M. A. Rouf Mian

Tall fescue EST-SSR markers with transferability across several grass species

Tall fescue (Festuca arundinacea Schreb.) is a major cool season forage and turf grass in the temperate regions of the world. It is also a close relative of other important forage and turf grasses, including meadow fescue and the cultivated ryegrass species. Until now, no SSR markers have been developed from the tall fescue genome. We designed 157 EST-SSR primer pairs from tall fescue ESTs and tested them on 11 genotypes representing seven grass species. Nearly 92% of the primer pairs produced characteristic simple sequence repeat (SSR) bands in at least one species. A large proportion of the primer pairs produced clear reproducible bands in other grass species, with most success in the close taxonomic relatives of tall fescue. A high level of marker polymorphism was observed in the outcrossing species tall fescue and ryegrass (66%). The marker polymorphism in the self-pollinated species rice and wheat was low (43% and 38%, respectively). These SSR markers were useful in the evaluation of genetic relationships among the Festuca and Lolium species. Sequencing of selected PCR bands revealed that the nucleotide sequences of the forage grass genotypes were highly conserved. The two cereal species, particularly rice, had significantly different nucleotide sequences compared to the forage grasses. Our results indicate that the tall fescue EST-SSR markers are valuable genetic markers for the Festuca and Lolium genera. These are also potentially useful markers for comparative genomics among several grass species.

Genetic linkage mapping of the soybean aphid resistance gene in PI 243540

The soybean aphid (Aphis glycines Matsumura) is a pest of soybean [Glycine max (L.) Merr.] in many soybean growing countries of the world, mainly in Asia and North America. A single dominant gene in PI 243540 confers resistance to the soybean aphid. The objectives of this study were to identify simple sequence repeat (SSR) markers closely linked to the gene in PI 243540 and to position the gene on the consensus soybean genetic map. One hundred eighty-four F2 plants and their F2:3 families from a cross between the susceptible cultivar Wyandot and PI 243540, and the two parental lines were screened with the Ohio biotype of soybean aphid using greenhouse choice tests. A SSR marker from each 10-cM section of the consensus soybean map was selected for bulked segregant analysis (BSA) to identify the tentative genomic location of the gene. The BSA technique was useful to localize the gene to a genomic region in soybean linkage group (LG) F. The entire F2 population was then screened with polymorphic SSR markers from this genomic region and a linkage map with nine SSR markers flanking the gene was constructed. The aphid resistance gene was positioned in the interval between SSR markers Satt334 and Sct_033 on LG F. These SSR markers will be useful for marker assisted selection of this gene. The aphid resistance gene from PI 243540 mapped to a different linkage group than the only named soybean aphid resistance gene, Rag1, from ‘Dowling’. Also, the responses of the two known biotypes of the soybean aphid to the gene from PI 243540 and Rag1 were different. Thus, the aphid resistance gene from PI 243540 was determined to be a new and independent gene that has been named Rag2.

Tall fescue genomic SSR markers: development and transferability across multiple grass species

Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies. Currently, a very limited number of SSR markers are available for tall fescue (Festuca arundinacea Schreb.) and other forage grass species. A tall fescue genomic library enriched in (GA/CT)n repeats was used to develop primer pairs (PPs) flanking SSRs and assess PP functionality across different forage, cereal, and turf grass species. A total of 511 PPs were developed and assessed for their utility in six different grass species. The parents and a subset of a tall fescue mapping population were used to select PPs for mapping in tall fescue. Survey results revealed that 48% (in rice) to 66% (in tall fescue) of the PPs produced clean SSR-type amplification products in different grass species. Polymorphism rates were higher in tall fescue (68%) compared to other species (46% ryegrass, 39% wheat, and 34% rice). A set of 194 SSR loci (38%) were identified which amplified across all six species. Loci segregating in the tall fescue mapping population were grouped as loci segregating from the female parent (HD28-56, 37%), the male parent (R43-64, 37%), and both parents (26%). Three percent of the loci that were polymorphic between parents were monomorphic in the pseudo F1 mapping population and the remaining loci segregated. Sequencing of amplified products obtained from PP NFFAG428 revealed a very high level of sequence similarity among the grass species under study. Our results are the first report of genomic SSR marker development from tall fescue and they demonstrate the usefulness of these SSRs for genetic linkage mapping in tall fescue and cross-species amplification.

Genetic mapping revealed two loci for soybean aphid resistance in PI 567301B

The soybean aphid (Aphis glycines Matsumura) is the most damaging insect pest of soybean [Glycine max (L.) Merr.] in North America. New soybean aphid biotypes have been evolving quickly and at least three confirmed biotypes have been reported in USA. These biotypes are capable of defeating most known aphid resistant soybean genes indicating the need for identification of new genes. Plant Introduction (PI) 567301B was earlier identified to have antixenosis resistance against biotype 1 and 2 of the soybean aphid. Two hundred and three F7:9 recombinant inbred lines (RILs) developed from a cross of soybean aphid susceptible cultivar Wyandot and resistant PI 567301B were used for mapping aphid resistance genes using the quantitative trait loci (QTL) mapping approach. A subset of 94 RILs and 516 polymorphic SNP makers were used to construct a genome-wide molecular linkage map. Two candidate QTL regions for aphid resistance were identified on this linkage map. Fine mapping of the QTL regions was conducted with SSR markers using all 203 RILs. A major gene on chromosome 13 was mapped near the previously identified Rag2 gene. However, an earlier study revealed that the detached leaves of PI 567301B had no resistance against the soybean aphids while the detached leaves of PI 243540 (source of Rag2) maintained aphid resistance. These results and the earlier finding that PI 243540 showed antibiosis resistance and PI 567301B showed antixenosis type resistance, indicating that the aphid resistances in the two PIs are not controlled by the same gene. Thus, we have mapped a new gene near the Rag2 locus for soybean aphid resistance that should be useful in breeding for new aphid-resistant soybean cultivars. Molecular markers closely linked to this gene are available for marker-assisted breeding. Also, the minor locus found on chromosome 8 represents the first reported soybean aphid-resistant locus on this chromosome.

Population Genetic Structure of Aphis glycines

ABSTRACT The soybean aphid (Aphis glycines Matsumura) is an invasive pest of cultivated soybean (Glycine max L.) in North America. After the initial invasion in 2000, the aphid has quickly spread across most of the United States and Canada, suggesting large-scale dispersal and rapid adaptation to new environments. Using microsatellite markers from closely related species, we compared the genetic diversity and the amount of genetic differentiation within and among 2 South Korean and 10 North American populations. Overall allelic polymorphism was low, never exceeding four alleles per locus. However, differences in genetic diversity were seen among South Korean and North American populations in terms of heterozygote excesses and genotypic richness. Within North America, two populations (Michigan and Ontario), had lower genetic diversities and exhibited high genetic differentiation compared with the remaining eight populations. The earlier collection time of Michigan and Ontario samples explained the genetic differences better than geographic subdivisions. These data indicate a pattern of small colonizing populations on soybeans, followed by rapid clonal amplification and subsequent large-scale dispersal across North America.

Detached Leaf and Whole Plant Assays for Soybean Aphid Resistance: Differential Responses Among Resistance Sources and Biotypes

ABSTRACT The soybean aphid, Aphis glycines Matsumura, is a pest of cultivated soybean, Glycine max (L.) Merr., in North America. Recent developments in host plant resistance studies have identified at least four soybean aphid resistance genes (Rog1–4) and two soybean aphid biotypes (biotype 1 and 2), defined by differential survivability on resistant soybean. Detached soybean leaves were tested as a more rapid and practical assay to assess host plant resistance and virulence. Two susceptible lines (‘Wyandot’ and ‘Williams 82’) and two resistant lines (PI 243540 and PI 567301B) were examined. Various life history characteristics were compared among aphids on whole plants and detached leaves. Results indicated that resistance to soybean aphid was lost using detached leaves of PI 567301B but retained with PI 243540. To test for aphid virulence, net fecundities were compared among biotype 1 and biotype 2 after rearing on detached leaves of the resistant ‘Jackson’ (to which biotype 2 is virulent). A significant difference was detected in net fecundities among biotypes on detached leaves of Jackson and used to predict growth rates and virulence from 30 field-collected individuals of unknown virulence. No field individuals matched biotype 2 predictions, but four individuals had higher net fecundities than biotype 2 predictions (13%) and could be considered moderately virulent. The results indicated that the retention of soybean aphid resistance in detached leaves is dependent on PI and resistant source, but if resistance is retained, detached leaves could be used to determine soybean aphid virulence.

Validation of Reference Genes for Gene Expression Studies in Aphis glycines (Hemiptera: Aphididae)

ABSTRACT Quantitative real-time polymerase chain reaction (qRT-PCR) is a common and robust tool for accurate quantification of mRNA transcripts. To normalize results, a housekeeping gene ([HKG], reference gene or endogenous control gene) is mandatory. Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a significant soybean, Glycine max (L.) Merr., pest, yet gene expression and functional genomics studies are hindered by a lack of stable HKGs. We evaluated seven potential HKGs (SDFS, succinate dehydrogenase flavoprotein subunit; EF1a, elongation factor-1&agr;; HEL, helicase; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; RPS9, ribosomal protein S9; TBP, TATA-box binding protein; and UBQ, ubiquitin-conjugating protein) to determine the most efficient HKGs that have stable expression among tissues, developmental stages, and aphids fed on susceptible and host plant—resistant soybean. HKG stability was determined using GeNorm and NormFinder. Results from three different experimental conditions revealed high stability of TBP compared with the other HKGs profiled across the samples assayed. RPS9 showed stable expression among aphids on susceptible and resistant plants, whereas EF1a showed stable expression in tissues and developmental stages. Therefore, we recommend the TBP as a suitable HKG for efficient normalization among treatments, tissues, and developmental stages of A. glycines. In addition, RPS9 may be used for host-plant resistance experiments and EF1a could be considered for testing differential expression across tissues or developmental stages. These results will enable a more accurate and reliable normalization of qRT-PCR data in A. glycines.

Microbiome diversity of Aphis glycines with extensive superinfection in native and invasive populations

Associations among insects and microbes can lead to beneficial or parasitic interactions. Using 454 sequencing of 16S RNA genes, we compared microbiome diversity and abundance among field-collected (F) and laboratory-reared (L) populations of the invasive soybean aphid (Aphis glycines), a pest of soybean. Additionally, we screened A. glycines populations from native (Japan, South Korea and China) and invasive regions (North America) to broadly determine the microbiome diversity. Our results suggested that Arsenophonus (relative abundance of 54.6%), Buchnera (38.7%) and Wolbachia (3.7%) were the major bacteria associated with A. glycines. Arsenophonus was the most abundant in F populations but was significantly reduced in L populations; additional bacteria species also had lower relative abundances in L populations. Native and invasive populations were largely similar in bacteria communities and revealed substantial superinfection of Arsenophonus and Wolbachia. The lone exception was a lack of Arsenophonus in A. glycines from Japan. Divergent selection pressures among natural and laboratory populations were inferred as factors driving the differential bacterial communities observed. Our results will allow for improved comparative aphid-symbiont research and broaden our understanding of the interactions among insects, endosymbionts and their environments.

Evolution of Soybean Aphid Biotypes: Understanding and Managing Virulence to Host-Plant Resistance

The soybean aphid (Aphis glycines Matsumura) has rapidly become one of the most significant insect pests of soybean (Glycine max) worldwide (Ragsdale et al., 2007). The rise of soybean aphid in importance is in large part due to the invasion of North America ca. 2000, presumably coming from its native range in Asia (Ragsdale et al., 2004). In the first few years of the North American invasion, the soybean aphid spread across much of the NorthCentral US and the provinces of Ontario and Quebec. Its current distribution includes over 80% of the soybean growing region of the US and Canada (Vennette & Ragsdale, 2004). Worldwide, the distribution includes much of East Asia (China, Japan, The Philippines, South Korea, Indonesia, Malaysia, Thailand, Vietnam, and Russia), all of which likely represents the ancestral range (Footit et al., 2006). Options for management and control of soybean aphid are limited. As an alternative to chemical insecticides, host-plant resistance is a common method of aphid control (Van Emden, 2007), which uses plant hosts with genetically inherited traits that enable the plant to withstand pest attack better than a plant lacking these traits (Smith, 2005). Although soybean aphid resistant varieties have been studied in China (Wu et al., 2004), new soybean varieties have been developed with resistance to the soybean aphid specifically for use in North America (Hill et al., 2004, 2006a,b; Mian et al., 2008; Zhang et al., 2009; Hill et al., 2010). Varieties for North American commercial use were available for the first time in 2010. However, the host-plant resistance strategy is complicated because of rapid evolution of soybean aphid populations (i.e. biotypes) that have overcome host-plant resistance (i.e. virulence). Biotypes can be defined as “populations within an arthropod species that differ in their ability to utilize a particular trait in a particular plant genotype” (Smith, 2005). The presence of soybean aphid biotypes with virulence to host-plant resistance varieties before these varieties were commercially released suggests that the soybean aphid can rapidly adapt to these new lines and thereby threaten the effectiveness and sustainability of the host-plant resistance strategy.

Analysis of tall fescue ESTs representing different abiotic stresses, tissue types and developmental stages

BackgroundTall fescue (Festuca arundinacea Schreb) is a major cool season forage and turf grass species grown in the temperate regions of the world. In this paper we report the generation of a tall fescue expressed sequence tag (EST) database developed from nine cDNA libraries representing tissues from different plant organs, developmental stages, and abiotic stress factors. The results of inter-library and library-specific in silico expression analyses of these ESTs are also reported.ResultsA total of 41,516 ESTs were generated from nine cDNA libraries of tall fescue representing tissues from different plant organs, developmental stages, and abiotic stress conditions. The Festuca Gene Index (FaGI) has been established. To date, this represents the first publicly available tall fescue EST database. In silico gene expression studies using these ESTs were performed to understand stress responses in tall fescue. A large number of ESTs of known stress response gene were identified from stressed tissue libraries. These ESTs represent gene homologues of heat-shock and oxidative stress proteins, and various transcription factor protein families. Highly expressed ESTs representing genes of unknown functions were also identified in the stressed tissue libraries.ConclusionFaGI provides a useful resource for genomics studies of tall fescue and other closely related forage and turf grass species. Comparative genomic analyses between tall fescue and other grass species, including ryegrasses (Lolium sp.), meadow fescue (F. pratensis) and tetraploid fescue (F. arundinacea var glaucescens) will benefit from this database. These ESTs are an excellent resource for the development of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) PCR-based molecular markers.