Isolation and characterization of genome-specific DNA sequences in Triticeae species

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), each representing more than 1 % of the genomes. These repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae. speltoides was a 763 by EcoRI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomerec chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280 bp, which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing-over or reciprocal translocation. In situ hybridization showed that the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 by DraI fragment from H. chilense, also tandemly repeated but more variable; loss of the DraI site resulting in a ladder pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H. chilense and hence was able to identify the origin of chromosomes in a H. chilense x Secale africanum hybrid by in situ hybridization. It has a high A + T content (66%), small internal duplications, and a 50 by degenerate inverted repeat. We speculate that it has dispersed by retrotransposition in association with other sequences carrying coding domains. The organization and evolution of such sequences are important in understanding long-range genome organization and the types of change that can occur on evolutionary and plant breeding timescales. Genome-specific sequences are also useful as markers for alien chromatin in plant breeding.     

Wheat genome structure: translocations during the course of polyploidization

The genomic organization of Triticum timopheevii (2n=28, A^tA^tGG) was compared with hexaploid wheat T. aestivum (2n=42, AABBDD) by comparative mapping using microsatellites derived from bread wheat. Genetic maps for the two crosses T. timopheevii var. timopheevii × T. timopheevii var. typica and T. timopheevii K-38555×T. militinae were constructed. On the first population, 121 loci were mapped, and on the second population 103 loci. The transferability of the wheat markers to T. timopheevii was generally better for the A genome-specific markers (76–78% produced amplification products; 26 and 29% were polymorphic) than for B genome-specific markers (54% produced amplification products; 14 and 16% were polymorphic). Of the D genome-specific markers, one third produced amplification products in T. timopheevii, but only 5 and 2% were polymorphic in the corresponding mapping populations. The maps constructed confirmed the previously described translocation between chromosome arms 6A^tS and 1GS and revealed at least two yet unknown rearrangements on chromosomes 4A^t and 6A^t. The presence of other translocations and rearrangements between T. timopheevii and T. aestivum was demonstrated by a variety of markers mapping to nonhomoeologous positions.     

Characterization of disomic addition lines Brassica napus-Brassica nigra by isozyme,fatty acid,and RFLP markers

Summary Six Brassica napus — B. nigra disomic addition lines were characterized by isozyme, fatty acid, and RFLP markers. The markers were arranged in six synteny groups, representing six of the eight chromosomes present in the B. nigra genome. Synteny group 1 displayed high levels of linoleic and linolenic acids in the seeds of the B. nigra parent. Synteny group 3 accumulated higher levels of eicosenoic and erucic acid than B. nigra. Three of the lines transmitted the alien chromosome to 100% of the progeny. The rest had variable transmission rates but all were above 50%. Most of the lines produced disomic addition plants in their progeny, suggesting pollen transmission of the alien chromosome. In addition to the marked lines, six others remained unmarked. These could be grouped into two classes according to their alien chromosome transmission. It is likely that they represent the two other B. nigra chromosomes that remained uncharacterized by the markers. No diploid individuals carrying B. nigra genome-specific markers were detected in the progenies studied.     

Genome-specific primer sets for starch biosynthesis genes in wheat

Common wheat (Triticum aestivum L., 2n=6x=42) is an allohexaploid composed of three closely related genomes, designated A, B, and D. Genetic analysis in wheat is complicated, as most genes are present in triplicated sets located in the same chromosomal regions of homoeologous chromosomes. The goal of this report was to use genomic information gathered from wheat–rice sequence comparison to develop genome-specific primer sets for five genes involved in starch biosynthesis. Intron locations in wheat were inferred through the alignment of wheat cDNA sequences with rice genomic sequence. Exon-anchored primers, which amplify across introns, allowed the sequencing of introns from the three genomes for each gene. Sequence variation within introns among the three wheat genomes provided the basis for genome-specific primer design. For three genes, ADP-glucose pyrophosphorylase (Agp-L), sucrose transporter (SUT), and waxy (Wx), genome-specific primer sets were developed for all three genomes. Genome-specific primers were developed for two of the three genomes for Agp-S and starch synthase I (SsI). Thus, 13 of 15 possible genome-specific primer sets were developed using this strategy. Seven genome-specific primer combinations were used to amplify alleles in hexaploid wheat lines for sequence comparison. Three single nucleotide polymorphisms (SNPs) were identified in a comparison of 5,093 bp among a minimum of ten wheat accessions. Two of these SNPs could be converted into cleaved amplified polymorphism sequence (CAPS) markers. Our results indicated that the design of genome-specific primer sets using intron-based sequence differences has a high probability of success, while the identification of polymorphism among alleles within a genome may be a challenge.     

Construction of a binary BAC library for an apomictic monosomic addition line of<Emphasis Type="Italic"> Beta corolliflora</Emphasis> in sugar beet and identification of the clones derived from the alien chromosome

A plant-transformation-competent binary BAC library was constructed from the genomic DNA of the chromosome 9 monosomic addition line of Beta corolliflora Zoss. in sugar beet (B. vulgaris. L). This monosomic addition line (designated M14) is characterized by diplosporic reproduction caused by the alien chromosome carrying the gene(s) responsible for diplospory. The library consists of 49,920 clones with an average insert size of 127 kb, representing approximately 7.5 haploid genome equivalents and providing a greater than 99% probability of isolating a single-copy DNA sequence from the library. To develop the scaffold of a physical map for the alien chromosome, B. corolliflora genome-specific dispersed repetitive DNA sequences were used as probes to isolate BAC clones derived from the alien chromosome in the library. A total of 2,365 positive clones were obtained and arrayed into a sublibrary specific for B. corolliflora chromosome 9 (designated bcBAC-IX). The bcBAC-IX sublibrary was further screened with a subtractive cDNA pool generated from the ovules of M14 and the floral buds of B. vulgaris by the suppression subtractive hybridization method. One hundred and three positive binary BACs were obtained, which potentially contain the genes of the alien chromosome specifically expressed during the ovule and embryo development of M14, and may be associated with apomictic reproduction. Thus, these binary BAC clones will be useful for identification of the genes for apomixis by genetic transformation.Communicated by H. C. Becker     

Molecular phylogeny of the genus Triticum L

The genus Triticum L. includes the major cereal crop, common or bread wheat (hexaploid Triticum aestivum L.), and other important cultivated species. Here, we conducted a phylogenetic analysis of all known wheat species and the closely related Aegilops species. This analysis was based on chloroplast matK gene comparison along with trnL intron sequences of some species. Polyploid wheat species are successfully divided only into two groups – Emmer (sections Dicoccoides and Triticum) and Timopheevii (section Timopheevii). Results reveal strictly maternal plastid inheritance of synthetic wheat amphiploids included in the study. A concordance of chloroplast origin with the definite nuclear genomes of polyploid species that were inherited at the last hybridization events was found. Our analysis suggests that there were two ancestral representatives of Aegilops speltoides Tausch that participated in the speciation of polyploid wheats with B and G genome in their genome composition. However, G genome species are younger in evolution than ones with B genome. B genome-specific PCR primers were developed for amplification of Acc-1 gene.     

Behavioural Reproductive Isolation in a Rotifer Hybrid Zone

A hybrid zone between two Brachionus plicatilis rotifer mitochondrial DNA (mtDNA) lineages was recently described in the Iberian Peninsula between a pond (Santed 2) and a lake (Gallocanta). The patterns of mitochondrial and nuclear genetic variation observed suggested that gene flow is mainly male-mediated from the lake to the pond. Here we test two hypotheses: (a) that male-mediated gene flow occurs through assortative mating between individuals from these ponds, (b) that behavioural isolation occurs between the two mtDNA lineages. We isolated, reared and genotyped rotifer clones from resting eggs collected in the sediments of these and two other distant ponds. We devised a quick, inexpensive RFLP method to discriminate between B. plicatilis and its sibling species B. ‘Manjavaeas’ and between both mtDNA B. plicatilis lineages. Behavioural no-choice tests using new-born, virgin males and females were performed between five clones. B. ‘Manjavacas’ and B. plicatilis were reproductively isolated. B. plicatilis clones did not show evidence of reproductive isolation, regardless of their mtDNA lineage, except Santed 2 males, which discriminated strongly against Gallocanta females. These results could help to explain the discrepancies between mitochondrial and nuclear genetic variation reported in the two populations.     

Evidence for a low level of genomic specificity of sequence-tagged-sites in Stylosanthes

Genome-specific DNA markers are of great value in many applications. Recent work on different plants and animal species indicated that PCR- (polymerase chain reaction) based genetic marker systems using specific primers are highly genome-specific. To test the genome specificity of sequence-tagged-sites (STSs) as genetic markers in Stylosanthes, 20 pairs of primers were generated. Fifteen were from randomly selected single-copy Pstl genomic clones, and the other five were from two known gene sequences. These primer pairs were analysed against a set of 24 genotypes representing 12 different Stylosanthes species. Thirteen of these primer pairs amplified successfully. Overall, there was a low level of genome specificity, suggesting a low degree of genomic divergence within this group of Stylosanthes species. Of the 312 entries (24 genotypes by 13 primer pairs), PCR amplifications were unsuccessful (little or no products) in only 16 cases. The number of banding patterns detected by each of these primer pairs varied from 2 to 12 with an average pair-wise polymorphism of 44.3%. The level of intraspecific variation detected on normal agarose gels was only 3.8%. Further evidence that diploid S. hamata and diploid S. humilis are progenitors of tetraploid S. hamata and that S. viscosa is a progenitor of S. scabra, was obtained.     

Dreb1 genes in wheat (Triticum aestivum L.): development of functional markers and gene mapping based on SNPs

The Dreb genes are involved in abiotic stress tolerances, such as drought, salinity, low temperature and ABA. The purpose of the present research was to establish protocols for the development of genome-specific and allele specific markers in common wheat (Triticum aestivum L.) using the Dreb1 genes as an example. Based on the available sequences of Dreb1 genes in common wheat and related species, five primer pairs were designed using Primer Premier 5.0. Two primers, P25F/PR and P21F/P21R, amplified 596- and 1113-bp fragments, respectively, from the A genome, P18F/P18R amplified a 717-bp fragment from the B genome, and primers P22F/PR and P20F/P20R amplified 596- and 1193-bp fragments, respectively, from the D genome. Using these genome-specific primers and the Chinese Spring using nulli-tetrasomic lines, the Dreb1 genes were located on chromosomes 3A, 3B and 3D. Two SNPs (S646 and S770) in Dreb-B1 distinguished the Opata 85 and W7984 parents of the ITMI mapping population, but there was no polymorphism between the orthologous Dreb-A1 and Dreb-D1 sequences. By assaying the genotypes of 115 RILs with the allele-specific primer P40 based on SNP S770, Dreb-B1 was mapped between markers Xmwg818 and Xfbb117 on chromosome 3BL. This genetic mapping of Dreb-B1 on chromosome 3B may be helpful in wheat breeding programs aimed at improving drought tolerance.     

Dominance of Lysobacter sp. in the rhizosphere of two coastal sand dune plant species, Calystegia soldanella and Elymus mollis

Bacterial diversity in the rhizosphere of beach morning glory (Calystegia soldanella) and wild rye (Elymus mollis), two of the major plant species inhabiting the coastal sane dune in Tae-An, Korea, was studied by the analysis of community 16S rRNA gene clones. The amplified rDNA restriction analysis (ARDRA) of the clones using HaeIII exhibited significant differences in the community composition between the two plant species as well as regional differences, but also identified a specific ARDRA pattern that was most common among the clones regardless of plant species. Subsequent sequence analysis indicated that the pattern was that of Lysobacter spp., which is a member of the family Xanthomonadaceae, class Gamma proteobacteria. The Lysobacter clones comprised 50.6% of the clones derived from C. soldanella and 62.5% of those from E. mollis. Other minor patterns included those of Pseudomonas spp., species of Rhizobium, Chryseobacterium spp. and Pantoea spp. among C. soldanella clones, and Pseudomonas sp. and Aeromonas hydrophila among E. mollis clones. It is not yet clear what kind of roles Lysobacter plays in association with sand dune plants, but its universal presence in the rhizosphere, together with the potential of this taxon for antagonistic activity against plant pathogens, suggests that Lysobacter might form a symbiotic relationship with its host plants.     

The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family

A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb.     

Isolation and characterization of two Brassica napus embryo acyl-ACP thioesterase cDNA clones

Acyl-ACP thioesterases are involved in regulating chain termination of fatty acid biosynthesis in plant systems. Previously, acyl-ACP thioesterase purified from Brassica napus seed tissue has been shown to have a high preference for hydrolysing oleoyl-ACP. Here, oligonucleotides derived from B. napus oleoyl-ACP thioesterase protein sequence data have been used to isolate two acyl-ACP thioesterase clones from a B. napus embryo cDNA library. The two clones, pNL2 and pNL3, contain 1642 bp and 1523 bp respectively and differ in the length of their 3 non-coding regions. Both cDNAs contain open reading frames of 366 amino acids which encode for 42 kDa polypeptides. Mature rape thioesterase has an apparent molecular weight of 38 kDa on SDS-PAGE and these cDNAs therefore encode for precursor forms of the enzyme. This latter finding is consistent with the expected plastidial location of fatty acid synthase enzymes. Northern blot analysis shows thioesterase mRNA size to be ca. 1.6 kb and for the thioesterase genes to be highly expressed in seed tissue coincident with the most active phase of storage lipid synthesis. There is some sequence heterogeneity between the two cDNA clones, but overall they are highly homologous sharing 95.7% identity at the DNA level and 98.4% identity at the amino acid level. Some sequence heterogeneity was also observed between the deduced and directly determined thioesterase protein sequences. Consistent with the observed sequence heterogeneity was Southern blot data showing B. napus thioesterase to be encoded by a small multi-gene family.     

The use of Agrobacterium rhizogenes transformed roots to obtain transgenic shoots of the apple rootstock Jork 9

The apple rootstock Jork 9 was transformed using four different Agrobacterium rhizogenes virulent strains. The mannopine strain 8196 gave the best results in the production of chimeric plants compared to two agropine strains (A4 and 15834) and one cucumopine strain. Shoot regeneration was performed on both untransformed and transformed roots. Optimum combination and concentration of thidiazuron (TDZ) and -naphtaleneacetic acid (NAA) was different between untransformed and transformed roots. From the transformed roots seven shoots were obtained and propagated as individual clones. All shoots from these clones rooted on a hormone-free medium contrary to untransformed shoots that did not root under similar culture conditions. Differences in the morphology of the leaves and stems were observed between the clones. The transformed status of the different clones was verified with mannopine tests, PCR and Southern blot analyses. Five clones contained the mas1', the ORF 13 and the rolB genes, whereas two clones contained only the rolB gene.     

Analysis of DNA from a Beta procumbens chromosome fragment in sugar beet carrying a gene for nematode resistance

Summary We have begun to apply techniques for the preparation and anaylsis of large DNA segments from sugar beet (Beta vulgaris) addition lines carrying a mitotically stable chromosome fragment from B. procumbens that confers monogenic resistance to the nematode Heterodera schachtii, with a view towards isolating the resistance gene. DNA probes specific for this chromosome fragment were selected, and various methods for cloning genome-specific fragments, including probes from megabase DNA separated in pulsed-field slab gels, are compared. Probes that display high homology to B. procumbens have been used for hybridization of a representative genomic library and for initial step in mapping the chromosome fragment via pulsed-field gel electrophoresis after restriction with infrequently cutting enzymes. Our data indicate that DNA molecules from the entire chomosome fragment can be separated from protoplast DNA lysates.     

Unique chromosome behavior and genetic control in Brassica×Orychophragmus wide hybrids: a review

Researchers recognized early that chromosome behavior, as other morphological characters, is under genetic control and gave some cytogenetical examples such as the homoeologous chromosome pairing in wheat. In the intergeneric sexual hybrids between cultivated Brassica species and another crucifer Orychophragmus violaceus, the phenomenon of parental genome separation was found under genetic control during mitosis and meiosis. The cytogenetics of these hybrids was species-specific for Brassica parents. The different chromosome behavior of hybrids with three Brassica diploids (B. rapa, B. nigra and B. oleracea) might contribute to the different cytology of hybrids with three tetraploids (B. napus, B. juncea and B. carinata). The finding that genome-specific retention or loss of chromosomes in hybrids of O. violaceus with B. carinata and synthetic Brassica hexaploids (2n=54, AABBCC) is likely related to nucleolar dominance gives new insight into the molecular mechanisms regarding the cytology in these hybrids. It is proposed that the preferential expressions of genes for centromeric proteins from one parent (such as the well presented centromeric histone H3) are related with chromosome stability in wide hybrids and nucleolar dominance is beneficial to the production of centromere-specific proteins of the rRNAs-donor parent and to the stability of its chromosomes.     

Identification of CED-3 substrates by a yeast-based screening method

Jpk, originally isolated as an associating factor with the position-specific regulatory element of Hoxa-7, was found to be toxic to Escherichia coli (1) and to F9 teratocarcinoma cells (2) when transiently transfected and expressed. To investigate the possibility of tumor gene therapy using Jpk, its effect was tested in B16F10 murine melanoma cells. Because Jpk reduces the viability of B16F10 cells when transiently expressed, the Jpk gene was cloned into a tetracycline-controlled gene expression vector, pRetro-On to circumvent the lethal effect in unwanted situations. The retroviral plasmid pRetroJpk purified from the packaging cell was infected into B16F10 melanoma cells and screened in the presence of puromycin. Out of a total of 53 stable clones selected with puromycin, two clones overexpressed Jpk at more than twice the level when induced by doxycycline, a tetracycline-derivative, which implies the amount of the Jpk exhibiting the toxicity is critical. Although these clones control only low levels of Jpk, overexpression of the established melanoma cell line may help us decipher the function of Jpk and apply it as a tumor therapeutic gene in the future.     

Construction of a rice bacterial artificial chromosome library and identification of clones linked to the Xa-21 disease resistance locus

A bacterial artificial chromosome (BAC) library consisting of 11 000 clones with an average DNA insert size of 125 kb was constructed from rice nuclear DNA. The BAC clones were stable in E. coli after 100 generations of serial growth. Transformation of the BAC clones by electroporation into E. coli was highly efficient and increased with decreasing size of the DNA inserts. The library was evaluated for the presence of organellar, repeated, and telomeric sequences. A very low percentage (<0.3%) of the library consisted of chloroplast and mitochondrial clones. Eighteen BACs were identified that hybridized with an Arabidopsis telomere repeat. Sixteen BACs hybridized with the AA genome-specific repetitive sequence pOs48. Twelve clones were isolated that hybridized with three DNA markers linked to the Xa-21 disease resistance locus. The results indicate that the BAC system can be used to clone and manipulate large pieces of plant DNA efficiently.     

不同年龄巴山木竹种群克隆结构

很多竹类植物是典型的克隆植物,也是大熊猫的食物。研究典型竹子种群克隆结构的形成和发展对竹林的生产和抚育具有理论和实践意义,可为预测该竹林群落的演替趋势和大熊猫保护提供科学依据。利用SSR标记研究不同年龄A(7龄)、B(30龄)和C(60龄)巴山木竹种群的克隆结构和多样性,探讨小尺度范围内不同年龄巴山木竹种群的克隆结构及斑块的建立和发展。8对SSR引物共扩增出了118个位点,3个种群样地的256个样本共检测到了49个克隆(基因型),A、B和C种群分别检测出31、10个和8个克隆。随着种群年龄的增长,巴山木竹克隆面积增加,克隆数量减少;A和B样地各克隆分布格局为团块状,而C样地克隆既有团块状又有离散状。这一结果显示出在幼苗定居的初期,基株可能以短距离的克隆延伸为主从而呈现出团块状;而随着年龄的增长,克隆面积不断扩大,当复轴混生型的巴山木竹克隆受到强大的压迫时,基株可能会进行较多的单轴和长距离克隆延伸,呈现出离散状。Mantel检测和空间自相关分析都支持3个样地在小尺度范围内存在明显的克隆空间遗传结构。3个样地在10 m等级下显著的正相关空间遗传结构距离为3.1、28、48 m,X-轴截距为9.051、30.698和50.536,空间自相关系数的范围分别为0.1—0.167、0.008—0.703和0.006—0.735。由此可推断,随着年龄的增长,巴山木竹克隆斑块的规模在不断地扩大,同一克隆的分株数量增加,在均匀取样情况下,正相关空间遗传结构距离范围内取到具有相同基因型的可能性越大。A、B和C 3样地的基因型比率(G/N)为1、0.14和0.055,Simpson多样性指数(D)分别为1、0.876和0.744。这说明巴山木竹幼苗期基因型比例远远高于成年的竹林,随着年龄的增长巴山木竹克隆多样性虽有所降低,但由于有性繁殖的作用仍然保持了较高的多样性。聚类和主坐标分析均表明总体上各样地的克隆被聚为一类,但不同样地少数克隆的基因型有重叠和聚集,可推断出不同巴山木竹种群之间可能存在着基因流动和近似的克隆起源。     

Construction and characterization of a half million clone BAC library of durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>)

Durum wheat (Triticum turgidum ssp. durum, 2n = 4x = 28, genomes AB) is an economically important cereal used as the raw material to make pasta and semolina. In this paper we present the construction and characterization of a bacterial artificial chromosome (BAC) library of tetraploid durum wheat cv. Langdon. This variety was selected because of the availability of substitution lines that facilitate the assignment of BACs to the A and B genome. The selected Langdon line has a 30-cM segment of chromosome 6BS from T. turgidum ssp. dicoccoides carrying a gene for high grain protein content, the target of a positional cloning effort in our laboratory. A total of 516,096 clones were organized in 1,344 384-well plates and blotted on 28 high-density filters. Ninety-eight percent of these clones had wheat DNA inserts (0.3% chloroplast DNA, 1.4% empty clones and 0.3% empty wells). The average insert size of 500 randomly selected BAC clones was 131 kb, resulting in a coverage of 5.1-fold genome equivalents for each of the two genomes, and a 99.4% probability of recovering any gene from each of the two genomes of durum wheat. Six known copy-number probes were used to validate this theoretical coverage and gave an estimated coverage of 5.8-fold genome equivalents. Screening of the library with 11 probes related to grain storage proteins and starch biosynthesis showed that the library contains several clones for each of these genes, confirming the value of the library in characterizing the organization of these important gene families. In addition, characterization of fingerprints from colinear BACs from the A and B genomes showed a large differentiation between the A and B genomes. This library will be a useful tool for evolutionary studies in one of the best characterized polyploid systems and a source of valuable genes for wheat. Clones and high-density filters can be requested at Communicated by P. LangridgeThe first two authors contributed equally to the investigation     

Selection of Burkholderia pseudomallei protease-binding peptides by phage display

Burkholderia pseudomallei is a causative agent of melioidosis, a fatal community acquired septicemia in Southeast Asia and Northern Australia. A protease has been proposed to be one of the major pathogenic factors to play a significant role in melioidosis. We have used phage display technology to identify peptides binding to B. pseudomallei protease. By screening a constrained cyclic heptapeptide library, five independent clones with affinity to this protease were isolated and the amino acid sequences were determined. The cyclic heptapeptides from two of the phage clones (Cys-Phe-Phe-Met-Pro-His-Thr-Phe-Cys) were identical and showed the strongest phage-protease interaction as detected by ELISA. Four of the five selected phages at the amount of 10¹³ phages could inhibit B. pseudomallei protease activity by approximately 50%.