Invited review: Genomic selection for small ruminants in developed countries: how applicable for the rest of the world?

Improved management and use of estimated breeding values in breeding programmes, have resulted in rapid genetic progress for small ruminants (SR) in Europe and other developed countries. The development of single nucleotide polymorphisms chips opened opportunities for genomic selection (GS) in SR in these countries. Initially focused on production traits (growth and milk), GS has been extended to functional traits (reproductive performance, disease resistance and meat quality). The GS systems have been characterized by smaller reference populations compared with those of dairy cattle and consisting mostly of cross- or multi-breed populations. Molecular information has resulted in gains in accuracy of between 0.05 and 0.27 and proved useful in parentage verification and the identification of QTLs for economically important traits. Except for a few established breeds with some degree of infrastructure, the basic building blocks to support conventional breeding programmes in small holder systems are lacking in most developing countries. In these systems, molecular data could offer quick wins in undertaking parentage verification and genetic evaluations using G matrix, and determination of breed composition. The development of next-generation molecular tools has prompted investigations on genome-wide signatures of selection for mainly adaptive and reproduction traits in SR in developing countries. Here, the relevance of the developments and application of GS and other molecular tools in developed countries to developing countries context is examined. Worth noting is that in the latter, the application of GS in SR will not be a 'one-size fits all' scenario. For breeds with some degree of conventional genetic improvement, classical GS may be feasible. In small holder systems, where production is key, community-based breeding programmes can provide the framework to implement GS. However, in fragile growth systems, for example those found in marginal environments, innovative GS to maximize adaptive diversity will be required. A cost-benefit analysis should accompany any strategy of implementing GS in these systems.

Genetic diversity and population structure of Urochloa grass accessions from Tanzania using simple sequence repeat (SSR) markers.

Urochloa (syn.-Brachiaria s.s.) is one of the most important tropical forages that transformed livestock industries in Australia and South America. Farmers in Africa are increasingly interested in growing Urochloa to support the burgeoning livestock business, but the lack of cultivars adapted to African environments has been a major challenge. Therefore, this study examines genetic diversity of Tanzanian Urochloa accessions to provide essential information for establishing a Urochloa breeding program in Africa. A total of 36 historical Urochloa accessions initially collected from Tanzania in 1985 were analyzed for genetic variation using 24 SSR markers along with six South American commercial cultivars. These markers detected 407 alleles in the 36 Tanzania accessions and 6 commercial cultivars. Markers were highly informative with an average polymorphic information content of 0.79. The analysis of molecular variance revealed high genetic variation within individual accessions in a species (92%), fixation index of 0.05 and gene flow estimate of 4.77 showed a low genetic differentiation and a high level of gene flow among populations. An unweighted neighbor-joining tree grouped the 36 accessions and six commercial cultivars into three main clusters. The clustering of test accessions did not follow geographical origin. Similarly, population structure analysis grouped the 42 tested genotypes into three major gene pools. The results showed the Urochloa brizantha (A. Rich.) Stapf population has the highest genetic diversity (I = 0.94) with high utility in the Urochloa breeding and conservation program. As the Urochloa accessions analyzed in this study represented only 3 of 31 regions of Tanzania, further collection and characterization of materials from wider geographical areas are necessary to comprehend the whole Urochloa diversity in Tanzania.

First Complete Genome Sequences of Porcine Bocavirus Strains from East Africa.

Here, we report the first complete genome sequences of two strains of porcine bocavirus (JOA_011 and JOA_015) detected in Uganda and Kenya, respectively. These data will help in understanding the molecular and evolutionary characteristics of the porcine bocaviruses in this region and the development of appropriate diagnostic and control tools.

Differential T-cell responses to a chimeric Plasmodium falciparum antigen; UB05-09, correlates with acquired immunity to malaria.

The development of a sterilizing and cost-effective vaccine against malaria remains a major problem despite recent advances. In this study, it is demonstrated that two antigens of P. falciparum UB05, UB09 and their chimera UB05-09 can serve as protective immunity markers by eliciting higher T-cell responses in malaria semi-immune subjects (SIS) than in frequently sick subjects (FSS) and could be used to distinguish these two groups. UB05, UB09 and UB05-09 were cloned, expressed in E. coli, purified and used to stimulate PBMCs isolated from 63 subjects in a malaria endemic area, for IFN-γ production, which was measured by the ELISpot assay. The polymorphism of UB09 gene in the malaria infected population was also studied by PCR/sequencing of the gene in P. falciparum field isolates. All three antigens were preferentially recognized by PBMCs from SIS. IFN-γ production induced by these antigens correlated with the absence of fever and parasitaemia. UB09 was shown to be relatively well-conserved in nature. It is concluded that UB05, UB09 and the chimera UB05-09 posses T-cell epitopes that are associated with protection against malaria and could thus be used to distinguish SIS from FSS eventhough acute infection with malaria has been shown to reduce cytokine production in some studies. Further investigations of these antigens as potential diagnostic and/or vaccine candidates for malaria are indicated.

Detection and genetic characterization of porcine group A rotaviruses in asymptomatic pigs in smallholder farms in East Africa: predominance of P[8] genotype resembling human strains.

Viral enteritis is a serious problem accounting for deaths in neonatal animals and humans worldwide. The absence of surveillance programs and diagnostic laboratory facilities have resulted in a lack of data on rotavirus associated diarrheas in pigs in East Africa. Here we describe the incidence of group A rotavirus (RVA) infections in asymptomatic young pigs in East Africa. Of the 446 samples examined, 26.2% (117/446) were positive for RVA. More nursing piglets (78.7%) shed RVA than weaned (32.9%) and grower (5.8%) pigs. RVA incidence was higher in pigs that were either housed_free-range (77.8%) or tethered_free-range (29.0%) than those that were free-range or housed or housed-tethered pigs. The farms with larger herd size (>10 pigs) had higher RVA prevalence (56.5%) than farms with smaller herd size (24.1-29.7%). This study revealed that age, management system and pig density significantly (p<0.01) influenced the incidence of RVA infections, with housed_free-range management system and larger herd size showing higher risks for RVA infection. Partial (811-1604nt region) sequence of the VP4 gene of selected positive samples revealed that different genotypes (P[6], P[8] and P[13]) are circulating in the study area with P[8] being predominant. The P[6] strain shared nucleotide (nt) and amino acid (aa) sequence identity of 84.4-91.3% and 95.1-96.9%, respectively, with known porcine and human P[6] strains. The P[8] strains shared high nt and aa sequence identity with known human P[8] strains ranging from 95.6-100% to 92-100%, respectively. The P[13] strains shared nt and aa sequence identity of 83.6-91.7% and 89.3-96.4%, respectively, only with known porcine P[13] strains. No P[8] strains yielded RNA of sufficient quality/quantity for full genome sequencing. However analysis of the full genome constellation of the P[6], two P[13] and one untypeable strains revealed that the P[6] strain (Ke-003-5) genome constellation was G26-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, P[13] strains (Ug-049 and Ug-453) had G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 while the untypeable strain (Ug-218) had G5-P[?]-I5-R1-C1-M1-A8-N1-T1-E1-H? In conclusion, P[6] and P[8] genotypes detected were genetically closely related to human strains suggesting the possibility of interspecies transmission. Further studies are required to determine the role of RVA in swine enteric disease burden and to determine the genetic/antigenic heterogeneity of the circulating strains for development of accurate diagnostic tools and to implement appropriate prophylaxis programs.

A database of expressed genes from Cochliomyia hominivorax (Diptera: Calliphoridae).

We used an expressed sequence tag and 454 pyrosequencing approach to initiate a study of the genome of the screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). Two normalized cDNA libraries were constructed from RNA isolated from embryos and second instar larvae from the Panama 95 strain. Approximately 5,400 clones from each library were sequenced from both the 5' and 3' directions using the Sanger method. In addition, double-stranded cDNA was prepared from random-primed polyA RNA purified from embryos, second-instar larvae, adult males, and adult females. These four cDNA samples were used for 454 pyrosequencing that produced approximately 300,000 independent sequences. Sequences were assembled into a database of assembled contigs and singletons and used to search public protein databases and annotate the sequences. The full database consists of 6,076 contigs and 58,221 singletons assembled from both the traditional expressed sequence tag (EST) and 454 sequences. Annotation of the data led to the identification of several gene coding regions with possible roles in sex determination in the screwworm. This database will facilitate the design of microarray and other experiments to study screwworm gene expression on a larger scale than previously possible.

RNA interference in Trypanosoma brucei: cloning of small interfering RNAs provides evidence for retroposon-derived 24-26-nucleotide RNAs.

In animals and protozoa, gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous cellular RNAs, a phenomenon known as RNA interference (RNAi). In vitro and in vivo dsRNA is processed by a nuclease to produce 21-25-nt small interfering RNAs (siRNAs) that guide target RNA degradation. Here we show that activation of RNAi in Trypanosoma bruceiby expression or electroporation of actin dsRNA results in production of actin siRNAs and that 10% of these RNAs sediment as high-molecular-weight complexes at 100,000 x g. To characterize actin siRNAs, we established a cloning and enrichment strategy starting from 20-30 nt RNAs isolated from high-speed pellet and supernatant fractions. Sequence analysis revealed that actin siRNAs are 24-26 nt long and their distribution relative to actin dsRNA was similar in the two fractions. By sequencing over 1,300 fragments derived from the high-speed pellet fraction RNA, we found abundant 24-26-nt-long fragments homologous to the ubiquitous retroposon INGI and the site-specific retroposon SLACS. Northern hybridization with strand-specific probes confirmed that retroposon-derived 24-26-nt RNAs are present in both supernatant and high-speed pellet fractions and that they are constitutively expressed. We speculate that RNAi in trypanosomes serves a housekeeping function and is likely to be involved in silencing retroposon transcripts.

Characterization of a candidate Trypanosoma brucei U1 small nuclear RNA gene.

We have previously shown that the poly(A) polymerase (PAP) gene of Trypanosoma brucei is interrupted by an intervening sequence. It was postulated that removing this intron by cis-splicing requires a yet unidentified U1 small nuclear RNA (snRNA), which in other organisms engages in base-pair interactions across the 5' splice site during early spliceosome assembly. Here we present a characterization of a 75 nucleotide long candidate T. brucei U1 snRNA. Immunoprecipitation studies indicate that a trimethylguanosine cap structure is present at the 5' end and that the RNA is bound to core proteins common to spliceosomal ribonucleoprotein particles. The U1 snRNA has the potential for extensive intermolecular base pairing with the PAP 5' splice site. We used block replacement mutagenesis to identify sequences necessary for in vivo expression of U1 snRNA. We found that at least two cis-acting elements, tRNA-like A and B boxes, located in the 5'-flanking region are necessary for U1 snRNA synthesis; no internal sequences close to the transcription start site are essential, suggesting a promoter architecture distinct from other trypanosome U-snRNA genes.

Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.

The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting a-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the alpha-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.

A new twist in trypanosome RNA metabolism: cis-splicing of pre-mRNA.

It has been known for almost a decade and a half that in trypanosomes all mRNAs are trans-spliced by addition to the 5' end of the spliced leader (SL) sequence. During the same time period the conviction developed that classical cis-splicing introns are not present in the trypanosome genome and that the trypanosome gene arrangement is highly compact with small intergenic regions separating one gene from the next. We have now discovered that these tenets are no longer true. Poly(A) polymerase (PAP) genes in Trypanosoma brucei and Trypanosoma cruzi are split by intervening sequences of 653 and 302 nt, respectively. The intervening sequences occur at identical positions in both organisms and obey the GT/AG rule of cis-splicing introns. PAP mRNAs are trans-spliced at the very 5' end as well as internally at the 3' splice site of the intervening sequence. Interestingly, 11 nucleotide positions past the actual 5' splice site are conserved between the T. bruceiand T. cruzi introns. Point mutations in these conserved positions, as well as in the AG dinucleotide of the 3' splice site, abolish intron removal in vivo. Our results, together with the recent discovery of cis-splicing introns in Euglena gracilis, suggest that both trans- and cis-splicing are ancient acquisitions of the eukaryotic cell.

Generation of expressed sequence tags as physical landmarks in the genome of Trypanosoma brucei.

Previous molecular genetic studies on the African trypanosome have focused on only a few genes and gene products, the majority of which are concerned with surface antigenic variation; consequently, an insignificant number of the genes of this organism have been characterized to date. In order to: (1) identify new genes and analyze their expression profile, (2) generate expressed sequence tags (ESTs) for derivation of a physical map of the trypanosome genome, and (3) make available the partial sequence information and the corresponding clones for general biomedical research on the parasite, we have performed single-pass sequencing of random, directionally cloned cDNAs from a bloodstream form Trypanosoma brucei rhodesiense library. Analysis of 2128 such ESTs sequenced so far in this study showed significant similarities [BLASTX P(n)-value < 10(-4), and a match > 10 amino acid residues] with proteins whose genes have been described in diverse organisms including man, rodents, kinetoplastids, yeasts and plants. A number of the ESTs encode homologues of proteins involved in various functions including signal reception and transduction, cell division, gene regulation, DNA repair and replication, general metabolism, and structural integrity. Although some of these genes may have been expected to be present in the African trypanosomes, the majority of them had not previously been described in these organisms. A large proportion, 768 individual ESTs (36%, representing 385 different transcripts), had a significant homology with genes described in organisms other than the African trypanosomes; however, 15% of the ESTs were from genes already described in trypanosomes. Among the ESTs analysed were 462 distinct known genes, only 77 of which have been described in T. brucei. Approximately 52% of the ESTs did not show any significant homology with the sequences in any of the public domain databases.