Genetic mapping and QTL analysis for peanut smut resistance.

BACKGROUND: Peanut smut is a disease caused by the fungus Thecaphora frezii Carranza & Lindquist to which most commercial cultivars in South America are highly susceptible. It is responsible for severely decreased yield and no effective chemical treatment is available to date. However, smut resistance has been identified in wild Arachis species and further transferred to peanut elite cultivars. To identify the genome regions conferring smut resistance within a tetraploid genetic background, this study evaluated a RIL population {susceptible Arachis hypogaea subsp. hypogaea (JS17304-7-B) × resistant synthetic amphidiploid (JS1806) [A. correntina (K 11905) × A. cardenasii (KSSc 36015)] × A. batizocoi (K 9484)4×} segregating for the trait. RESULTS: A SNP based genetic map arranged into 21 linkage groups belonging to the 20 peanut chromosomes was constructed with 1819 markers, spanning a genetic distance of 2531.81 cM. Two consistent quantitative trait loci (QTLs) were identified qSmIA08 and qSmIA02/B02, located on chromosome A08 and A02/B02, respectively. The QTL qSmIA08 at 15.20 cM/5.03 Mbp explained 17.53% of the phenotypic variance, while qSmIA02/B02 at 4.0 cM/3.56 Mbp explained 9.06% of the phenotypic variance. The combined genotypic effects of both QTLs reduced smut incidence by 57% and were stable over the 3 years of evaluation. The genome regions containing the QTLs are rich in genes encoding proteins involved in plant defense, providing new insights into the genetic architecture of peanut smut resistance. CONCLUSIONS: A major QTL and a minor QTL identified in this study provide new insights into the genetic architecture of peanut smut resistance that may aid in breeding new varieties resistant to peanut smut.

Genetic fingerprinting and aflatoxin production of Aspergillus section Flavi associated with groundnut in eastern Ethiopia.

BACKGROUND: Aspergillus species cause aflatoxin contamination in groundnut kernels, being a health threat in agricultural products and leading to commodity rejection by domestic and international markets. Presence of Aspergillus flavus and A. parasiticus colonizing groundnut in eastern Ethiopia, as well as presence of aflatoxins have been reported, though in this region, no genetic studies have been done of these species in relation to their aflatoxin production. RESULTS: In this study, 145 Aspergillus isolates obtained from groundnut kernels in eastern Ethiopia were genetically fingerprinted using 23 Insertion/Deletion (InDel) markers within the aflatoxin-biosynthesis gene cluster (ABC), identifying 133 ABC genotypes. Eighty-four isolates were analyzed by Ultra-Performance Liquid Chromatography (UPLC) for in vitro aflatoxin production. Analysis of genetic distances based on the approximately 85 kb-ABC by Neighbor Joining (NJ), 3D-Principal Coordinate Analysis (3D-PCoA), and Structure software, clustered the isolates into three main groups as a gradient in their aflatoxin production. Group I, contained 98% A. flavus, including L- and non-producers of sclerotia (NPS), producers of B1 and B2 aflatoxins, and most of them collected from the lowland-dry Babile area. Group II was a genetic admixture population of A. flavus (NPS) and A. flavus S morphotype, both low producers of aflatoxins. Group III was primarily represented by A. parasiticus and A. flavus S morphotype isolates both producers of B1, B2 and G1, G2 aflatoxins, and originated from the regions of Darolabu and Gursum. The highest in vitro producer of aflatoxin B1 was A. flavus NPS N1436 (77.98 µg/mL), and the highest producer of aflatoxin G1 was A. parasiticus N1348 (50.33 µg/mL), these isolates were from Gursum and Darolabu, respectively. CONCLUSIONS: To the best of our knowledge, this is the first study that combined the use of InDel fingerprinting of the ABC and corresponding aflatoxin production capability to describe the genetic diversity of Aspergillus isolates from groundnut in eastern Ethiopia. Three InDel markers, AFLC04, AFLC08 and AFLC19, accounted for the main assignment of individuals to the three Groups; their loci corresponded to aflC (pksA), hypC, and aflW (moxY) genes, respectively. Despite InDels within the ABC being often associated to loss of aflatoxin production, the vast InDel polymorphism observed in the Aspergillus isolates did not completely impaired their aflatoxin production in vitro.

Genetic diversity of aflatoxin-producing Aspergillus flavus isolated from selected groundnut growing agro-ecological zones of Uganda.

BACKGROUND: Groundnut pre- and post-harvest contamination is commonly caused by fungi from the Genus Aspergillus. Aspergillus flavus is the most important of these fungi. It belongs to section Flavi; a group consisting of aflatoxigenic (A. flavus, A. parasiticus and A. nomius) and non-aflatoxigenic (A. oryzae, A. sojae and A. tamarii) fungi. Aflatoxins are food-borne toxic secondary metabolites of Aspergillus species associated with severe hepatic carcinoma and children stuntedness. Despite the well-known public health significance of aflatoxicosis, there is a paucity of information about the prevalence, genetic diversity and population structure of A. flavus in different groundnut growing agro-ecological zones of Uganda. This cross-sectional study was therefore conducted to fill this knowledge gap. RESULTS: The overall pre- and post-harvest groundnut contamination rates with A. flavus were 30.0 and 39.2% respectively. Pre- and post-harvest groundnut contamination rates with A. flavus across AEZs were; 2.5 and 50.0%; (West Nile), 55.0 and 35.0% (Lake Kyoga Basin) and 32.5 and 32.5% (Lake Victoria Basin) respectively. There was no significant difference (χ2 = 2, p = 0.157) in overall pre- and post-harvest groundnut contamination rates with A. flavus and similarly no significant difference (χ2 = 6, p = 0.199) was observed in the pre- and post-harvest contamination of groundnut with A. flavus across the three AEZs. The LKB had the highest incidence of aflatoxin-producing Aspergillus isolates while WN had no single Aspergillus isolate with aflatoxin-producing potential. Aspergillus isolates from the pre-harvest groundnut samples had insignificantly higher incidence of aflatoxin production (χ2 = 2.667, p = 0.264) than those from the post-harvest groundnut samples. Overall, A. flavus isolates exhibited moderate level (92%, p = 0.02) of genetic diversity across the three AEZs and low level (8%, p = 0.05) of genetic diversity within the individual AEZs. There was a weak positive correlation (r = 0.1241, p = 0.045) between genetic distance and geographic distance among A. flavus populations in the LKB, suggesting that genetic differentiation in the LKB population might be associated to geographic distance. A very weak positive correlation existed between genetic variation and geographic location in the entire study area (r = 0.01, p = 0.471), LVB farming system (r = 0.0141, p = 0.412) and WN farming system (r = 0.02, p = 0.478). Hierarchical clustering using the unweighted pair group method with arithmetic means (UPGMA) revealed two main clusters of genetically similar A. flavus isolates. CONCLUSIONS: These findings provide evidence that genetic differentiation in A. flavus populations is independent of geographic distance. This information can be valuable in the development of a suitable biocontrol management strategy of aflatoxin-producing A. flavus.

New tools to screen wild peanut species for aflatoxin accumulation and genetic fingerprinting.

BACKGROUND: Aflatoxin contamination in peanut seeds is still a serious problem for the industry and human health. No stable aflatoxin resistant cultivars have yet been produced, and given the narrow genetic background of cultivated peanuts, wild species became an important source of genetic diversity. Wild peanut seeds, however, are not abundant, thus, an effective method of screening for aflatoxin accumulation using minimal seeds is highly desirable. In addition, keeping record of genetic fingerprinting of each accession would be very useful for breeding programs and for the identification of accessions within germplasm collections. RESULTS: In this study, we report a method of screening for aflatoxin accumulation that is applicable to the small-size seeds of wild peanuts, increases the reliability by testing seed viability, and records the genetic fingerprinting of the samples. Aflatoxin levels observed among 20 wild peanut species varied from zero to 19000 ng.g-1 and 155 ng.g-1 of aflatoxin B1 and B2, respectively. We report the screening of 373 molecular markers, including 288 novel SSRs, tested on 20 wild peanut species. Multivariate analysis by Neighbor-Joining, Principal Component Analysis and 3D-Principal Coordinate Analysis using 134 (36 %) transferable markers, in general grouped the samples according to their reported genomes. The best 88 markers, those with high fluorescence, good scorability and transferability, are reported with BLAST results. High quality markers (total 98) that discriminated genomes are reported. A high quality marker with UPIC score 16 (16 out of 20 species discriminated) had significant hits on BLAST2GO to a pentatricopeptide-repeat protein, another marker with score 5 had hits on UDP-D-apiose synthase, and a third one with score 12 had BLASTn hits on La-RP 1B protein. Together, these three markers discriminated all 20 species tested. CONCLUSIONS: This study provides a reliable method to screen wild species of peanut for aflatoxin resistance using minimal seeds. In addition we report 288 new SSRs for peanut, and a cost-effective combination of markers sufficient to discriminate all 20 species tested. These tools can be used for the systematic search of aflatoxin resistant germplasm keeping record of the genetic fingerprinting of the accessions tested for breeding purpose.

Study of the genetic diversity of the aflatoxin biosynthesis cluster in Aspergillus section Flavi using insertion/deletion markers in peanut seeds from Georgia, USA.

Aflatoxins are among the most powerful carcinogens in nature. The major aflatoxin-producing fungi are Aspergillus flavus and A. parasiticus. Numerous crops, including peanut, are susceptible to aflatoxin contamination by these fungi. There has been an increased use of RNA interference (RNAi) technology to control phytopathogenic fungi in recent years. In order to develop molecular tools targeting specific genes of these fungi for the control of aflatoxins, it is necessary to obtain their genome sequences. Although high-throughput sequencing is readily available, it is still impractical to sequence the genome of every isolate. Thus, in this work, the authors proposed a workflow that allowed prescreening of 238 Aspergillus section Flavi isolates from peanut seeds from Georgia, USA. The aflatoxin biosynthesis cluster (ABC) of the isolates was fingerprinted at 25 InDel (insertion/deletion) loci using capillary electrophoresis. All isolates were tested for aflatoxins using ultra-high-performance liquid chromatography. The neighbor-joining, three-dimension (3D) principal coordinate, and Structure analyses revealed that the Aspergillus isolates sampled consisted of three main groups determined by their capability to produce aflatoxins. Group I comprised 10 non-aflatoxigenic A. flavus; Group II included A. parasiticus; and Group III included mostly aflatoxigenic A. flavus and the three non-aflatoxigenic A. caelatus. Whole genomes of 10 representative isolates from different groups were sequenced. Although InDels in Aspergillus have been used by other research groups, this is the first time that the cluster analysis resulting from fingerprinting was followed by whole-genome sequencing of representative isolates. In our study, cluster analysis of ABC sequences validated the results obtained with fingerprinting. This shows that InDels used here can predict similarities at the genome level. Our results also revealed a relationship between groups and their capability to produce aflatoxins. The database generated of Aspergillus spp. can be used to select target genes and assess the effectiveness of RNAi technology to reduce aflatoxin contamination in peanut.

Development of a Large Set of Microsatellite Markers in Zapote Mamey (Pouteria sapota (Jacq.) H.E. Moore & Stearn) and Their Potential Use in the Study of the Species.

Pouteria sapota is known for its edible fruits that contain unique carotenoids, as well as for its fungitoxic, anti-inflammatory and anti-oxidant activity. However, its genetics is mostly unknown, including aspects about its genetic diversity and domestication process. We did high-throughput sequencing of microsatellite-enriched libraries of P. sapota, generated 5223 contig DNA sequences, 1.8 Mbp, developed 368 microsatellites markers and tested them on 29 individuals from 10 populations (seven wild, three cultivated) from Mexico, its putative domestication center. Gene ontology BLAST analysis of the DNA sequences containing microsatellites showed potential association to physiological functions. Genetic diversity was slightly higher in cultivated than in the wild gene pool (HE = 0.41 and HE = 0.35, respectively), although modified Garza-Williamson Index and Bottleneck software showed evidence for a reduction in genetic diversity for the cultivated one. Neighbor Joining, 3D Principal Coordinates Analysis and assignment tests grouped most individuals according to their geographic origin but no clear separation was observed between wild or cultivated gene pools due to, perhaps, the existence of several admixed populations. The developed microsatellites have a great potential in genetic population and domestication studies of P. sapota but additional sampling will be necessary to better understand how the domestication process has impacted the genetic diversity of this fruit crop.

Genetic diversity, disease resistance, and environmental adaptation of Arachis duranensis L.: New insights from landscape genomics.

The genetic diversity that exists in natural populations of Arachis duranensis, the wild diploid donor of the A subgenome of cultivated tetraploid peanut, has the potential to improve crop adaptability, resilience to major pests and diseases, and drought tolerance. Despite its potential value for peanut improvement, limited research has been focused on the association between allelic variation, environmental factors, and response to early (ELS) and late leaf spot (LLS) diseases. The present study implemented a landscape genomics approach to gain a better understanding of the genetic variability of A. duranensis represented in the ex-situ peanut germplasm collection maintained at the U.S. Department of Agriculture, which spans the entire geographic range of the species in its center of origin in South America. A set of 2810 single nucleotide polymorphism (SNP) markers allowed a high-resolution genome-wide characterization of natural populations. The analysis of population structure showed a complex pattern of genetic diversity with five putative groups. The incorporation of bioclimatic variables for genotype-environment associations, using the latent factor mixed model (LFMM2) method, provided insights into the genomic signatures of environmental adaptation, and led to the identification of SNP loci whose allele frequencies were correlated with elevation, temperature, and precipitation-related variables (q < 0.05). The LFMM2 analysis for ELS and LLS detected candidate SNPs and genomic regions on chromosomes A02, A03, A04, A06, and A08. These findings highlight the importance of the application of landscape genomics in ex situ collections of peanut and other crop wild relatives to effectively identify favorable alleles and germplasm for incorporation into breeding programs. We report new sources of A. duranensis germplasm harboring adaptive allelic variation, which have the potential to be utilized in introgression breeding for a single or multiple environmental factors, as well as for resistance to leaf spot diseases.

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds.

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate economically important agricultural commodities, including peanuts, posing a high risk to human and animal health. Due to the narrow genetic base, peanut cultivars demonstrate limited resistance to fungal pathogens. Therefore, numerous wild peanut species with tolerance to Aspergillus have received substantial consideration by scientists as sources of disease resistance. Exploring plant germplasm for resistance to aflatoxins is difficult since aflatoxin accumulation does not follow a normal distribution, which dictates the need for the analyses of thousands of single peanut seeds. Sufficiently hydrated peanut (Arachis spp.) seeds, when infected by Aspergillus species, are capable of producing biologically active stilbenes (stilbenoids) that are considered defensive phytoalexins. Peanut stilbenes inhibit fungal development and aflatoxin production. Therefore, it is crucial to analyze the same seeds for peanut stilbenoids to explain the nature of seed resistance/susceptibility to the Aspergillus invasion. None of the published methods offer single-seed analyses for aflatoxins and/or stilbene phytoalexins. We attempted to fulfill the demand for such a method that is environment-friendly, uses inexpensive consumables, and is sensitive and selective. In addition, the method is non-destructive since it uses only half of the seed and leaves the other half containing the embryonic axis intact. Such a technique allows germination and growth of the peanut plant to full maturity from the same seed used for the aflatoxin and stilbenoid analysis. The integrated part of this method, the manual challenging of the seeds with Aspergillus, is a limiting step that requires more time and labor compared to other steps in the method. The method has been used for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus and to determine and characterize novel sources of genetic resistance to this fungal pathogen.

Draft genome sequence data of Nothopassalora personata, peanut foliar pathogen from Argentina.

Late leaf spot (LLS) caused by the Ascomycete Nothopassalora personata (N.p.) (Syn. Cercosporidium personatum) is the main foliar disease of peanuts in Argentina and in peanut producing areas of the world, causing up to 70% yield losses. The extremely slow growth of this fungus in culture, that takes around one month to form a 1 cm colony (0.45 mm/day), and the lack of adequate young tissues from where to extract nucleic acids, have hindered genetic studies of this pathogen. Here, we report the first genome sequence of a N. personata isolate from South America, as well as genetic variants on its conserved genes, and the complete sequence of its mating-type locus MAT1-2 idiomorph. The N. personata isolate IPAVE 0302 was obtained from peanut leaves in Córdoba, Argentina. The whole genome sequencing of IPAVE 0302 was performed as paired end 150 bp NovaSeq 6000 and de novo assembled. Clean reads were mapped to the reference genome for this species NRRL 64463 and the genetic variants on highly conserved genes and throughout the genome were analyzed. Sequencing data were submitted to NCBI GenBank Bioproject PRJNA948451, accession number SRR23957761. Additional Fasta files are available from Harvard Dataverse (https://doi.org/10.7910/DVN/9AGPMG and https://doi.org/10.7910/DVN/YDO3V6). The data reported here will be the basis for the analysis of genetic diversity of the LLS pathogen of peanut in Argentina, information that is critical to make decisions on management strategies.

First draft genome and transcriptome of Cercosporidium personatum, causal agent of late leaf spot disease of peanut.

OBJECTIVE: Two main fungal leaf spot diseases occur in peanut, namely early leaf spot (ELS) and late leaf spot (LLS), these cause a yearly average of $44 million losses. Limited genetic information, 3534 bp of sequencing, exists about the causal agent of LLS, Cercosporidium personatum (syn. Nothopassalora personata, syn. Phaeoisariopsis personata). The extremely slow growth of this fungus, approximately 1 cm colony in 6 months, and challenges in nucleic acid extractions have hindered research on LLS. Our goal in this work is to provide a reference genome for research on this pathogen. RESULTS: Whole genome and transcriptome sequencing of the LLS fungus were obtained. A total of 233,542,110 reads of the genome were de novo assembled resulting in 1061 scaffolds, and estimated genome size 27,597,787 bp. RNA sequencing resulted in 11,848,198 reads that were de novo assembled into 13,343 contigs. Genome annotation resulted in 10,703 putative genes. BUSCO analysis of the genome and annotation resulted in 91.1% and 89.5% completeness, respectively. Phylogenetic dendrograms for 5442 bp and 4401 bp of RNA Polymerase II largest and second largest subunits, and for 5474 bp of the ribosomal RNA cistron of C. personatum are presented in relation to closely related fungi.

First draft genome of Thecaphora frezii, causal agent of peanut smut disease.

OBJECTIVES: The fungal pathogen Thecaphora frezii Carranza & Lindquist causes peanut smut, a severe disease currently endemic in Argentina. To study the ecology of T. frezii and to understand the mechanisms of smut resistance in peanut plants, it is crucial to know the genetics of this pathogen. The objective of this work was to isolate the pathogen and generate the first draft genome of T. frezii that will be the basis for analyzing its potential genetic diversity and its interaction with peanut cultivars. Our research group is working to identify peanut germplasm with smut resistance and to understand the genetics of the pathogen. Knowing the genome of T. frezii will help analyze potential variants of this pathogen and contribute to develop enhanced peanut germplasm with broader and long-lasting resistance. DATA DESCRIPTION: Thecaphora frezii isolate IPAVE 0401 (here referred as T.f.B7) was obtained from a single hyphal-tip culture, its DNA was sequenced using Pacific Biosciences Sequel II (PacBio) and Illumina NovaSeq6000 (Nova). Data from both sequencing platforms were combined and the de novo assembling estimated a 29.3 Mb genome size. Completeness of the genome examined using Benchmarking Universal Single-Copy Orthologs (BUSCO) showed the assembly had 84.6% of the 758 genes in fungi_odb10.

Transformation of Major Peanut (Arachis hypogaea) Stilbenoid Phytoalexins Caused by Selected Microorganisms.

The peanut plant accumulates defensive stilbenoid phytoalexins in response to the presence of soil fungi, which in turn produce phytoalexin-detoxifying enzymes for successfully invading the plant host. Aspergillus spp. are opportunistic pathogens that invade peanut seeds; most common fungal species often produce highly carcinogenic aflatoxins. The purpose of the present research was to evaluate the in vitro dynamics of peanut phytoalexin transformation/detoxification by important fungal species. This work revealed that in feeding experiments, Aspergillus spp. from section Flavi were capable of degrading the major peanut phytoalexin, arachidin-3, into its hydroxylated homolog, arachidin-1, and a benzenoid, SB-1. However, Aspergillus niger from section Nigri as well as other fungal and bacterial species tested, which are not known to be involved in the infection of the peanut plant, were incapable of changing the structure of arachidin-3. The results of feeding experiments with arachidin-1 and resveratrol are also reported. The research provided new knowledge on the dynamics of peanut stilbenoid transformations by essential fungi. These findings may contribute to the elucidation of the phytoalexin detoxification mechanism involved in the infection of peanut by important toxigenic Aspergillus spp.

Gibberellin-associated cisgenes modify growth, stature and wood properties in Populus.

We studied the effects on plant growth from insertion of five cisgenes that encode proteins involved in gibberellin metabolism or signalling. Intact genomic copies of PtGA20ox7, PtGA2ox2,Pt RGL1_1, PtRGL1_2 and PtGAI1 genes from the genome-sequenced Populus trichocarpa clone Nisqually-1 were transformed into Populus tremula × alba (clone INRA 717-1B4), and growth, morphology and xylem cell size characterized in the greenhouse. Each cisgene encompassed 1-2 kb of 5' and 1 kb of 3' flanking DNA, as well as all native exons and introns. Large numbers of independent insertion events per cisgene (19-38), including empty vector controls, were studied. Three of the cisgenic modifications had significant effects on plant growth rate, morphology or wood properties. The PtGA20ox7 cisgene increased rate of shoot regeneration in vitro, accelerated early growth, and variation in growth rate was correlated with PtGA20ox7 gene expression. PtRGL1_1 and PtGA2ox2 caused reduced growth, while PtRGL1_2 gave rise to plants that grew normally but had significantly longer xylem fibres. RT-PCR studies suggested that the lack of growth inhibition observed in PtRGL1_2 cisgenic plants was a result of co-suppression. PtGAI1 slowed regeneration rate and both PtGAI1 and PtGA20ox7 gave rise to increased variance among events for early diameter and volume index, respectively. Our work suggests that cisgenic insertion of additional copies of native genes involved in growth regulation may provide tools to help modify plant architecture, expand the genetic variance in plant architecture available to breeders and accelerate transfer of alleles between difficult-to-cross species.

Genotyping tools and resources to assess peanut germplasm: smut-resistant landraces as a case study.

Peanut smut caused by Thecaphora frezii is a severe fungal disease currently endemic to Argentina and Brazil. The identification of smut resistant germplasm is crucial in view of the potential risk of a global spread. In a recent study, we reported new sources of smut resistance and demonstrated its introgression into elite peanut cultivars. Here, we revisited one of these sources (line I0322) to verify its presence in the U.S. peanut germplasm collection and to identify single nucleotide polymorphisms (SNPs) potentially associated with resistance. Five accessions of Arachis hypogaea subsp. fastigiata from the U.S. peanut collection, along with the resistant source and derived inbred lines were genotyped with a 48K SNP peanut array. A recently developed SNP genotyping platform called RNase H2 enzyme-based amplification (rhAmp) was further applied to validate selected SNPs in a larger number of individuals per accession. More than 14,000 SNPs and nine rhAmp assays confirmed the presence of a germplasm in the U.S. peanut collection that is 98.6% identical (P < 0.01, bootstrap t-test) to the resistant line I0322. We report this germplasm with accompanying genetic information, genotyping data, and diagnostic SNP markers.

Sixteen Draft Genome Sequences Representing the Genetic Diversity of Aspergillus flavus and Aspergillus parasiticus Colonizing Peanut Seeds in Ethiopia.

Draft genomes of 16 isolates of Aspergillus flavus Link and Aspergillus parasiticus Speare, identified as the predominant genotypes colonizing peanuts in four farming regions in Ethiopia, are reported. These data will allow mining for sequences that could be targeted by RNA interference to prevent aflatoxin accumulation in peanut seeds.

Draft genome assembly of Passalora sequoiae a needle blight pathogen on Leyland cypress.

OBJECTIVE: Passalora sequoiae (family Mycosphaerellaceae) causes a twig blight on Leyland cypress that requires numerous fungicide applications annually to minimize economic losses for ornamental plant nursery and Christmas tree producers. The objective was to generate a high-quality draft assembly of the genome of P. sequoiae as a resource for primer development to investigate genotype diversity. DATA DESCRIPTION: We report here the genome sequence of P. sequoiae 9LC2 that was isolated from Leyland cypress 'Leighton Green' in 2017 in southern Mississippi, USA. The draft genome was obtained using Pacific Biosciences (PacBio) SMRT and Illumina HiSeq 2500 sequencing. Illumina reads were mapped to PacBio assembled contigs to determine base call consistency. Based on a total of 44 contigs with 722 kilobase (kb) average length (range 9.4 kb to 3.4 Mb), the whole genome size was estimated at 31,768,716 bp. Mapping of Illumina reads to PacBio contigs resulted in a 1000 × coverage and were used to confirm accuracy of the consensus sequences.

Analysis of small RNA populations generated in peanut leaves after exogenous application of dsRNA and dsDNA targeting aflatoxin synthesis genes.

Previously, we have shown that RNA interference (RNAi) can prevent aflatoxin accumulation in transformed peanuts. To explore aflatoxin control by exogenous delivery of double-strand RNA (dsRNA) it is necessary to understand the generation of small RNA (sRNA) populations. We sequenced 12 duplicate sRNA libraries of in-vitro-grown peanut plants, 24 and 48 h after exogenous application of five gene fragments (RNAi-5x) related to aflatoxin biosynthesis in Aspergillus flavus. RNAi-5x was applied either as double-stranded RNA (dsRNA) or RNAi plasmid DNA (dsDNA). Small interfering RNAs (siRNAs) derived from RNAi-5x were significantly more abundant at 48 h than at 24 h, and the majority mapped to the fragment of aflatoxin efflux-pump gene. RNAi-5x-specific siRNAs were significantly, three to fivefold, more abundant in dsDNA than dsRNA treatments. Further examination of known micro RNAs related to disease-resistance, showed significant down-regulation of miR399 and up-regulation of miR482 in leaves treated with dsDNA compared to the control. These results show that sRNA sequencing is useful to compare exogenous RNAi delivery methods on peanut plants, and to analyze the efficacy of molecular constructs to generate siRNAs against specific gene targets. This work lays the foundation for non-transgenic delivery of RNAi in controlling aflatoxins in peanut.

Microsatellites reveal genetic diversity in Rotylenchulus reniformis populations.

Rotylenchulus reniformis is the predominant parasitic nematode of cotton in the Mid South area of the United States. Although variable levels of infection and morphological differences have been reported for this nematode, genetic variability has been more elusive. We developed microsatellite-enriched libraries for R. reniformis, produced 1152 clones, assembled 694 contigs, detected 783 simple sequence repeats (SSR) and designed 192 SSR-markers. The markers were tested on six R. reniformis cultures from four states, Texas, Louisiana, Mississippi and Georgia, in the USA. Based on performance we selected 156 SSR markers for R. reniformis from which 88 were polymorphic across the six reniform nematode populations, showing as the most frequent motif the dinucleotide AG. The polymorphic information content of the markers ranged from 0.00 to 0.82, and the percentage of multiallelic loci of the isolates was between 40.9 and 45.1%. An interesting finding in this study was the genetic variability detected among the three Mississippi isolates, for which 22 SSR markers were polymorphic. We also tested the level of infection of these isolates on six cotton genotypes, where significant differences were found between the Texas and Georgia isolates. Coincidentally, 62 polymorphic markers were able to distinguish these two populations. Further studies will be necessary to establish possible connections, if any, between markers and level of pathogenicity of the nematode. The SSR markers developed here will be useful in the assessment of the genetic diversity of this nematode, could assist in management practices for control of reniform nematode, be used in breeding programs for crop resistance, and help in detecting the origin and spread of this nematode in the United States.

Introgression of peanut smut resistance from landraces to elite peanut cultivars (Arachis hypogaea L.).

Smut disease caused by the fungal pathogen Thecaphora frezii Carranza & Lindquist is threatening the peanut production in Argentina. Fungicides commonly used in the peanut crop have shown little or no effect controlling the disease, making it a priority to obtain peanut varieties resistant to smut. In this study, recombinant inbred lines (RILs) were developed from three crosses between three susceptible peanut elite cultivars (Arachis hypogaea L. subsp. hypogaea) and two resistant landraces (Arachis hypogaea L. subsp. fastigiata Waldron). Parents and RILs were evaluated under high inoculum pressure (12000 teliospores g-1 of soil) over three years. Disease resistance parameters showed a broad range of variation with incidence mean values ranging from 1.0 to 35.0% and disease severity index ranging from 0.01 to 0.30. Average heritability (h2) estimates of 0.61 to 0.73 indicated that resistance in the RILs was heritable, with several lines (4 to 7 from each cross) showing a high degree of resistance and stability over three years. Evidence of genetic transfer between genetically distinguishable germplasm (introgression in a broad sense) was further supported by simple-sequence repeats (SSRs) and Insertion/Deletion (InDel) marker genotyping. This is the first report of smut genetic resistance identified in peanut landraces and its introgression into elite peanut cultivars.

Efficiency of gene silencing in Arabidopsis: direct inverted repeats vs. transitive RNAi vectors.

We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required.