Inheritance of inflorescence architecture in sorghum.

The grass inflorescence is the primary food source for humanity, and has been repeatedly shaped by human selection during the domestication of different cereal crops. Of all major cultivated cereals, sorghum [Sorghum bicolor (L.) Moench] shows the most striking variation in inflorescence architecture traits such as branch number and branch length, but the genetic basis of this variation is little understood. To study the inheritance of inflorescence architecture in sorghum, 119 recombinant inbred lines from an elite by exotic cross were grown in three environments and measured for 15 traits, including primary, secondary, and tertiary inflorescence branching. Eight characterized genes that are known to control inflorescence architecture in maize (Zea mays L.) and other grasses were mapped in sorghum. Two of these candidate genes, Dw3 and the sorghum ortholog of ramosa2, co-localized precisely with QTL of large effect for relevant traits. These results demonstrate the feasibility of using genomic and mutant resources from maize and rice (Oryza sativa L.) to investigate the inheritance of complex traits in related cereals.

Alignment of genetic maps and QTLs between inter- and intra-specific sorghum populations.

To increase the value of associated molecular tools and also to begin to explore the degree to which interspecific and intraspecific genetic variation in Sorghum is attributable to corresponding genetic loci, we have aligned genetic maps derived from two sorghum populations that share one common parent (Sorghum bicolor L. Moench accession BTx623) but differ in morphological and evolutionarily distant alternate parents (S. propinquum or S. bicolor accession IS3620C). A total of 106 well-distributed DNA markers provide for map alignment, revealing only six nominal differences in marker order that are readily explained by sampling variation or mapping of paralogous loci. We also report a total of 61 new QTLs detected from 17 traits in these crosses. Among eight corresponding traits (some new, some previously published) that could be directly compared between the two maps, QTLs for two (tiller height and tiller number) were found to correspond in a non-random manner (P<0.05). For several other traits, correspondence of subsets of QTLs narrowly missed statistical significance. In particular, several QTLs for leaf senescence were near loci previously mapped for 'stay-green' that have been implicated by others in drought tolerance. These data provide strong validation for the value of molecular tools developed in the interspecific cross for utilization in cultivated sorghum, and begin to separate QTLs that distinguish among Sorghum species from those that are informative within the cultigen (S. bicolor).

Diversity and selection in sorghum: simultaneous analyses using simple sequence repeats.

Although molecular markers and DNA sequence data are now available for many crop species, our ability to identify genetic variation associated with functional or adaptive diversity is still limited. In this study, our aim was to quantify and characterize diversity in a panel of cultivated and wild sorghums (Sorghum bicolor), establish genetic relationships, and, simultaneously, identify selection signals that might be associated with sorghum domestication. We assayed 98 simple sequence repeat (SSR) loci distributed throughout the genome in a panel of 104 accessions comprising 73 landraces (i.e., cultivated lines) and 31 wild sorghums. Evaluation of SSR polymorphisms indicated that landraces retained 86% of the diversity observed in the wild sorghums. The landraces and wilds were moderately differentiated (F st=0.13), but there was little evidence of population differentiation among racial groups of cultivated sorghums (F st=0.06). Neighbor-joining analysis showed that wild sorghums generally formed a distinct group, and about half the landraces tended to cluster by race. Overall, bootstrap support was low, indicating a history of gene flow among the various cultivated types or recent common ancestry. Statistical methods (Ewens-Watterson test for allele excess, lnRH, and F st) for identifying genomic regions with patterns of variation consistent with selection gave significant results for 11 loci (approx. 15% of the SSRs used in the final analysis). Interestingly, seven of these loci mapped in or near genomic regions associated with domestication-related QTLs (i.e., shattering, seed weight, and rhizomatousness). We anticipate that such population genetics-based statistical approaches will be useful for re-evaluating extant SSR data for mining interesting genomic regions from germplasm collections.

Microsatellite identification and characterization in peanut ( A. hypogaea L.).

A major constraint to the application of biotechnology to the improvement of the allotetraploid peanut, or groundnut ( Arachis hypogaea L.), has been the paucity of polymorphism among germplasm lines using biochemical (seed proteins, isozymes) and DNA markers (RFLPs and RAPDs). Six sequence-tagged microsatellite (STMS) markers were previously available that revealed polymorphism in cultivated peanut. Here, we identify and characterize 110 STMS markers that reveal genetic variation in a diverse array of 24 peanut landraces. The simple-sequence repeats (SSRs) were identified with a probe of two 27648-clone genomic libraries: one constructed using PstI and the other using Sau3AI/ BamHI. The most frequent, repeat motifs identified were ATT and GA, which represented 29% and 28%, respectively, of all SSRs identified. These were followed by AT, CTT, and GT. Of the amplifiable primers, 81% of ATT and 70.8% of GA repeats were polymorphic in the cultivated peanut test array. The repeat motif AT showed the maximum number of alleles per locus (5.7). Motifs ATT, GT, and GA had a mean number of alleles per locus of 4.8, 3.8, and 3.6, respectively. The high mean number of alleles per polymorphic locus, combined with their relative frequency in the genome and amenability to probing, make ATT and GA the most useful and appropriate motifs to target to generate further SSR markers for peanut.

Simple sequence repeat marker diversity in cassava landraces: genetic diversity and differentiation in an asexually propagated crop.

Cassava (Manihot esculenta) is an allogamous, vegetatively propagated, Neotropical crop that is also widely grown in tropical Africa and Southeast Asia. To elucidate genetic diversity and differentiation in the crop's primary and secondary centers of diversity, and the forces shaping them, SSR marker variation was assessed at 67 loci in 283 accessions of cassava landraces from Africa (Tanzania and Nigeria) and the Neotropics (Brazil, Colombia, Peru, Venezuela, Guatemala, Mexico and Argentina). Average gene diversity (i.e., genetic diversity) was high in all countries, with an average heterozygosity of 0.5358 +/- 0.1184. Although the highest was found in Brazilian and Colombian accessions, genetic diversity in Neotropical and African materials is comparable. Despite the low level of differentiation [F(st)(theta) = 0.091 +/- 0.005] found among country samples, sufficient genetic distance (1-proportion of shared alleles) existed between individual genotypes to separate African from Neotropical accessions and to reveal a more pronounced substructure in the African landraces. Forces shaping differences in allele frequency at SSR loci and possibly counterbalancing successive founder effects involve probably spontaneous recombination, as assessed by parent-offspring relationships, and farmer-selection for adaptation.

Identifying genes of agronomic importance in maize by screening microsatellites for evidence of selection during domestication.

Crop species experienced strong selective pressure directed at genes controlling traits of agronomic importance during their domestication and subsequent episodes of selective breeding. Consequently, these genes are expected to exhibit the signature of selection. We screened 501 maize genes for the signature of selection using microsatellites or simple sequence repeats (SSRs). We applied the Ewens-Watterson test, which can reveal deviations from a neutral-equilibrium model, as well as two nonequilibrium tests that incorporate the domestication bottleneck. We investigated two classes of SSRs: those known to be polymorphic in maize (Class I) and those previously classified as monomorphic in maize (Class II). Fifteen SSRs exhibited some evidence for selection in maize and 10 showed evidence under stringent criteria. The genes containing nonneutral SSRs are candidates for agronomically important genes. Because demographic factors can bias our tests, further independent tests of these candidates are necessary. We applied such an additional test to one candidate, which encodes a MADS box transcriptional regulator, and confirmed that this gene experienced a selective sweep during maize domestication. Genomic scans for the signature of selection offer a means of identifying new genes of agronomic importance even when gene function and the phenotype of interest are unknown.

Solid-phase PCR in microwells: effects of linker length and composition on tethering, hybridization, and extension.

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells are suitable for enzymatic detection of immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer. The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.

Evaluation of Hbr (MITE) markers for assessment of genetic relationships among maize ( Zea mays L.) inbred lines.

Recently, a new type of molecular marker has been developed that is based on the presence or absence of the miniature inverted repeat transposable element (MITE) family Heartbreaker ( Hbr) in the maize genome. These so-called Hbr markers have been shown to be stable, highly polymorphic, easily mapped, and evenly distributed throughout the maize genome. In this work, we used Hbr-derived markers for genetic characterization of a set of maize inbred lines belonging to Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups. In total, 111 markers were evaluated across 62 SS and NSS lines. Seventy six markers (68%) were shared between the two groups, and 25 of the common markers occurred at fairly low frequency (

Microsatellites in Zea - variability, patterns of mutations, and use for evolutionary studies.

To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees.

Characterization of RFLP probe sequences for gene discovery and SSR development in Sorghum bicolor (L.) Moench.

In this study, we collected and analyzed DNA sequence data for 789 previously mapped RFLP probes from Sorghum bicolor (L.) Moench. DNA sequences, comprising 894 non-redundant contigs and end sequences, were searched against three GenBank databases, nucleotide (nt), protein (nr) and EST (dbEST), using BLAST algorithms. Matching ESTs were also searched against nt and nr. Translated DNA sequences were then searched against the conserved domain database (CDD) to determine if functional domains/motifs were congruent with the proteins identified in previous searches. More than half (500/894 or 56%) of the query sequences had significant matches in at least one of the GenBank searches. Overall, proteins identified for 148 sequences (17%) were consistent among all searches, of which 66 sequences (7%) contained congruent coding domains. The RFLP probe sequences were also evaluated for the presence of simple sequence repeats (SSRs) and 60 SSRs were developed and assayed in an array of sorghum germplasm comprising inbreds, landraces and wild relatives. Overall, these SSR loci had lower levels of polymorphism ( D = 0.46, averaged over 51 polymorphic loci) compared with sorghum SSRs that were isolated by library hybridization screens ( D = 0.69, averaged over 38 polymorphic loci). This result was probably due to the relatively small proportion of di-nucleotide repeat-containing markers (42% of the total SSR loci) obtained from the DNA sequence data. These di-nucleotide markers also contained shorter repeat motifs than those isolated from genomic libraries. Based on BLAST results, 24 SSRs (40%) were located within, or near, previously annotated or hypothetical genes. We determined the location of 19 of these SSRs relative to putative coding regions. In general, SSRs located in coding regions were less polymorphic ( D = 0.07, averaged over three loci) than those from gene flanking regions, UTRs and introns ( D = 0.49, averaged over 16 loci). The sequence information and SSR loci generated through this study will be valuable for application to sorghum genetics and improvement, including gene discovery, marker-assisted selection, diversity and pedigree analyses, comparative mapping and evolutionary genetic studies.

Structure of linkage disequilibrium and phenotypic associations in the maize genome.

Association studies based on linkage disequilibrium (LD) can provide high resolution for identifying genes that may contribute to phenotypic variation. We report patterns of local and genome-wide LD in 102 maize inbred lines representing much of the worldwide genetic diversity used in maize breeding, and address its implications for association studies in maize. In a survey of six genes, we found that intragenic LD generally declined rapidly with distance (r(2) < 0.1 within 1500 bp), but rates of decline were highly variable among genes. This rapid decline probably reflects large effective population sizes in maize during its evolution and high levels of recombination within genes. A set of 47 simple sequence repeat (SSR) loci showed stronger evidence of genome-wide LD than did single-nucleotide polymorphisms (SNPs) in candidate genes. LD was greatly reduced but not eliminated by grouping lines into three empirically determined subpopulations. SSR data also supplied evidence that divergent artificial selection on flowering time may have played a role in generating population structure. Provided the effects of population structure are effectively controlled, this research suggests that association studies show great promise for identifying the genetic basis of important traits in maize with very high resolution.

Dwarf8 polymorphisms associate with variation in flowering time.

Historically, association tests have been used extensively in medical genetics, but have had virtually no application in plant genetics. One obstacle to their application is the structured populations often found in crop plants, which may lead to nonfunctional, spurious associations. In this study, statistical methods to account for population structure were extended for use with quantitative variation and applied to our evaluation of maize flowering time. Mutagenesis and quantitative trait locus (QTL) studies suggested that the maize gene Dwarf8 might affect the quantitative variation of maize flowering time and plant height. The wheat orthologs of this gene contributed to the increased yields seen in the 'Green Revolution' varieties. We used association approaches to evaluate Dwarf8 sequence polymorphisms from 92 maize inbred lines. Population structure was estimated using a Bayesian analysis of 141 simple sequence repeat (SSR) loci. Our results indicate that a suite of polymorphisms associate with differences in flowering time, which include a deletion that may alter a key domain in the coding region. The distribution of nonsynonymous polymorphisms suggests that Dwarf8 has been a target of selection.

The MITE family heartbreaker (Hbr): molecular markers in maize.

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 x Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

An EST-enriched comparative map of Brassica oleracea and Arabidopsis thaliana.

A detailed comparative map of Brassica oleracea and Arabidopsis thaliana has been established based largely on mapping of Arabidopsis ESTs in two Arabidopsis and four Brassica populations. Based on conservative criteria for inferring synteny, "one to one correspondence" between Brassica and Arabidopsis chromosomes accounted for 57% of comparative loci. Based on 186 corresponding loci detected in B. oleracea and A. thaliana, at least 19 chromosome structural rearrangements differentiate B. oleracea and A. thaliana orthologs. Chromosomal duplication in the B. oleracea genome was strongly suggested by parallel arrangements of duplicated loci on different chromosomes, which accounted for 41% of loci mapped in Brassica. Based on 367 loci mapped, at least 22 chromosomal rearrangements differentiate B. oleracea homologs from one another. Triplication of some Brassica chromatin and duplication of some Arabidopsis chromatin were suggested by data that could not be accounted for by the one-to-one and duplication models, respectively. Twenty-seven probes detected three or more loci in Brassica, which represent 25.3% of the 367 loci mapped in Brassica. Thirty-one probes detected two or more loci in Arabidopsis, which represent 23.7% of the 262 loci mapped in Arabidopsis. Application of an EST-based, cross-species genomic framework to isolation of alleles conferring phenotypes unique to Brassica, as well as the challenges and opportunities in extrapolating genetic information from Arabidopsis to Brassica and to more distantly related crops, are discussed.

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis.

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.

A multiplex PCR for identifying Shiga-like toxin-producing Escherichia coli O157:H7.

A multiplex PCR assay specifically detecting Escherichia coli O157:H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157:H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157:H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55:H7 and E. coli O55:NM, and other non-O157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.

The complete nucleotide sequence and genome organization of the M RNA segment of peanut bud necrosis tospovirus and comparison with other tospoviruses.

The M RNA of peanut bud necrosis virus (PBNV; synonym groundnut bud necrosis virus) is 4801 nucleotides in length. It comprised two ORFs in an ambisense organization and terminal inverted repeats. The 3' large ORF (3363 nucleotides in the virus-complementary strand) encoded a protein with a predicted size of 127.2 kDa which was identified as the glycoprotein precursor (GP) of the G1 and G2 glycoproteins. A comparison of the deduced amino acid sequence of GP revealed 37% identity and 58-59% similarity with that of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), and 21-23% identity and 44-47% similarity with those of other members of the genus Bunyavirus. The 5' small ORF (924 nucleotides in the virussense strand) encoded a 34.2 kDa protein which was identified as the non-structural (NSm) protein based on 41-43% identity and 60-63% similarity with that of TSWV and INSV. Defective RNA molecules derived from the genomic M RNA were detected during continuous passage of the virus by sap inoculations.

Polymerase chain reaction for detecting Escherichia coli O157: H7.

Escherichia coli O157:H7 is known as an important cause of hemorrhagic colitis and hemolytic uremic syndrome. Real-time procedures that are sensitive for detecting small populations of this bacterium in food are lacking and needed. An expression library was constructed by ligation of BamHI-EcoRI DNA fragments of E. coli O157:H7 to plasmid vector pUC19 and transformation of recombinant plasmids to E. coli JM109. A clone that contained a specific DNA fragment of E. coli O157:H7 was identified by colony immunoblot assay using monoclonal antibody MAb 4E8C12 that uniquely links to E. coli O157:H7 and a few other serotypes of verotoxin-producing E. coli. The DNA sequence of the clone consisted of 110 bp of 5' region of enterohemorrhagic E. coli (EHEC) eae gene and a 688 bp DNA fragment adjacent to 5' end of the eae gene, including an unknown function gene encoding 156 amino acids. A pair of oligonucleotide primers was synthesized based on the sequence of the 688 bp fragment. The primers were used in a polymerase chain reaction (PCR) to amplify a target DNA of 633 bp. The primers amplified 1 ng of DNA from 67 strains of E. coli O157:H7, two strains of E. coli O157:NM, and 7 of 11 E. coli O55:H7 and O55:NM strains, but not 50 ng of DNA from 34 strains of 29 other E. coli serotypes and 25 strains of 8 other bacterial species. Annealing temperatures from 60 to 63 degrees C could be used for the PCR without loss of specificity. The minimum amount of target DNA detected by the PCR was 5 pg. When a boiling method and GeneReleaser were used, the PCR was able to detect as few as 25 and 38 CFU of E. coli O157:H7, respectively, in 3 h.

Peanut bud necrosis tospovirus S RNA: complete nucleotide sequence, genome organization and homology to other tospoviruses.

The complete nucleotide sequence of the S RNA of peanut bud necrosis virus (PBNV) has been determined. The RNA is 3 057 nucleotides in length, contains inverted repeats and two open reading frames (ORFs) with an ambisense coding strategy that are separated by an A+U-rich intergenic region. One ORF (1 320 nucleotides in the viral sense strand) encodes a Mr 49.5 kDa protein, identified as the nonstructural (NSs) protein based on similarity to published tospovirus sequences. The second ORF (831 nucleotides in virus complementary strand) encodes a Mr 30.6 kDa protein. This protein was identified as the nucleocapsid (N) protein based on sequence similarities. Amino acid sequence comparison of N and NSs proteins revealed identities of 22-34% with the reported tospovirus isolates of serogroups I, II, and III, whereas it had 82-86% identity with viruses in serogroup IV, watermelon silver mottle virus (WSMV) and tomato isolate of peanut bud necrosis (PBNV-To). Two subgenomic RNA species detected in PBNV infected tissue corresponded to the predicted sizes (1.65 and 1.4 kb) of the NSs and N mRNAs. The data presented show conclusively that PBNV should be included in serogroup IV, along with WSMV and PBNV-To.