Noelle A. Barkley

Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population

BackgroundSorghum [Sorghum bicolor (L.) Moench] is ranked as the fifth most important grain crop and serves as a major food staple and fodder resource for much of the world, especially in arid and semi-arid regions. The recent surge in sorghum research is driven by its tolerance to drought/heat stresses and its strong potential as a bioenergy feedstock. Completion of the sorghum genome sequence has opened new avenues for sorghum functional genomics. However, the availability of genetic resources, specifically mutant lines, is limited. Chemical mutagenesis of sorghum germplasm, followed by screening for mutants altered in important agronomic traits, represents a rapid and effective means of addressing this limitation. Induced mutations in novel genes of interest can be efficiently assessed using the technique known as Targeting Induced Local Lesion IN Genomes (TILLING).ResultsA sorghum mutant population consisting of 1,600 lines was generated from the inbred line BTx623 by treatment with the chemical agent ethyl methanesulfonate (EMS). Numerous phenotypes with altered morphological and agronomic traits were observed from M2 and M3 lines in the field. A subset of 768 mutant lines was analyzed by TILLING using four target genes. A total of five mutations were identified resulting in a calculated mutation density of 1/526 kb. Two of the mutations identified by TILLING and verified by sequencing were detected in the gene encoding caffeic acid O-methyltransferase (COMT) in two independent mutant lines. The two mutant lines segregated for the expected brown midrib (bmr) phenotype, a trait associated with altered lignin content and increased digestibility.ConclusionTILLING as a reverse genetic approach has been successfully applied to sorghum. The diversity of the mutant phenotypes observed in the field, and the density of induced mutations calculated from TILLING indicate that this mutant population represents a useful resource for members of the sorghum research community. Moreover, TILLING has been demonstrated to be applicable for sorghum functional genomics by evaluating a small subset of the EMS-induced mutant lines.

The genome sequences of Arachis duranensis and Arachis ipaensis , the diploid ancestors of cultivated peanut

Cultivated peanut (Arachis hypogaea) is an allotetraploid with closely related subgenomes of a total size of ∼2.7 Gb. This makes the assembly of chromosomal pseudomolecules very challenging. As a foundation to understanding the genome of cultivated peanut, we report the genome sequences of its diploid ancestors (Arachis duranensis and Arachis ipaensis). We show that these genomes are similar to cultivated peanuts A and B subgenomes and use them to identify candidate disease resistance genes, to guide tetraploid transcript assemblies and to detect genetic exchange between cultivated peanuts subgenomes. On the basis of remarkably high DNA identity of the A. ipaensis genome and the B subgenome of cultivated peanut and biogeographic evidence, we conclude that A. ipaensis may be a direct descendant of the same population that contributed the B subgenome to cultivated peanut.

Genetic diversity and population structure analysis of accessions in the US historic sweet sorghum collection

Sweet sorghum has the potential to become a versatile feedstock for large-scale bioenergy production given its sugar from stem juice, cellulose/hemicellulose from stalks, and starch from grain. However, for researchers to maximize its feedstock potential a first step includes additional evaluations of the 2,180 accessions with varied origins in the US historic sweet sorghum collection. To assess genetic diversity of this collection for bioenergy breeding and population structure for association mapping, we selected 96 accessions and genotyped them with 95 simple sequence repeat markers. Subsequent genetic diversity and population structure analysis methods identified four subpopulations in this panel, which correlated well with the geographic locations where these accessions originated or were collected. Model comparisons for three quantitative traits revealed different levels of population structure effects on flowering time, plant height, and brix. Our results suggest that diverse germplasm accessions curated from different geographical regions should be considered for plant breeding programs to develop sweet sorghum cultivars or hybrids, and that this sweet sorghum panel can be further explored for association mapping.

Population structure and marker–trait association analysis of the US peanut (Arachis hypogaea L.) mini-core collection

Peanut (Arachis hypogaea L.) is one of the most important oilseed and nutritional crops in the world. To efficiently utilize the germplasm collection, a peanut mini-core containing 112 accessions was established in the United States. To determine the population structure and its impact on marker–trait association, this mini-core collection was assessed by genotyping 94 accessions with 81 SSR markers and two functional SNP markers from fatty acid desaturase 2 (FAD2). Seed quality traits (including oil content, fatty acid composition, flavonoids, and resveratrol) were obtained through nuclear magnetic resonance (NMR), gas chromatography (GC), and high-performance liquid chromatography (HPLC) analysis. Genetic diversity and population structure analysis identified four major subpopulations that are related to four botanical varieties. Model comparison with different levels of population structure and kinship control was conducted for each trait and association analyses with the selected models verified that the functional SNP from the FAD2A gene is significantly associated with oleic acid (C18:1), linoleic acid (C18:2), and oleic-to-linoleic (O/L) ratio across this diverse collection. Even though the allele distribution of FAD2A was structured among the four subpopulations, the effect of FAD2A gene remained significant after controlling population structure and had a likelihood-ratio-based R2 (RLR2) value of 0.05 (oleic acid), 0.09 (linoleic acid), and 0.07 (O/L ratio) because the FAD2A alleles were not completely fixed within subpopulations. Our genetic analysis demonstrated that this peanut mini-core panel is suitable for association mapping. Phenotypic characterization for seed quality traits and association testing of the functional SNP from FAD2A gene provided information for further breeding and genetic research.

Transfer of simple sequence repeat (SSR) markers from major cereal crops to minor grass species for germplasm characterization and evaluation

A major challenge for the molecular characterization and evaluation of minor grass species germplasm is the lack of sufficient DNA markers. A set of 210 simple sequence repeat (SSR) markers developed from major cereal crops (self-pollinated wheat and rice, mainly self-pollinated sorghum and out-crossing maize) were evaluated for their transferability to minor grass species (finger millet, Eleusine coracana ; seashore paspalum, Paspalum vaginatum ; and bermudagrass, Cynodon dactylon ). In total, 412 cross-species polymorphic amplicons were identified. Over half of the primers generated reproducible cross-species or cross-genus amplicons. The transfer rate of SSR markers was correlated with the phylogenetic relationship (or genetic relatedness) of these species. The average transfer rate of genomic SSR markers was different from the average transfer rate of expressed sequence tag (EST)-SSR markers. The level of polymorphism was significantly higher among species (67%) than within species (34%), and was related to the degree of out-crossing for each species. The level of polymorphism detected within species was 57% from self-incompatible species, 39% from out-crossing species and 20% from self-pollinated species. Genomic SSRs detected a higher level of polymorphism than EST-SSRs. The use of transferred polymorphic SSR markers for the characterization and evaluation of germplasm is discussed.

A Simple Allele-Specific PCR Assay for Detecting FAD2 Alleles in Both A and B Genomes of the Cultivated Peanut for High-Oleate Trait Selection

In cultivated tetraploid peanut (2n = 4x = 40, AABB), the conversion of oleic acid to linoleic acid is mainly catalyzed by the Δ12 fatty acid desaturase (FAD). Two homoeologous genes (FAD2A and FAD2B) encoding for the desaturase are located on the A and B genomes, respectively. Abolishing or reducing the desaturase activity by gene mutation can significantly increase the oleic acid/linoleic acid ratio. F435-derived high-oleate peanut cultivars contain two key mutations within the Δ12 fatty acid desaturase gene which include a 1-bp substitution of G:C→A:T in the A genome and a 1-bp insertion of A:T in the B genome. Both of these mutations contribute to abolishing or reducing the desaturase activity, leading to accumulation of oleate versus linoleate. Currently, detection of FAD2 alleles can be achieved by a cleaved amplified polymorphic sequence marker for the A genome and a real-time polymerase chain reaction (PCR) marker for the B genome; however, detection of these key mutations has to use different assay platforms. Therefore, a simple PCR assay for detection of FAD2 alleles on both genomes was developed by designing allele-specific primers and altering PCR annealing temperatures. This assay was successfully used for detecting FAD2 alleles in peanut. Gas chromatography (GC) was used to determine fatty acid composition of PCR-assayed genotypes. The results from the PCR assay and GC analysis were consistent. This PCR assay is quick, reliable, economical, and easy to use. Implementation of this PCR assay will greatly enhance the efficiency of germplasm characterization and marker-assisted selection of high oleate in peanut.

Genetic diversity of cultivated and wild-type peanuts evaluated with M13-tailed SSR markers and sequencing

Thirty-one genomic SSR markers with a M13 tail attached were used to assess the genetic diversity of the peanut mini core collection. The M13-tailed method was effective in discriminating almost all the cultivated and wild accessions. A total of 477 alleles were detected with an average of 15.4 alleles per locus. The mean polymorphic information content (PIC) score was 0.687. The cultivated peanut (Arachis hypogaea L.) mini core produced a total of 312 alleles with an average of 10.1 alleles per locus. A neighbour-joining tree was constructed to determine the interspecific and intraspecific relationships in this data set. Almost all the peanut accessions in this data set classified into subspecies and botanical varieties such as subsp. hypogaea var. hypogaea, subsp. fastigiata var. fastigiata, and subsp. fastigiata var. vulgaris clustered with other accessions with the same classification, which lends further support to their current taxonomy. Alleles were sequenced from one of the SSR markers used in this study, which demonstrated that the repeat motif is conserved when transferring the marker across species borders. This study allowed the examination of the diversity and phylogenetic relationships in the peanut mini core which has not been previously reported.

Development of a real-time PCR genotyping assay to identify high oleic acid peanuts ( Arachis hypogaea L.)

Oleic acid, a monounsaturated, omega-9 fatty acid found in peanut (Arachis hypogaea L.) oil is an important seed quality trait because it provides increased shelf life, improved flavor, enhanced fatty acid composition, and has a beneficial effect on human health. Hence, a concentrated effort has been put forth on developing peanut cultivars that have high oleic acid (>74%) and a low amount (<10%) of linoleic acid, a polyunsaturated omega-6 fatty acid. A main bottleneck, however, in breeding research is fast selection of the trait(s) of interest. Therefore, in an effort to expedite breeding efforts, a real-time PCR genotyping assay was developed to rapidly identify the wild type and the mutant allele that are responsible for normal or high levels of oleic acid, respectively in peanut seeds. This test utilizes two TaqMan® probes to detect the presence of an indel (insertion/deletion) in FAD2B and can be employed on DNA extracted from either seeds or leaves. The presence of the insertion (mutant allele) in fad2B causes a frameshift downstream in the coding sequence that ultimately alters the mRNA transcript level, and thus, decreases the activity of microsomal oleoyl-PC desaturase enzyme which converts oleic acid (C18:1) to linoleic acid (C18:2). Validation of the real-time assay was carried out by quantitatively evaluating the fatty acid composition by gas chromatography (GC). Overall, this real-time PCR assay facilitates the identification of progeny carrying the high oleic acid alleles, and thus, allows early elimination of undesirable non-high oleic acid lines in segregating populations.

Discovering and verifying DNA polymorphisms in a mung bean [V. radiata (L.) R. Wilczek] collection by EcoTILLING and sequencing

BackgroundVigna radiata, which is classified in the family Fabaceae, is an important economic crop and a dietary staple in many developing countries. The species radiata can be further subdivided into varieties of which the variety sublobata is currently acknowledged as the putative progenitor of radiata. EcoTILLING was employed to identify single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELS) in a collection of Vigna radiata accessions.FindingsA total of 157 DNA polymorphisms in the collection were produced from ten primer sets when using V. radiata var. sublobata as the reference. The majority of polymorphisms detected were found in putative introns. The banding patterns varied from simple to complex as the number of DNA polymorphisms between two pooled samples increased. Numerous SNPs and INDELS ranging from 4–24 and 1–6, respectively, were detected in all fragments when pooling V. radiata var. sublobata with V. radiata var. radiata. On the other hand, when accessions of V. radiata var. radiata were mixed together and digested with CEL I relatively few SNPs and no INDELS were detected.ConclusionEcoTILLING was utilized to identify polymorphisms in a collection of mung bean, which previously showed limited molecular genetic diversity and limited morphological diversity in the flowers and pod descriptors. Overall, EcoTILLING proved to be a powerful genetic analysis tool providing the rapid identification of naturally occurring variation.

Genetic Mapping of QTLs Controlling Fatty Acids Provided Insights into the Genetic Control of Fatty Acid Synthesis Pathway in Peanut (Arachis hypogaea L.)

Peanut, a high-oil crop with about 50% oil content, is either crushed for oil or used as edible products. Fatty acid composition determines the oil quality which has high relevance to consumer health, flavor, and shelf life of commercial products. In addition to the major fatty acids, oleic acid (C18:1) and linoleic acid (C18:2) accounting for about 80% of peanut oil, the six other fatty acids namely palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), gadoleic acid (C20:1), behenic acid (C22:0), and lignoceric acid (C24:0) are accounted for the rest 20%. To determine the genetic basis and to improve further understanding on effect of FAD2 genes on these fatty acids, two recombinant inbred line (RIL) populations namely S-population (high oleic line ‘SunOleic 97R’ × low oleic line ‘NC94022’) and T-population (normal oleic line ‘Tifrunner’ × low oleic line ‘GT-C20’) were developed. Genetic maps with 206 and 378 marker loci for the S- and the T-population, respectively were used for quantitative trait locus (QTL) analysis. As a result, a total of 164 main-effect (M-QTLs) and 27 epistatic (E-QTLs) QTLs associated with the minor fatty acids were identified with 0.16% to 40.56% phenotypic variation explained (PVE). Thirty four major QTLs (>10% of PVE) mapped on five linkage groups and 28 clusters containing more than three QTLs were also identified. These results suggest that the major QTLs with large additive effects would play an important role in controlling composition of these minor fatty acids in addition to the oleic and linoleic acids in peanut oil. The interrelationship among these fatty acids should be considered while breeding for improved peanut genotypes with good oil quality and desired fatty acid composition.