Mycologia, 107(1), 2015, pp. 32–38. DOI: 10.3852/13-186 # 2015 by The Mycological Society of America, Lawrence, KS 66044-8897
Genetic diversity of Kenyan native oyster mushroom (Pleurotus) Ojwang D. Otieno1 Calvin Onyango
population (0.25) and commercial cultivars (0.24) did not differ significantly. However, diversity was greater within (89%; P . 0.001) populations than among populations. Homology search analysis against the GenBank database using 16 rDNA ITS sequences randomly selected from the two clades of AFLP dendrogram revealed three mushroom species: P. djamor, P. floridanus and P. sapidus; the three mushrooms form part of the diversity of Pleurotus species in Kenya. The broad diversity within the Kenyan Pleurotus species suggests the possibility of obtaining native strains suitable for commercial cultivation. Key words: DNA polymorphism, genetic variability, molecular phylogeny, oyster mushroom
Food Technology Division, Kenya Industrial Research and Development Institute, P.O. Box 30650-00100 Nairobi, Kenya
Justus Mungare Onguso Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, P.O Box 62000-00200 Nairobi, Kenya
Lexa G. Matasyoh Department of Biological Sciences, University of Eldoret, P.O. Box 1125-30100 Eldoret, Kenya
Bramwel W. Wanjala Biotechnology Institute, Kenya Agricultural and Livestock Research Organization, P.O. Box 1473300800, Nairobi, Kenya
INTRODUCTION
Mark Wamalwa Jagger J.W. Harvey
Members of the genus Pleurotus commonly known as oyster mushroom are widely distributed throughout the world. The high nutritional and medicinal properties of Pleurotus species make it a beneficial dietary item (Alam et al. 2008, Barros et al. 2008). The species also is able to grow and mature in 2–3 mo. Consequently, Pleurotus is the second most popular cultivated mushroom species in the world market (Royse et al. 2004). Mushroom farming in Kenya is a relatively small, and the information on mushroom cultivation in the country is limited. However, the mushroom industry in the country is rapidly growing, and production cannot currently meet an increasing local demand. Local farmers rely on the importation of mother cultures from outside Africa for spawn production. Some farmers also prefer importing spawn instead of mother cultures for cultivation. The imported mushrooms lineages are faced with numerous challenges including poor regional adaptability, increased susceptibility to pests and diseases and low yields. Exploitation of native strains of mushroom species is therefore likely to provide strains with desirable characteristics for commercial cultivation. Characterization and identification at the species level is an important first step in systematic exploitation of any fungal strain in specific applications. The traditional phenotypic approach is still being used; however, this method has been criticized widely for its high subjectivity to environmental conditions (Jang et al. 2003). Characterization with molecular tools such as the AFLP markers and ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequences has proven
Biosciences eastern and central Africa –International Livestock Research Institute (BecA-ILRI Hub, ILRI, P.O. Box 30709, Nairobi, Kenya
Abstract: Members of the genus Pleurotus, also commonly known as oyster mushroom, are well known for their socioeconomic and biotechnological potentials. Despite being one of the most important edible fungi, the scarce information about the genetic diversity of the species in natural populations has limited their sustainable utilization. A total of 71 isolates of Pleurotus species were collected from three natural populations: 25 isolates were obtained from Kakamega forest, 34 isolates from Arabuko Sokoke forest and 12 isolates from Mount Kenya forest. Amplified fragment length polymorphism (AFLP) was applied to thirteen isolates of locally grown Pleurotus species obtained from laboratory samples using five primer pair combinations. AFLP markers and internal transcribed spacer (ITS) sequences of the ribosomal DNA were used to estimate the genetic diversity and evaluate phylogenetic relationships, respectively, among and within populations. The five primer pair combinations generated 293 polymorphic loci across the 84 isolates. The mean genetic diversity among the populations was 0.25 with the population from Arabuko Sokoke having higher (0.27) diversity estimates compared to Mount Kenya population (0.24). Diversity between the isolates from the natural Submitted 5 Jun 2013; accepted for publication 3 Oct 2014. 1 Corresponding author. E-mail:
[email protected]
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OTIENO ET AL.: PLEUROTUS to be more reliable in biodiversity studies. Different mushroom lineages, including members belonging to the genus Pleurotus have been successfully discriminated with molecular tools (Meng et al. 2003). In the present study we evaluated the genetic variability and phylogenetic relationships of Kenyan native Pleurotus species with AFLP markers and rDNA ITS sequences. This is the first time, to the best of our knowledge, that such a study was carried out in Kenya. MATERIALS AND METHODS Sample collection and preparation.—Sampling of Pleurotus species was conducted in three major indigenous forests in Kenya: Arabuko Sokoke Forest in the southern part, Mount Kenya Forest in the central part and Kakamega Forest in the western part of the country. These forests are located more than 350 km a part from each other. The altitude of Arabuko Sokoke Forest is 150–198 ft, whereas Kakamega and Mount Kenya Forest are 1500–1700 ft and 5100–7780 ft, respectively. Thirty-four fruiting bodies of Pleurotus species were collected from the Arabuko Sokoke Forest. The fruiting bodies were collected from either dead or decaying trunks of tree species including Brachystegia speciformis, Afzelia quansensis, Jubalnadiaa magnistipulata and Paramacfolobium coereleum. A total of 25 fruiting bodies of Pleurotus species were obtained from the Kakamega Forest. They were collected from either dead or decaying trunks of different of Croton megalocapus, Sequoiadendron sp., Maesopsis eminii, Vitex kiniensis, Albizia gumifera and Prunus africana. The 12 fruiting bodies of Pleurotus species were collected from either dead or decaying trunks of Vitex kiniensis, Copressus lustanica, Croton megalocapus and Pondocapus milajianus. Each isolate was obtained at least 10 m apart to avoid sampling repeats. Thirteen isolates of cultivated Pleurotus species were obtained from Jomo Kenyatta University of Agriculture and Technology for comparative data analysis. Preparation of tissue cultures.—Young and healthy fruit bodies were prepared by inoculating small interior tissues in Petri dishes containing potato dextrose agar. Mycelium from each sample was subcultured at 25 C several times until pure cultures were obtained. Isolation of genomic DNA.— Total genomic DNA was extracted from the mycelia with the modified cetyl trimethyl ammonium bromide method (Izumitsu et al. 2012). Mycelium (0.1 g) was homogenized in 400 mL CTAB buffer in a 2000 Genogrinder (Troemner Inc., Beirut, Lebanon). The crushed mycelia were resuspended in 0.5 mL extraction buffer (100 mM Tris-HCl [pH 8], 2% [wt/vol] CTAB, 50 mM EDTA, 0.7 M Nacl, 1% [vol/vol] b-mercaptoethanol and 1% [w/v] PVP) and incubated 1 h at 65 C. Solution (0.5 mL) of chloroform-isoamyl alcohol (24 : 1 vol/vol) was added to the mixture of extraction buffer and the two phases were mixed several times by gently inverting tubes. The resulting emulsion was centrifuged at 4500 3 g, , 20 C (room temperature) for 5 min with Beckman Coulter, AllegraTM 25R centrifuge (Beckman Coulter Inc., Califor-
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nia). The upper aqueous phase was removed and mixed with 50 mL 3M sodium acetate and 400 mL 100% isopropanol in 1.2 mL tubes. Samples of DNA were left to precipitate for 12 h at 4 C and centrifuged at 3500 3 g, , 20 C for 5 min. Supernatant was discarded and pellets washed two times with 400 mL 70% ethanol. The pellets were air-dried on a clean paper towel in the hood for 1 h before washing two times with an equal volume of 70% ethanol. DNA pellets were resuspended in a low-salt TE0.1 buffer (15 mM Tris-HCl pH 8.0, EDTA 0.5M) and incubated at 37 C for 30 min with 2 mL DNAse-free RNAseA (10 mg/mL; New England Biolabs, category No. EN0531. The quantity and quality of isolated DNA were determined with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, Delaware) and agarose gel electrophoresis, respectively. Restriction digestion of genomic DNA and adaptor ligation.— Restriction digestion of genomic DNA and adaptor ligation were performed as described by Vos et al. (1995) with minor modifications. One microgram (25 ng/mL) of template DNA was digested with 5 U EcoRI and MseI enzymes and ligated to 1 mL of each adaptor from the AFLP Core Reagent Kit (Applied Biosystems, Foster City, California; catalog No. 402005). The restriction digestion-ligation mixture was diluted 10-fold with low salt TE0.1 buffer (15 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) before preselective amplification. A 20 mL total reaction volume containing 4 mL diluted DNA from the digestion and ligation step, 5 mM EcoRI (E) primer (59–GAC TGC GTA CCA ATT C–39), 1 mM MseI (M) primer (59–GAT GAG TCC TGAGTAA–39) and 15 mL AFLP amplification core mix (ABI, Foster City, California) was used. The thermal-cycling program for pre-amplification was: 2 min at 72 C; 20 cycles consisting of 20 s denaturation at 94 C, 30 s primer annealing at 56 C, 2 min at 72 C extension and a final 30 min hold at 60 C. AFLP preselective amplification.—Preselected reaction mixture was diluted 20-fold using 15 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM EDTA. A total 3 mL was used as DNA template for selective amplification. The EcoRI (1 mM) selective primers labeled with fluorescent dyes JOE, FAM or NAD were used in combination with five MseI (5 mM) selective primers and 15 mL AFLP amplification core mix as described by Vos et al. (1995). AFLP selective amplification.—PCR was performed with these parameters: an initial 2 min at 94 C followed by one cycle of 94 C for 20 s, 66 C for 30 s and 72 C for 2 min. This cycle was repeated eight times by lowering the annealing temperature 1 C each successive cycle. This was followed by 20 cycles of 94 C for 20 s, 56 C for 30 s and 72 C for 2 min and a further hold of 30 min at 60 C. The fragments were detected using an ABI 3730 3 l genetic analyzer (ABI Inc., Foster City, California). Gene Scan 500 LIZ (ABI Inc., Foster City, California) was used as a molecular size standard. AFLP data analysis.—Isolates were scored for presence (1) and absence (0) of AFLP bands with GeneMapper 4.1 software (Applied Biosystem Inc., Foster City, California). Electropherogram peaks with high relative fluorescence units (rfu) with low background noise were scored to generate a data matrix. Category bins were created to group
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peaks based on the sizes of the allele. A threshold peak height was set at 50–500 rfu. Alleles present in all accessions were not scored. A pairwise genetic distance matrix for all genotypes was constructed from the AFLP data matrix with Euclidean distance method (Kaufman and Rousseeuw 1990). A dendrogram based on the distance matrix was constructed by an unweighted pair group of arithmetic averages (UPGMA) clustering algorithm (Sokal and Michener 1958) implemented in the Tools for Population Genetic Analysis (TPGA) software (Miller 1997). Measures of genetic diversity, including Nei’s unbiased measure of genetic diversity (h) and Shannon’s Information index (I) at each locus were determined with TPGA software and GenAlEx 6.5. Analysis of internal transcribed spacer sequences.— Sixteen isolates randomly collected from each of the two clusters generated by AFLP dendrogram were used for further analysis with ITS sequencing. DNA fragments containing the ITS1 and ITS2 region of rDNA were amplified with ITS1 (59–TCC GTA GGT GAA CCT GCG G–39) forward and ITS-4 (59–TCC TCC GCT TAT TGA TAT GC–39) reverse primers (White et al. 1990). Amplification was performed with this PCR program: 95 C for 1 min; 35 cycles at 94 C for 30 s, 60 C for 30 s, 72 C for 2 min; and a final elongation at 72 C for 10 min. PCR products were treated with ExoSAP-IT (USB Corp., Cleveland, Ohio) and electrophoresed on a 1.5% (w/v) agarose gel prepared in 13 TBE (0.1M Tris-HCl pH 8.0; 0.1M boric acid; 0.5M EDTA) buffer. PCR product was visualized with GelRedTM (Biotium Inc., UK) staining. The PCR product was measured with a 1 kb DNA ladder as a marker. Amplified DNA was sequenced with the ABI PRISM 3.1 BigDye terminator kit (Perkin Elmer, USA) and electrophoresed in an ABI PRISM 3700 Genetic Analyzer. Sequencing was carried out for both strands with the forward and reverse primers used for initial amplification. A phylogenetic tree based on ITS sequences was inferred with the maximum likelihood method and the Jukes-Cantor substitution model as implemented in the molecular evolutionary genetics analysis (MEGA) 5.0. Support for phylogenetic groupings was assessed by bootstrap analysis (1000 replicates) with random addition of sequences during each heuristic search (Felsenstein 1985). Only significant bootstrap replication frequencies above 50% were indicated. Resulting ITS sequences were edited and aligned with Bioedit software (Hall 1999). The nucleotide sequences from the most homologous BLASTn alignments were retrieved from the GeneBank database (http://www. ncbi.nlm.nih.gov/blast) and used as reference representatives for phylogenetic analysis. Sequences have been deposited in GenBank under the submission ID BankIt1720168 (NCBI). The trees were rooted with Hemicola grisea (Peck) Singer as outgroup taxa.
RESULTS AFLP polymorphism.—A total of 14 primer pairs were screened for their ability to generate polymorphic fragments. Only five primer pairs generated the most polymorphic and scorable fragment numbers
TABLE I. AFLP primers pairs and polymorphism across 84 isolates of Pleurotus species Primer pair Ea+AAC/ Mb+CTC E+ACA/ M+CAT E+AT/ M+CTG E+AGG/ M+CTG E+AGG/ M+CAT Total a b
Total No. of fragments
Polymorphic Polymorphism fragments (%)
228
116
51
115
102
89
160
40
25
20
16
80
120 643
56 293
47 51
E 5 EcoR1 primers. M 5 Mse1 primers.
(TABLE I). The selected primer pairs produced 293 polymorphic fragments accounting for 51% of the total numbers of fragments. The fragments were 51— 497 base pairs (bp). The number of polymorphic loci produced by each primer pair varied from 16 (for primer pair 59Eco +AGG 2 Mse +CTG 39) to 116 (for primer pair 59Eco +AAC 2 Mse +CTC 39). Genetic variability.— Genetic diversity among the four populations of Pleurotus species are revealed (TABLE II). The diversity was lowest (h 5 0.24) among both the populations of MounttKenya (MK) and cultivated species (JK) and highest (h 5 0.27) among the population of Arabuko Sokoke (AS). The same order of diversity was revealed by Shannon’s information index (I) and percentage polymorphism (%loci). The mean diversity estimates between the isolates from the natural populations (h 5 0.25) and cultivated species (h 5 0.24) was small and did not have any significant difference. However, the analysis of molecular variance (AMOVA) revealed that the majority of the total observed allele frequency variations were found from within populations (89%). The remaining 11% could be attributed to frequency variations among populations. This was confirmed further by low gene differentiation between the pairs of populations in terms of allele frequency (FST). The contributions from each of these sources were statistically significant (P , 0.001). Genetic distances.—The genetic distances among the four populations of Pleurotus species are illustrated (TABLE III). The population of cultivated species (JK) was closely (0.2028) related to population in Arabuko Sokoke forest and distantly (0.0464) related to population in Kakamega Forest. The population of Pleurotus species in Kakamega Forest was closely
OTIENO ET AL.: PLEUROTUS TABLE II.
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Genetic diversity estimates among four populations of Pleurotus species
Pop ID
Latitude
Longitude
Sample size
naa
neb
hc
I
% locid
ASe KKe MKe JKf
3u209S 0u169N 0u129S —
39u509E 34u459E 37u189E —
34 25 12 13
1.98 1.88 1.73 1.72
1.42 1.39 1.37 1.37
0.27 0.25 0.24 0.24
0.41 0.39 0.37 0.35
99.1 92.1 83.1 82.1
a
na 5 observed number of alleles. ne 5 effective number of alleles. c h 5 Nei’s unbiased measure of genetic diversity. d % loci 5 percentage polymorphic loci. e AS, KK and MK represent populations of Pleurotus species from Arabuko Sokoke, Kakamega and Mount Kenya Forests, respectively. f JK represents cultivated Pleurotus species. b
related to population in Mount Kenya Forest and distantly related to population in Arabuko Sokoke. The genetic distances among the populations were small and are not statistically significant. Cluster analysis.—An AFLP dendrogram constructed by UPGMA analysis is illustrated (FIG. 1). The 84 isolates formed two major clades with bootstrap values of 64% and 67%, respectively. Clade 1 included 24 isolates of Pleurotus species, 13 from Arabuko Sokoke (AS) and five from Kakamega (KK) and six isolates of cultivated species (JK). Five isolates of cultivated species formed most of distinct subgroup (b) within the first clade. The second clade consisted of 60 isolates distributed across the 8 (c-j) subgroups. This clade consisted mainly of isolates from the natural populations: 12 isolates from Mount Kenya Forest, 20 isolates from Kakamega Forest, 21 isolates from Arabuko Sokoke Forest and seven isolates of cultivated species. Isolates in these two clades showed high homology as revealed by high bootstrap values (.59). The neighbor joining analysis of ITS sequence data derived from 16 randomly selected isolates together with six reference strains from the GenBank revealed two distinct clades (FIG. 2), which were largely consistent with the AFLP dendrogram. Phylogenetic analysis.—Based on homology searches against the GenBank database, we retrieved a total of nine ITS sequences with high sequence identity (.90%) to our sequences. These nine sequences all had associated species identification in the GenBank database, belonging to the genus Pleurotus. Many other ITS sequences (.20) in GenBank had similar sequence identities (,90%) to the ones characterized in this study. Such sequences were not included in our analyses because they were from direct environmental DNA sampling and sequencing and had no species designation associated with them. Based on an initial phylogenetic analysis of all nine ITS sequences from GenBank and comparison with our own 16 ITS
sequences, six representative GenBank sequences were chosen. The selection of the six sequences was based on their comparable sequence lengths to our sequences, their phylogenetic positions, as well as whether there were two or more strains for the same species. The respective GenBank accession numbers for ITS sequences and the associated species in the GenBank are provided (FIG. 2). DISCUSSION Edible mushrooms represent a promising tool in efforts to increase food and nutritional security in developing countries. In Kenya only exotic Pleurotus species is being cultivated. This activity supports the livelihoods of many resource-poor families living in both rural and peri-urban areas. This study explored the diversity of Kenyan native Pleurotus species and provided the first insight into their genetic relationships with the exotic cultivated species. AFLP markers generated 293 polymorphic loci across the 84 isolates of Pleurotus species studied. The high number of polymorphic loci generated by the five AFLP primer pairs confirmed the suitability of AFLP markers to discriminate mushroom lineages. Remote relationship of the outgroup taxa (H.grisea) to the 84 isolates in the AFLP dendrogram further indicates the validity of using AFLP for the classification of mushroom lineages including closely related species. The primer pairs E + ACA/M + CTC, E + AT/M + CAT, E + ACA/M + CTA, E + ACC/M + CTC and E + AT/M + CTC are therefore good candidate primer combinations to verify genetic diversity of other wild Pleurotus species. Similarly, Meng et al. (2003) reported the suitability of AFLP markers in biodiversity studies and genotyping of different mushroom lineages. The diversity among the natural populations of Pleurotus species (h 5 0.25) was slightly higher than that of cultivated species (h 5 0.24) as illustrated (TABLE II). The low diversity within the cultivated
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FIG. 2. The evolutionary tree of 16 isolates and six reference strains of Pleurotus species based on ITS sequences. The tree was inferred by with the maximum likelihood method based on the Jukes-Cantor model. KK, AS and MK represent Pleurotus species from Kakamega, Arabuko Sokoke and Mount Kenya forests, respectively; JK represents isolates of cultivated Pleurotus species in Kenya. Bootstrap values above 50% are shown above the nodes. a–j represent subclades.
FIG. 1. Unweighted pair-group method with arithmetic clustering (UPGMA) dendrogram based on data from AFLP analysis of 71 isolates of Pleurotus species. KK, AS and MK represent Pleurotus species from Kakamega, Arabuko Sokoke and Mt. Kenya forests, respectively; JK represents cultivated Pleurotus species in Kenya. Numbers at the branches are bootstrap values.
species could be attributed to the fact that cultivated species have restricted distribution and/or are usually derived from a limited number of elite lines, which often are used in the production of many cultivars. Multiple production of cultivars from limited lines results in an increasingly narrow genetic base (Ramanatha Rao and Hodgkin 2002). A similar study reported low variation in mushroom species
OTIENO ET AL.: PLEUROTUS that have long been cultivated for commercial purposes (Old et al. 1984, Burdon and Roelfs 1985). The analysis of molecular variance (AMOVA) revealed high variability within (89%) than among (11%) populations. This was consistent with the low degree of gene differentiation (FST 5 0.125) between the pairs of populations, suggesting that a large amount of genetic diversity lies within populations. The observed distribution pattern of genetic variation within populations seems to be the result of minimal gene flow, probably by inefficient basidiospore dispersal and outcrossing mechanisms, which promote inbreeding within populations. The high genetic variation is generally expected for wild organisms that reproduce sexually and have broad ecological niches and a wide geographical distribution (James et al. 1999). Diverse strains suitable for commercial production could be obtained from within the natural populations present in Kenyan forests. The AFLP cluster analysis revealed that isolates of Pleurotus species obtained from different locations did not regroup according to their geographical origins. Some species originally collected from distant areas displayed high similarity values; conversely some species from the same geographical locations did not cluster. Long-distance spore dispersal could be responsible for observed distribution patterns. Evidence for similar gene flow was found between populations of ectomycorrhizal mushroom as far as 2000 km apart (Xu et al. 1998). It was hypothesized that wind dispersal of sexual basidiospores could be responsible for the observed gene flow. Another probable explanation of the observed distribution patterns could be that the distribution of Pleurotus species in the natural population predates forest fragmentation and/or is promoted by human movement. It is likely to be a combination of these factors, considering the presence of the Rift Valley (geographic separation) and deforestation, and movement of mushrooms by people. A similar phylogenetic relationship pattern was used to provide a framework for understanding the biogeographic history of speciation in Pleurotus (Vilgalys and Sun 1994). The dendrogram based on the rDNA ITS sequences of the 16 isolates was largely consistent with the AFLP dendrogram. The two distinct clades with 100% bootstrap support formed by the rDNA ITS sequences suggest close relationships among them, indicating that they might be distributed from a common ancestor. This also suggests the likelihood of a high degree of similarity in terms of growth characteristics, color, size and Biological Efficiency (BE) among these strains. Similar phylogenetic analyses were used to determine characteristics among the Pleurotaceae that shared a common ancestry (Thorn et al. 2000).
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ITS phylogenies have been used to explain geographic speciation of Pleurotus species and allied taxa (Zervakis 2004). In this study the distribution of isolates in the two ITS clades did not correspond to their geographical origin as is the case with AFLP analysis, revealing that the diversity of Pleurotus species is shared across the country. Homology search analysis using 16 ITS sequences against the GenBank database allowed the identification of three mushroom species belonging to the genus Pleurotus: P. floridanus, P. djamor and P. sapidus. All the commercial strains (JK07, JK13, JK10, JK01) were 100% identical to their Genebank homolog P. floridanus. This confirms the suitability of rDNA ITS sequences to investigate fungal relationships at different taxonomic levels (Ravash et al. 2010). Therefore, this study has identified these three mushroom species as part of the diversity of Pleurotus species in Kenya. Conclusions.—This study confirms that AFLP markers are suitable tool for genetic assessment of Pleurotus species. The analysis of molecular variance suggested that the native strains of Pleurotus species in Kenya are genetically more diverse than their exotic counterparts now being cultivated there. Further taxonomic investigations involving microscopic and macroscopic features coupled with the analyses of multiple additional genes of more Pleurotus species/isolates from different regions are needed to fully understand the diversity of Kenyan Pleurotus species. ACKNOWLEDGMENTS This study was made possible through the financial support from the National Council for Science and Technology (grant reference number NCST/5/003/INN1st) and Kenya Industrial Research and Development Institute. The authors thank the Kenya Forest Service for providing mushroom genetic materials. We also thank the Biosciences for eastern and central Africa-International Livestock Research Institute (BecA-ILRI) Hub in Nairobi, Kenya, for guidance, collaboration and laboratory support. Support from the Australian government through the BecA-Commonwealth Scientific and Industrial Research Organization is also gratefully acknowledged.
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