Fine Mapping and Characterization of Candidate Genes that Control Resistance to Cercospora sojina K. Hara in Two Soybean Germplasm Accessions

Frogeye leaf spot (FLS), caused by the fungus Cercospora sojina K. Hara, may cause a significant yield loss to soybean growers in regions with a warm and humid climate. Two soybean accessions, PI 594891 and PI 594774, were identified to carry a high level of resistance similar to that conditioned by the Rcs3 gene in ''Davis''. Previously, we reported that the resistance to FLS in these two plant introductions (PIs) was controlled by a novel gene (s) on chromosome 13 that is different from Rcs3. To fine-map the novel FLS resistance gene(s) in these two PIs, F_{2: 3} seeds from the crosses between PI 594891 and PI 594774, and the FLS susceptible genotype ''Blackhawk'' were genotyped with SNP markers that were designed based on the SoySNP50k iSelect BeadChip data to identify recombinant events and locate candidate genes. Analysis of lines possessing key recombination events helped narrow down the FLS-resistance genomic region in PI 594891 from 3.3 Mb to a 72.6 kb region with five annotated genes. The resistance gene in PI 594774 was fine-mapped into a 540 kb region that encompasses the 72.6 kb region found in PI 594891. Sequencing five candidate genes in PI 594891 identified three genes that have several mutations in the promoter, intron, 5'', and 3'' UTR regions. qPCR analysis showed a difference in expression levels of these genes in both lines compared to Blackhawk in the presence of C. sojina. Based on phenotype, genotype and haplotype analysis results, these two soybean accessions might carry different resistance alleles of the same gene or two different gene(s). The identified SNPs were used to develop Kompetitive Allele Specific PCR (KASP) assays to detect the resistance alleles on chromosome 13 from the two PIs for marker-assisted selection.     

Characterization of HMW-GSs and their gene inaction in tetraploid wheat

In this study, we report the expression of HMW-GSs in 87 accessions of tetraploid wheat, the characterization of three inactive and one active HMW glutenin genes, and the functional verification of HMW-GSs by promoter–GUS expression. SDS-PAGE profiles revealed that tetraploid wheat has many different combinations of HMW-GSs and the number of subunits varies from 1 to 4. HMW glutenin genes at the Glu-A1x, Glu-A1y and Glu-B1y loci exhibited different frequencies of inaction while the Glu-B1x allele was expressed in all 87 accessions. Gene cloning showed that only 1Bx (Tdu-e) could express a full-length protein and its deduced protein sequence has the typical primary structure but with fewer cysteine residues. The expression of the other three HMW glutenin genes has been disrupted by stop codons in their repetitive domains. Besides short indels or mutations of one or more bases, an 85-bp deletion and a 185-bp insertion were found in the promoter regions of 1Ay (Tdu-s) and 1Bx (Tdu-e). The transient expression of promoter–GUS constructs indicated that the 1Ay promoter can drive expression of the GUS gene. We conclude that defects (stop codons or the insertion of large transposon-like elements) in the coding regions may be the most probable cause for the inaction of the HMW glutenin genes.     

Polyploidy origin of wheatgrass Douglasdeweya wangii (Triticeae, Poaceae): evidence from nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL–F sequences

To study hybrid speciation in wheatgrass Douglasdeweya wangii and to investigate the evolutionary pattern of nuclear ribosomal DNA (nrDNA) internal transcribed spacer sequences (ITSs) in allotetraploids, DNA sequence variation of ITSs and chloroplast trnL–F sequences from D. wangii and its putative donors were analyzed. The ITSs revealed that D. wangii had an StP genome composition. Most accessions of D. wangii had one parental ITS copy in their genome, one accession had two parental ITSs. The trnL–F sequences revealed an especially close relationship of Pseudoroegneria to all D. wangii individuals included, and the two accessions of Pseudoroegneria tauri (PI401324 and PI401331) were maternal candidates of the studied D. wangii individuals. Both of ITS and trnL–F trees suggested multiple origins and recurrent hybridization of D. wangii. Thus, the results suggested that: (1) the St and P genome in allotetraploid D. wangii were donated by Pseudoroegneria and Agropyron, respectively;(2) Pseudoroegneria was the maternal donor of D. wangii, and P. tauri 26 (accession PI401324) and P. tauri 27 (accession PI401331) were most likely the potential candidates of maternal donors; (3) D. wangii individuals studied here showed multiple origins and experienced recurrent hybridization; and (4) bidirectional interlocus concerted evolution of ITSs had occurred in most D. wangii accessions, while in one accession concerted evolution among homeologous loci did not occur.     

Characterization of Imcrop, a Mutator-like MITE family in the rice genome

Sequence comparisons of ammonium transporter 1?C2 genes (OsAMT1-2) in different rice accessions revealed a MITE insertion in the upstream region of the gene. The 391-bp MITE, classified as a Mutator superfamily member and named Imcrop, included terminal inverted repeat (TIR) and 9-bp target site duplication (TSD) sequences. We identified 151 Imcrop elements dispersed on 12 chromosomes of the japonica reference genome. Of these, 12.6% were found in genic regions and 33.1% were located within 1.5 kb of annotated rice genes. We constructed comparative insertion maps with 111 and 102 intact Imcrop elements in the japonica and indica reference genomes, respectively. The Imcrop elements showed relatively even distribution across all chromosomes although their frequency was higher on chromosomes 1, 3, and 4 in both genomes. Seventy seven Imcrop elements were detected in both subspecies, whereas 34 and 25 insertions were found only in the japonica or indica genome, respectively. We compared insertion polymorphisms of 19 Imcrop elements found inside genes in 48 Korean rice cultivars, consisting of 42 japonica and six Tongil-types (indica-japonica cross). Thirteen insertions were common to all cultivars indicating these elements were present before indica-japonica divergence. The six other elements showed insertion polymorphisms among accessions, showing their recent insertion history or no critical positive effect of their insertion on the rice genome.     

Identification of Rotylenchulus reniformis Resistant Glycine Lines

Identification of resistance to reniform nematode (Rotylenchulus reniformis) is the first step in developing resistant soybean (Glycine max) cultivars that will benefit growers in the mid-South region of the United States. This study was conducted to identify soybean (G. max and G. soja) lines with resistance to this pathogen. Sixty-one wild and domestic soybean lines were evaluated in replicated growth chamber tests. Six previously untested soybean lines with useful levels of resistance to reniform nematode were identified in both initial screening and subsequent confirmation tests: released germplasm lines DS4-SCN05 (PI 656647) and DS-880 (PI 659348); accession PI 567516 C; and breeding lines DS97-84-1, 02011-126-1-1-2-1 and 02011-126-1-1-5-1. Eleven previously untested moderately susceptible or susceptible lines were also identified: released germplasm lines D68-0099 (PI 573285) and LG01-5087-5; accessions PI 200538, PI 416937, PI 423941, PI 437697, PI 467312, PI 468916, PI 594692, and PI 603751 A; and cultivar Stafford (PI 508269). Results of previously tested lines evaluated in the current study agreed with published reports 69.6% of the time for resistant lines and 87.5% of the time for susceptible lines. Soybean breeders may benefit from incorporating the newly identified resistant lines into their breeding programs.     

IS1549 from Mycobacterium smegmatis Forms Long Direct Repeats upon Insertion

A new insertion element, IS1549, was identified serendipitously from Mycobacterium smegmatis LR222 during experiments using a vector designed to detect the excision of IS6110 from between the promoter region and open reading frame (ORF) of an aminoglycoside phosphotransferase gene. Six of the kanamycin-resistant isolates had a previously unidentified insertion element upstream of the ORF of the aph gene. The 1,634-bp sequence contained a single ORF of 504 amino acids with 85% G+C content in the third codon position. The putative protein sequence showed a distant relationship to the transposase of IS231, which is a member of the IS4 family of insertion elements. IS1549 contains 11-bp terminal inverted repeats and is characterized by the formation of unusually long and variable-length (71- to 246-bp) direct repeats of the target DNA during transposition. Southern blot analysis revealed that five copies of IS1549 are present in LR222, but not all M. smegmatis strains carry this element. Only strains with a 65-kDa antigen gene with a PCR-restriction fragment length polymorphism type identical to that of M. smegmatis 607 contain IS1549. None of 13 other species of Mycobacterium tested by PCR with two sets of primers specific for IS1549 were positive for the expected amplified product.     

A novel 9-bp insertion detected in steroid 21-hydroxylase gene (CYP21A2): prediction of its structural and functional implications by computational methods

Background

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH). Detection of underlying mutations in CYP21A2 gene encoding steroid 21-hydroxylase enzyme is helpful both for confirmation of diagnosis and management of CAH patients. Here we report a novel 9-bp insertion in CYP21A2 gene and its structural and functional consequences on P450c21 protein by molecular modeling and molecular dynamics simulations methods.

Methods

A 30-day-old child was referred to our laboratory for molecular diagnosis of CAH. Sequencing of the entire CYP21A2 gene revealed a novel insertion (duplication) of 9-bp in exon 2 of one allele and a well-known mutation I172N in exon 4 of other allele. Molecular modeling and simulation studies were carried out to understand the plausible structural and functional implications caused by the novel mutation.

Results

Insertion of the nine bases in exon 2 resulted in addition of three valine residues at codon 71 of the P450c21 protein. Molecular dynamics simulations revealed that the mutant exhibits a faster unfolding kinetics and an overall destabilization of the structure due to the triple valine insertion was also observed.

Conclusion

The novel 9-bp insertion in exon 2 of CYP21A2 genesignificantly lowers the structural stability of P450c21 thereby leading to the probable loss of its function.     

A comparison study of Agrobacterium-mediated transformation methods for root-specific promoter analysis in soybean

Key message

Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.

Abstract

An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.     

Physiological determinants of growth rate in response to phosphorus supply in wild and cultivated Hordeum species

Summary Under favorable nutrition, accessions of the weedy barleygrass (Hordeum leporinum and H. glaucum) had a higher relative growth rate (RGR) than did accessions of cultivated barley (H. vulgare) or its wild progenitor (H. spontaneum). RGR was not positively correlated with the presumed level of soil fertility at the collection site of an accession either within or among species. RGR was reduced more strongly by low-P supply in the progenitor than in the crop or weed, indicating that selection of cultivars to grow in fertile soils had not reduced their potential to grow effectively under low-P conditions. Seed and embryo masses were more important than RGR in determining plant size. Relative differences among assessions in plant size declined with time, because (1) accessions with small seeds had a higher RGR, and (2) RGR of large-seeded accessions declined with time. Absolute growth rate correlated positively with leaf area and negatively with photosynthetic rate per unit leaf area. Under favorable nutrition, maximum photosynthetic rate correlated negatively with leaf length and therefore was higher in the weeds than in the crop or progenitor accessions. P absorption potential did not differ consistently among species but generally increased in response to P stress. Cultivars produced a few tall tillers, whereas weeds and progenitors produced many small tillers. The cultivar had a larger proportion of reproductive tillers, allocated a larger proportion of biomass to grain, and produced larger grains than did the weedy accession. By contrast, the weed began maturing seeds sooner, produced more reproductive tillers, and produced more grains per car and per plant than did the cultivar. The study suggests two major conclusions: (1) A low RGR is not an adaptation to low P supply in annual Hordeum species. (2) Seed size is the major determinant of early plant size between accessions in these Hordeum species under favorable nutrition. However, large seed size indirectly results in a low RGR because of the inverse relationship between plant size and RGR and results in a low photosynthetic rate because of the inverse relationship between leaf size and photosynthesis.     

Prion protein gene polymorphism in healthy and BSE-affected Slovak cattle

Variation of thePrP gene was examined in healthy and BSE-affected Slovak cattle. According to previous studies, the 23-bp indel polymorphism is supposed to be associated with higher susceptibility to BSE. We investigated 301 samples from healthy cattle of various Slovak breeds and 24 samples obtained from tissues of BSE-affected cattle in Slovakia. We examined thePrP gene for the 23-bp indel polymorphism in the putative promoter region, 12-bp indel polymorphism in the first intron of thePrP gene, variations in number of octapeptide repeat units, and presence of the silent AAC>AAT transition in codon 192 within the protein-coding region of thePrP gene. Altogether we found 23 different genotypes in the group of healthy cattle and only 6 genotypes in the group of BSE-affected cattle. Comparison of homozygotes for the 23-bp insertion and heterozygotes showed significant differences (P < 0.05) in genotype distribution between the examined groups. Thereby the homozygous insertion genotype at the 23-bp indel polymorphism site in the promoter region of the prion protein gene seems to have a protective effect against BSE.     

Characterization and genetics of multiple soybean aphid biotype resistance in five soybean plant introductions

Key message

Five soybean plant introductions expressed antibiosis resistance to multiple soybean aphid biotypes. Two introductions had resistance genes located in the Rag1, Rag2, and Rag3 regions; one introduction had resistance genes located in the Rag1, Rag2, and rag4 regions; one introduction had resistance genes located in the Rag1 and Rag2 regions; and one introduction had a resistance gene located in the Rag2 region.

Abstract

Soybean aphid (Aphis glycines Matsumura) is the most important soybean [Glycine max (L.) Merr.] insect pest in the USA. The objectives of this study were to characterize the resistance expressed in five plant introductions (PIs) to four soybean aphid biotypes, determine the mode of resistance inheritance, and identify markers associated with genes controlling resistance in these accessions. Five soybean PIs, from an initial set of 3000 PIs, were tested for resistance against soybean aphid biotypes 1, 2, 3, and 4 in choice and no-choice tests. Of these five PIs, PI 587663, PI 587677, and PI 587685 expressed antibiosis against all four biotypes, while PI 587972 and PI 594592 expressed antibiosis against biotypes 1, 2, and 3. F₂ populations derived from PI 587663 and PI 587972 were evaluated for resistance against soybean aphid biotype 1, and populations derived from PIs 587677, 587685, and 594592 were tested against biotype 3. In addition, F_2:3 plants were tested against biotypes 2 and 3. Genomic DNA from F₂ plants was screened with markers linked to Rag1, Rag2, Rag3, and rag4 soybean aphid-resistance genes. Results showed that PI 587663 and PI 594592 each had three genes with variable gene action located in the Rag1, Rag2, and Rag3 regions. PI 587677 had three genes with variable gene action located in the Rag1, Rag2 and rag4 regions. PI 587685 had one dominant gene located in the Rag1 region and an additive gene in the Rag2 region. PI 587972 had one dominant gene located in the Rag2 region controlling antixenosis- or antibiosis-type resistance to soybean aphid biotypes 1, 2, or 3. PIs 587663, 587677, and 587685 also showed antibiosis-type resistance against biotype 4. Information on multi-biotype aphid resistance and resistance gene markers will be useful for improving soybean aphid resistance in commercial soybean cultivars.

     

DNA sequence analysis of host range mutants of the promiscuous IncP-1 plasmids R18 and R68 with Tn7 insertions in oriV

Transposon Tn7 insertions in the origin of vegetative replication (oriV) result in host range mutants of the promiscuous IncP-1 plasmids R18 and R68 which affect plasmid replication in Escherichia coli but not in Pseudomonas aeruginosa. The sites of these insertions have been analyzed by DNA sequence analysis. In two mutants, the insertions generated direct duplications of 5′GTATT3′ at the target site which included the first base at the 5′ end of the fourth 17-bp direct repeat in oriV. In a third mutant the duplication of 5′GACAC3′ also involved the same direct repeat also at the 5′ end but contiguous with the previous duplication. DNA sequence analysis of another Tn7-induced host range mutant of R18, characterized by reduced conjugational transmissibility into P. stutzeri while retaining normal transmissibility within P. aeruginosa, showed that the insertion generated a 474-bp deletion which brought the insertion 20 bp 5′ to the 17-bp direct repeat between oriV and the oxytetracycline hydrochloride-resistant gene. The analysis of the DNA sequence data at the site of the Tn7 insertions shows that particular segments of the DNA sequence in oriV are differentially required for the replication of these plasmids in different bacterial hosts and thus of importance to the promiscuity of these plasmids.     

Influence of Phosphorus Nutrition on Growth and Carbon Partitioning in Glycine max

Soybean plants (Glycine max [L.] Merr var Amsoy 71) were grown in growth chambers with high-phosphorus (high-P) and low-phosphorus (low-P) culture solutions. Low-P treatment reduced shoot growth significantly 7 days after treatment began. Root growth was much less affected by low-P, there being no significant reduction in root growth rate until 17 days had elapsed. The results suggest that low-P treatment decreased soybean growth primarily through an effect on the expansion of the leaf surface which was diminished by 85%, the main effect of low-P being on the rate of expansion of individual leaves. Low-P had a lesser effect on photosynthesis; light saturated photosynthetic rates at ambient and saturating CO₂ levels were lowered by 55 and 45%, respectively, after 19 days of low-P treatment. Low-P treatment increased starch concentrations in mature leaves, expanding leaves and fibrous roots; sucrose concentrations, however, were reduced by low-P in leaves and increased in roots. Foliar F-2,6-BP levels were not affected by P treatment in the light but in darkness they increased with high-P and decreased with low-P. The increase in the starch/sucrose ratio in low-P leaves was correlated primarily with changes in the total activities of enzymes of starch and sucrose metabolism.