for G. elata, a traditional Chinese medicine herb. RESULTS: Microsatellite primer sets were developed from G. flabilabella using the modified AFLP and magnetic bead enrichment method. In total, 257 microsatellite loci were obtained from a magnetic bead enrichment SSR library. Of the 28 microsatellite loci, 16 were polymorphic, in which the number of alleles ranged from 2 to 15, with the observed heterozygosity ranging from 0.02 to 1.00. In total, 15, 13, and 7 of the loci were found to be interspecifically amplifiable to G. confusoides, G. elata, and G. javanica, respectively. CONCLUSIONS: Amplifiable and transferable microsatellite loci are potentially useful for future studies in investigating intraspecific genetic variation, reconstructing phylogeographic patterns among closely related species, and establishing the standard operating system of molecular identification in Gastrodia.

Isolation and characterization of microsatellite loci for a bioenergy grass, Miscanthus sacchariflorus (Poaceae).

PREMISE OF THE STUDY: Microsatellite loci were developed for the biomass C4 grass, Miscanthus sacchariflorus, and proved to be suitable markers for population genetic studies and germplasm management of this species. • METHODS AND RESULTS: Twenty-three polymorphic microsatellite loci were identified from an enriched genomic library of M. sacchariflorus. The polymorphism was assessed in 50 individuals from two populations in China. The number of alleles per locus varied from two to 18, with a mean of 8.13. The observed and expected heterozygosities ranged from 0.2 to 1.0 and from 0.198 to 0.898, respectively. • CONCLUSIONS: These new markers will be useful for further investigation of genetic diversity and population genetic structure as well as molecular breeding of Miscanthus species.

Cost-effective microsatellite markers for Banksia integrifolia (Proteaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed to assess the level of genetic variation and population structure in Banksia integrifolia, a widespread species endemic to eastern Australia. • METHODS AND RESULTS: We used next-generation sequencing approaches to identify and develop 11 polymorphic microsatellite markers with perfect tri- and tetranucleotide repeats. We tested these markers with 71 specimens from three populations. Observed and expected heterozygosities ranged from 0.0 to 0.875 and 0.0 to 0.763, respectively. • CONCLUSIONS: The developed markers will be valuable for studies of the population structure, mating system, and selection of provenances for restoration projects involving B. integrifolia.

Development and characterization of polymorphic microsatellite loci in Phellodendron amurense (Rutaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the rare species Phellodendron amurense to assess the genetic diversity and population structure of this plant. • METHODS AND RESULTS: In total, 27 microsatellite markers were developed for P. amurense by using an enriched genomic library and hybridization; all of these primers successfully amplified DNA fragments in P. amurense. These markers were screened in 74 individuals from four populations in China; 15 loci were found to be polymorphic, with the number of alleles per locus ranging from one to nine. • CONCLUSIONS: The microsatellite markers developed here represent a useful tool for studying the population genetic structure of P. amurense and to inform toward the development of effective conservation programs for this species.