bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 The five Urochloa spp. used in development of tropical forage cultivars originate from
2 defined subpopulations with differentiated gene pools
3
4 J Higgins1, P Tomaszewska2, TK Pellny3, V Castiblanco4, J Arango4, J Tohme4, T
5 Schwarzacher2, RA Mitchell3, JS Heslop-Harrison2, JJ De Vega1
6
7 1 Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK.
8 2 Department of Genetics and Genome Biology, University of Leicester, Leicester LE1 7RH,
9 UK.
10 3 Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK.
11 4 International Center for Tropical Agriculture (CIAT), 6713 Cali, Colombia.
12
13 HIGHLIGHT
14 We clarified the genetic make-up and population structure of 111 Urochloa spp. forage
15 grasses to inform cultivar development.
16
17 ABSTRACT
18 Background and Aims
19 Urochloa (syn. Brachiaria, and including some Panicum and Megathyrus) is a genus of tropical
20 and subtropical grasses widely sown as forage to feed ruminants in the tropics. A better
21 understanding of the diversity among Urochloa spp. allow us to leverage its varying ploidy
22 levels and genome composition to accelerate its improvement, following the example from
23 other crop genera.
24 Methods
25 We explored the genetic make-up and population structure in 111 accessions, which comprise
26 the five Urochloa species used for the development of commercial cultivars. These accessions
27 are conserved from wild materials from collection sites at their centre of origin in Africa. We
28 used RNA-seq, averaging 40M reads per accession, to generate 1,167,542 stringently
1 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
29 selected SNP markers that tentatively encompassed the complete Urochloa gene pool used
30 in breeding.
31 Key Results
32 We identified ten subpopulations, which had no relation with geographical origin and
33 represented ten independent gene pools, and two groups of admixed accessions. Our results
34 support a division in U. decumbens by ploidy, with a diploid subpopulation closely related to
35 U. ruziziensis, and a tetraploid subpopulation closely related to U. brizantha. We observed
36 highly differentiated gene pools in U. brizantha, which were not related with origin or ploidy.
37 Particularly, one U. brizantha subpopulation clustered distant from the other U. brizantha and
38 U. decumbens subpopulations, so likely containing unexplored alleles. We also identified a
39 well-supported subpopulation containing both polyploid U. decumbens and U. brizantha
40 accessions; this was the only group containing more than one species and tentatively
41 constitutes an independent “mixed” gene pool for both species. We observed two gene pools
42 in U. humidicola. One subpopulation, “humidicola-2”, was much less common but likely
43 includes the only known sexual accession in the species.
44 Conclusions
45 Our results offered a definitive picture of the available diversity in Urochloa to inform breeding
46 and resolve questions raised by previous studies. It also allowed us identifying prospective
47 founders to enrich the breeding gene pool and to develop genotyping and genotype-
48 phenotype association mapping experiments.
49
50 Keywords: Brachiaria, grassland, breeding, forage, RNA-seq, population structure,
51 Poaceae
2 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
52 INTRODUCTION
53 Urochloa (syn. Brachiaria, and including some Panicum and Megathyrus) is a genus of tropical
54 and subtropical grasses widely sown as forage to feed ruminants in the American and African
55 tropics, particularly in areas with marginal soils. Urochloa grasses exhibit good resilience and
56 low nutritional needs (Miles, 2007, Gracindo et al., 2014, Maass et al., 2015). Five species, U.
57 ruziziensis, U. decumbens, U. brizantha, U. humidicola, and U. maxima are broadly used as
58 fodder plants, covering over 100M hectares in Brazil alone. Such an enormous area, about
59 half that of each of the most widely grown cereals, wheat or maize, has a huge environmental
60 impact in terms of displacement of native species, water usage, and provision of ecosystem
61 services. In addition to extensive pasture systems in Latin America, Urochloa is also planted
62 in intensive smallholder systems in Africa and Asia (Keller-Grein et al., 1996, Maass et al.,
63 2015). Breeding programmes in different countries have exploited the diversity among
64 Urochloa spp. for the development of commercial forage cultivars by recurrent selection over
65 many years (Jank et al., 2014, Tsuruta et al., 2015, Worthington and Miles, 2015).
66
67 The genus Urochloa includes species previously classified under Brachiaria, Megathyrsus,
68 Eriochloa and Panicum (Torres González and Morton, 2005, Kellogg, 2015). Joint missions
69 between 1984 and 1985 conducted by the CGIAR (Consultative Group on International
70 Agricultural Research) centres in several African countries collected wild materials from the
71 species in the genus, mostly as live plant cuttings or ramets (Keller-Grein et al., 1996). These
72 activities built a global grass collection with ~ 700 Urochloa accessions that are held at CIAT
73 (Centro Internacional de Agricultura Tropical), ILRI (International Livestock Research
74 Institute), and EMBRAPA (Brazilian Agricultural Research Corporation).
75
76 Three Urochloa species (U. brizantha, U. decumbens, and U. ruziziensis) have been arranged
77 in an agamic (apomictic) group or complex (Do Valle and Savidan, 1996, Renvoize et al.,
78 1996, Ferreira et al., 2016, Triviño et al., 2017). Crosses between ~ 10 founders from these
79 three species were completed in the late 1980s and their progeny constitutes the basis of the
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80 current recurrent selection breeding programmes at CIAT and EMBRAPA (Miles et al., 2006).
81 On the other hand, U. humidicola and U. dictyoneura have been arranged in the “humidicola
82 complex” (Lutts et al., 1991, Renvoize et al., 1996, Triviño et al., 2017). More recently,
83 independent U. humidicola breeding programs have also been established at CIAT and
84 EMBRAPA after the discovery in the mid-2000s of a natural sexual polyploid germplasm
85 accession that could be crossed with other apomictic polyploid U. humidicola pollen donors
86 (Jungmann et al., 2010, Vigna et al., 2011a).
87
88 Urochloa spp. show varying ploidy levels and sub-genome compositions (Do Valle and
89 Savidan, 1996, Keller-Grein et al., 1996, Tomaszewska et al., 2021b, Tomaszewska et al.,
90 2021a), which likely result in a highly diverse gene pool that can be leveraged for continued
91 improvement through breeding. Exploiting sub-genome variability among species and ploidy
92 levels has been highly successful in the improvement of other crop tribes, such as Triticeae
93 and Brassicaceae (Gale and Miller, 1987, Burton et al., 2004, Ali et al., 2016). However,
94 genetic composition and relationships in Urochloa are poorly understood; studies from
95 countries with active Urochloa breeding programmes have explored the phylogeny in these
96 species to inform breeding, but projects leveraged of a limited number of markers, such as
97 ITS, RAPD, SSR, ISSR, and microsatellites (Torres González and Morton, 2005, Jungmann
98 et al., 2010, Vigna et al., 2011a, Vigna et al., 2011b, Ferreira et al., 2016, Triviño et al., 2017)
99
100 Urochloa spp. with apomictic or mixed reproduction have particularly resulted in odd levels of
101 ploidy and contribute to increased intraspecific variability. Polyploidy has many benefits for
102 plants, namely heterosis, gene redundancy, and loss of self-incompatibility and gain of
103 asexual reproduction. In a recent work (Tomaszewska et al., 2021b), we used flow cytometry
104 to determine the ploidy of over 350 Urochloa accessions from these collections and propose
105 an evolutionary model. This work extended and corrected some previous studies (Penteado
106 et al., 2000, Mendes-Bonato et al., 2002). We also concluded ploidy was not related to
107 geographical origin, which agrees with previous results (Jungmann et al., 2010, Vigna et al.,
4 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
108 2011a, Vigna et al., 2011b, Triviño et al., 2017). In another recent work (Worthington et al.,
109 2021), we have made available a genome assembly and gene annotation of a diploid
110 accession of U. ruziziensis (GCA_003016355), which has allowed a greater use of genomics
111 to characterise these materials. For example, we identified loss-of-function (LOF) genes
112 related to forage quality and environmental impact using allele mining (Hanley et al., 2020).
113
114 Here, we have characterised the genetic make-up and population structure of 111 accessions,
115 which are representative of the collections of wild materials in Africa in 1984 and 1985. These
116 111 accessions belong to the five Urochloa spp. that are used in the development of
117 commercial forage cultivars. We used RNA-seq from total RNA, so tentatively encompassing
118 the complete Urochloa gene pool used in breeding to obtain a definitive picture of the available
119 diversity, resolve questions raised by previous studies, and identify prospective founders to
120 improve the breeding gene pool.
121
122 METHODS
123 RNA extraction and sequencing
124 We sequenced 111 accessions from five Urochloa (syn. Brachiaria) species. 104 accessions
125 were sampled at the single time from the in-situ field collection maintained by the Genebank
126 at the International Center for Tropical Agriculture (CIAT) in Cali, Colombia. Accessions
127 sourced from CIAT are named as e.g. “CIAT 26146”, but we have removed “CIAT” from our
128 text. Fresh leaf material was collected and immediately frozen in liquid nitrogen. Samples were
129 ground in liquid nitrogen and lyophilised. Total RNA was extracted as described in Hanley et
130 al. (2020) with the difference that prior to DNAse treatment the pellets were dried in a rotary
131 evaporator (Eppendorf, USA) and stored at room temperature. Another seven accessions
132 were obtained from the United States Department of Agriculture (USDA, GA, USA) as seeds.
133 These seven accessions include “PI” at the beginning of their ID. These seven accessions
134 were sampled at a different time than the other accessions after growing in glasshouses at
135 University of Leicester, UK. We generated one single sample from each accession, and we
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136 use “sample” and “accessions” as synonyms in our case through the text. For all samples,
137 Illumina sequencing using standard RNA-seq library preparations with 150 bp paired reads
138 was conducted by Novogene Europe (Cambridge, UK). The raw reads were deposited in SRA
139 under Bioproject PRJNA513453.
140
141 Read alignment and SNP calling
142 Raw reads were pre-processed using Trim galore v. 0.5 (Krueger, 2015) with the options for
143 Illumina paired reads and trimming 13 bps at the 5’ end in both reads. Processed reads were
144 aligned to the available Urochloa genome (Worthington et al., 2021), which corresponded to
145 a U. ruziziensis accession. RNA to DNA alignments were done using STAR v. 2.6.0c (Dobin
146 et al., 2013) with a minimum overlap of 30 % and a maximum mismatch of 3 bp per alignment,
147 in order to allow for mapping from more distant species to the genome. Alignment coverage
148 was calculated using BEDTools genomecov. SNP calling was done using GATK v. 3.7.0 and
149 the recommended pipeline for RNA-seq (Van der Auwera et al., 2013). Firstly, we used
150 PicardTools v. 2.1.1 to annotated duplicate reads using the option MarkDuplicates. Later, we
151 used GATK’s tool SplitNCigarReads with the options “-rf ReassignOneMappingQuality -RMQF
152 255 -RMQT 60 -U ALLOW_N_CIGAR_READS” to reformat some alignments that span introns
153 to match conventions for the final step. The final step was SNP calling using GATK’s tool
154 HaplotypeCaller with all the samples at the same time (multisample mode). SNP calling was
155 run with the options “-ploidy 6 -dontUseSoftClippedBases -stand_call_conf 20 -
156 maxNumHaplotypesInPopulation 128” to obtain a good quality calling from RNA alignments.
157 GATK identified 6,461,493 variants, which included 5,757,116 SNPs. These were filtered for
158 a minimal allele frequency (MAF) of 1% to give a set of 4,722,195 SNPs. Sites with a depth of
159 lower than 5 were set to missing, then sites with more than 40% missing data were removed
160 to give a final set of 1,167,542 SNPs. Two additional subsets were obtained by filtering out
161 either the 67 samples (895,667 SNPs) in the agamic group or the U. humidicola samples
162 (512,611 SNPs). These subsets were filtered for MAF of 1%.
163
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164 Population analysis
165 Population structure analysis was performed through ADMIXTURE (Alexander and Lange,
166 2011) using K = 3 to K = 10 for the 111 samples, K = 2 to K = 8 for the 67 samples and K = 2
167 to K = 8 for the 28 samples. Each value of K was run 10 times, the cross-validation error was
168 averaged over the 10 runs. The 10 output files were combined using CLUMPP within the R
169 package POPHELPER v.2.2.7 (Francis, 2017). The PCA (principal component analysis) was
170 carried out using Tassel v5.2.41 (Bradbury et al., 2007).
171
172 RESULTS
173
174 Sequencing, aligning and SNP calling in a panel of Urochloa accessions from five
175 species
176 We sequenced 111 accessions from five Urochloa (syn. Brachiaria) species: U. ruziziensis,
177 U. brizantha, U. decumbens, U. humidicola and U. maxima (syn. Megathyrsus maximus).
178 Species identity and ploidy were previously determined using plant architecture traits and flow
179 cytometry of fluorescently stained nuclei (Tomaszewska et al., 2021a, Tomaszewska et al.,
180 2021b). The country of origin of 92 accessions was known and for 75 accessions we also
181 knew the collection coordinates (Fig. 1). Accessions were collected in a broad range of
182 latitudes (20.08S to 11.37N) but not of longitudes (26.98E to 42.05E), except for one U.
183 brizantha accession from Cameroon. Annotations were summarised in Table 1 and detailed
184 in Suppl. Table 1.
185
186 Samples were aligned to the available Urochloa genome assembly and annotation
187 (Worthington et al., 2021), which corresponds to a diploid U. ruziziensis sample. Two well-
188 defined groups of species were observed based on aligning metrics (Fig. 2); over 70 % of the
189 reads from U. ruziziensis, U. decumbens and U. brizantha (all but one) accessions had more
190 than 70 % of reads that aligned in the reference genome once (uniquely-mapping reads). On
191 the contrary, accessions from U. maxima and U. humidicola showed a percentage of uniquely-
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192 mapping reads under 70 % (Fig. 2A). The grouping was correlated to the genetic distance to
193 the reference genome (reference bias).
194
195 The percentage of reads mapping in multiple loci (multi-mapping reads) increased with ploidy
196 (Fig. 2B) for the group of the accessions belonging to the species U. ruziziensis, U.
197 decumbens and U. brizantha; diploids had a percentage of multi-mapping reads under 5 %,
198 while it was over 5 % in most polyploid accessions. However, the percentage of multi-mapping
199 reads in the other species, which are more distant species to the reference, was directly
200 proportional to the total number of mapped reads (Fig. 2B), i.e. independent of ploidy.
201
202 RNA-seq reads covered 268.84 Mbps (~36.7 % of the 732.5 Mbps genome assembly). The
203 covered regions are more than 2.5 times the original gene annotation from the U. ruziziensis
204 genome (43,152 genes comprising 102 Mbps). The median read coverage was 25 reads in
205 the covered regions, and the average read coverage in these regions was 2587 ± 54293
206 reads. After SNP calling and filtering, the average SNP density in the genome was 7.3
207 SNPs/Kbp, or 17.9 SNPs/Kbp if only considering the read covered part of the genome. Using
208 the 43,152 genes and 202,681 exons annotated in the genome reference, the median was 69
209 and 13 SNPs in each gene and exon (average was 95 and 36 per gene and exon,
210 respectively). 34,981 of the annotated genes had at least one SNP.
211
212 Admixture analysis
213 We employed genetic admixture analysis for defining subpopulations. To assign 111 Urochloa
214 accessions to subpopulations, the admixture (Fig. 3) and principal component (Fig. 4) analysis
215 were considered together. The “admixture model” assumes that each individual has ancestry
216 from one or more of “K” genetically distinct sources. An estimation of four subpopulations
217 (K = 4) was selected based on the CV error (Suppl. Fig. 1A) and population structure (Fig. 4).
218 A minimum threshold of 50% genetic composition was used to assign accessions to groups.
219 This allowed us to place the accessions in four groups (Fig. 3): U. humidicola (28 accessions),
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220 U. maxima (13 accessions), “agamic group 1” (54 accessions from the three remaining
221 species) and a closely related “agamic group 2” (that corresponded with the “brizantha-1”
222 subpopulation). Three samples obtained from USDA and identified simply as “Urochloa sp.”
223 showed an admixture of these four groups and were annotated as “admixed”. Accession
224 26438 (sample 86) was received as U. humidicola. Since it clustered with the U. maxima
225 accessions, we reassigned it into that species. When we reduced the number of groups (K =
226 3), the U. humidicola and U. maxima species clustered together, but the agamic groups 1 and
227 2 were consistent (Suppl. Figure 2). When we increased the number of groups (K = 5), a new
228 group split from the “agamic group 1” (that corresponded with the “brizantha-2”
229 subpopulation). The twenty-eight accessions in the U. humidicola group had a basic
230 chromosome number of 9 and high ploidy levels ranging from 6 to 9. The twelve accessions
231 in the U. maxima group had a basic chromosome number of 8 and are tetraploid. The 67
232 accessions in the agamic groups had a basic chromosome number of 9 and ploidy levels
233 ranging from 2 to 6 (Tomaszewska et al., 2021b).
234
235 The admixture analysis was subsequently carried out using only the 67 accessions in the
236 agamic group (Fig. 3B). An estimation of six groups (K = 6) was selected based on the CV
237 error (Suppl. Fig. 1B) and population structure (Fig. 4). A minimum threshold of 70 % shared
238 genetic composition was used to assign accessions to each of the six groups. As shown in
239 fig. 3B, the group “ruziziensis” was composed of all eleven U. ruziziensis accessions. It
240 included one accession wrongly classified as U. decumbens. Within it, five samples showed
241 shared ancestry (1-25%) with diploid U. decumbens. All seven diploid U. decumbens
242 accessions composed the group “decumbens-P2” and were pure accessions with no shared
243 ancestry with any other group. Similarly, ten tetraploid U. decumbens formed the group
244 “decumbens-P4” with pure accessions with no shared ancestry with any other group.
245 However, another six tetraploid U. decumbens composed a different group together with five
246 U. brizantha accessions, which was called “decumbens/brizantha”. This group of eleven
247 accessions was the only one composed by more than one species. Despite this mix, these
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248 accessions showed clear shared ancestry among them and no shared ancestry with any other
249 group (except two samples with minor components). Finally, the group “brizantha-1” and
250 “brizantha-2” were formed by eight and thirteen U. brizantha accessions, respectively. The
251 group “brizantha-2” has pure accessions with no shared ancestry with other groups (with one
252 minor exception under 5%); while most samples in “brizantha-1” have shared ancestry with
253 “decumbens-P4”. The group “brizantha-1” corresponds to the previous “agamic group 2”. The
254 “brizantha-2” subpopulation was only observed in Ethiopia, while “brizantha-1” was observed
255 in a broad range of latitudes. When we reduced the number of groups (K = 5), the “brizantha-
256 decumbens” merged with the “decumbens-P4”. When we increased the number of groups (K
257 = 7), five “brizantha-1” split into an independent subpopulation (Suppl. Fig. 3).
258
259 The admixture analysis was finally completed using only the twenty-eight U. humidicola
260 accessions (Fig. 3C). An estimation of two groups (K = 2) was selected based on the CV error
261 (Suppl. Fig. 1C) and population structure (Fig. 4). A minimum threshold of 70 % shared genetic
262 composition was used to assign accessions to a group. The twenty-eight samples into the two
263 groups: 23 accessions into “humidicola-1” and four accessions into “humidicola-2”. Accession
264 16878 was an equal mixed from both U. humidicola groups and annotated as “humidicola-
265 admixed”. When we increased the number of groups (K = 3 and K = 4), we obtained a small
266 subpopulation with the accessions with higher admixture (16878 and 26155) and an artificial
267 split with some “humidicola-1” accessions in an additional group (Suppl. Fig. 4).
268
269 A smaller number of thirteen U. maxima accessions showed little genetic diversity compared
270 to the other species. Because of the low diversity, we assigned all the U. maxima to a single
271 subpopulation, named “maxima”.
272
273 Population structure by principal component analysis
274 A principal component analysis (PCA) showed the relationship between the 111 accessions,
275 species and admixture groups (Fig. 4A and 4C). The PCA was also done for the 67 accessions
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276 in the agamic group alone (Fig. 4B and 4D). The PCA analysis allowed us to define 12 clusters
277 in total, which easily corresponded with the 10 subpopulations and two admixed groups. The
278 distribution of accessions into subpopulations according to the species and ploidy annotations
279 is represented in figure 5.
280
281 All subpopulations contained accessions from a single species, except subpopulation
282 “decumbens/brizantha”. Remarkably, this subpopulation contained samples that showed
283 greater similarity to each other -in spite of species- than to accessions from the same species
284 in different subpopulations. The two diploid subpopulations, “decumbens-P2” and “ruziziensis”
285 clustered together and apart from polyploid subpopulations. Subpopulation “brizantha-1” was
286 more distant to other agamic subpopulations than “brizantha-2” despite accessions in
287 “brizantha-1” showed shared admixture with tetraploid U. decumbens, while accessions in
288 “brizantha-2” did not.
289
290 Two groups of accessions contained hybrids, one containing hybrids between the distant
291 Urochloa species (“admixed” subpopulation) and another contained hybrids within the three
292 species in the agamic group (“agamic-admixed”), which readily interbreed in control
293 conditions.
294
295 DISCUSSION
296 We clarified the relationship between the gene pools from five Urochloa spp. that are used in
297 the development of commercial forage cultivars. By using RNA-seq, we leveraged in an
298 unprecedented number of markers, over 1.1M SNPs, that virtually encompassed the complete
299 transcriptome from the accessions based on the total genome length covered by the reads (~
300 269 Mbp or 37 % of the genome). We obtained a median of 69 and 13 SNP sites per gene
301 and exon, respectively, which makes this dataset a valuable resource for breeders and
302 researchers (e.g. to design screening markers). The greater length compared to the annotated
303 gene models (Worthington et al., 2021) was probably because the later only used
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304 transcriptomic data from U. ruziziensis. However compared with our dataset, that
305 transcriptomic data was from multiple tissues and included stress conditions. The genus
306 Urochloa includes species previously classified under other taxonomic groups. We have opted
307 to annotate all as Urochloa supported by recent work (Tomaszewska et al., 2021b). E.g., we
308 did observed the same distance between U. maxima and the agamic group than between U.
309 humidicola and the agamic group.
310
311 We observed two subpopulations of U. brizantha, two subpopulations of U. decumbens, and
312 one subpopulation with accessions from both U. brizantha and U. decumbens. The two U.
313 decumbens subpopulations were divided by ploidy. Diploid U. decumbens clustered with U.
314 ruziziensis, while tetraploid U. decumbens clustered with U. brizantha. This split in two U.
315 decumbens subpopulations by ploidy was previously reported using microsatellites (Triviño et
316 al., 2017). In previous studies, the relation of U. decumbens with the other two species has
317 been discussed, as it was alternatively found closely related to U. ruziensis (Ferreira et al.,
318 2016) or U. brizantha (Ambiel et al., 2008). In fact, both observations were correct depending
319 on the ploidy of the accessions under consideration.
320
321 Based on our results, U. brizantha diversity is complex and divided in several gene pools. A
322 group of eleven U. brizantha accessions was different enough to the rest of the agamic group
323 to form an independent cluster (“agamic group 2”; Fig. 3A and 4A). This group of eleven
324 accessions later formed the subpopulation “brizantha-1”. Despite of “brizantha-1” being
325 distant, we observed admixture between “brizantha-1” and “decumbens-P4”, “brizantha-2”
326 and “decumbens/brizantha” subpopulations (Fig. 3B). Among the possible evolutive scenarios
327 that would explain the multiple shared ancestry in “brizantha-1” despite being more distant,
328 previous studies have proposed a single polyploidization event taking place to establish both
329 the tetraploid U. brizantha and U. decumbens (Pessoa-Filho et al., 2017, Tomaszewska et al.,
330 2021b). The “brizantha-1” subpopulation was observed in a broad range of latitudes (e.g. in
331 Ethiopia and Zimbabwe), while “brizantha-2” was only observed in Ethiopia.
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332
333 We obtained a subpopulation, named “decumbens/brizantha”, that included an almost equal
334 number of U. decumbens and U. brizantha accessions. This is the only subpopulation with
335 more than one species, and most accessions did not show shared ancestry with the other
336 subpopulations from either of these species (Fig. 3B). Furthermore, the PCA also showed
337 “decumbens/brizantha” clustered independently to other groups (in the top right corner of Fig.
338 4D). Remarkably, the “decumbens/brizantha” merged with the “decumbens-P4” when we did
339 the admixture analysis with less subpopulations (K = 5). At the same time, two accessions
340 (16173 and PI226049) shared ancestry with “brizantha-2” and were situated between the
341 subpopulations “decumbens/brizantha” and “brizantha-2” in the PCA (Fig. 4D). Consequently,
342 we concluded “decumbens/brizantha” cannot be merged with either “decumbens-P4” or
343 “brizantha-2”, but on the contrary, evidence supported it is an independent subpopulation.
344
345 Vigna et al. (2011b) observed three clusters in U. brizantha after evaluating 172 accessions
346 from EMBRAPA’s collection (so resulting from the same field work in 1980s than our dataset)
347 using 20 SSR markers. Eleven accessions are common between both studies, and our
348 subpopulations “brizantha-1” and “brizantha-2” corresponded with clusters II and I,
349 respectively (Vigna et al., 2011b). Notably, their cluster III appears to include additional
350 “brizantha-1” and “brizantha-2” accessions (16122, 16480), so does not correspond with our
351 “decumbens-brizantha” subpopulation. Triviño et al. (2017) did not discussed a division among
352 U. brizantha accessions, but included a tree resulting from UPGMA clustering based on 39
353 microsatellites that would also support at least two gene pools in U. brizantha.
354
355 In the centre of the agamic group, we identified the “agamic-admixed” accessions (Fig. 4D).
356 This cluster of accessions included hybrid accessions resulting from interspecific species
357 within the agamic group, and should not be confused with the “admixed” accessions (Fig. 4C),
358 which resulted from crosses between more distant Urochloa species. Our analysis supports
359 that the “agamic-admixed” are either progeny from crosses between U. decumbens and either
13 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
360 U. decumbens or U. brizantha (16505, PI210724, PI292187); or between U. ruziziensis and
361 either U. decumbens or U. brizantha (26175, 16494, 26110, 1752).
362
363 U. maxima is also known as Panicum maximum or Megathyrsus maximus. All U. maxima
364 accessions (including accession 26438, which was incorrectly annotated as U. humidicola)
365 showed very limited diversity (Fig. 4) and were assigned to a single subpopulation (“maxima”).
366 This could reflect lower diversity in the species, or be a consequence of original collection and
367 sampling strategy, but it suggests there would be limited gains from including multiple
368 accessions from our study in breeding programmes.
369
370 We observed two different subpopulations in U. humidicola, named “humidicola-1” and
371 “humidicola-2”, plus a single accession (16878) that was an equal mix from both
372 subpopulations. In the initial admixture analysis with all the 111 accessions (Fig. 3A), the
373 “humidicola-2” and “humidicola-admixed” accessions had shared ancestry to the “agamic
374 group 1”, while the “humidicola-1” did not. Triviño et al. (2017) also observed a large group of
375 U. humidicola accessions including all but three of their accessions. These three distant U.
376 humidicola accessions were 675, 679 and 26146. Accession 679 is a “humidicola-2”
377 subpopulation in our study. Remarkably, 26146 is the sexual U. humidicola accession that
378 allowed the establishment of breeding programmes in mid-2000s, and combining the results
379 from both studies supports that the sexual 26146 accession is likely a “humidicola-2”
380 accession. We only had collection information for one “humidicola-2” accession, but it was
381 close to a “humidicola-1” accession, so it is not likely a geographical division. Vigna et al.
382 (2011a) analysed 26 U. humidicola accessions and used UPGMA clustering based on 38
383 microsatellites to divide U. humidicola in two branches in the resulting tree. All seven common
384 accessions with our study were “humidicola-1” and appeared in the top branch of the tree. The
385 bottom branch may be “humidicola-2”, since it included the sexual accession 26146, one
386 accession (26149) not sequenced in our dataset, and the progeny from their crossing. On the
387 other hand, two studies from the same group (Jungmann et al., 2010, Vigna et al., 2011a)
14 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
388 identified five clusters in U. humidicola accessions using ~ 50 SSR markers. While multiple
389 accessions were common to both studies, we did not find correlation between our results and
390 the five clusters; i.e. “humidicola-1” accessions were evenly divided among the multiple
391 clusters. In a similar conclusion, when we increased the number of tentative U. humidicola
392 subpopulations (K = 3 and K = 4), we found the additional groups to be artificial splits from
393 “humidicola-1” and not supported by the PCA.
394
395 CONCLUSION
396 We clarified the relationship between the gene pools from five Urochloa spp. that are used in
397 the development of commercial forage cultivars in different countries. We identified ten
398 subpopulations in total, which had no relation with geographical collection, and represent ten
399 independent gene pools (excluding the two admixed subpopulations). Our results support the
400 division in U. decumbens by ploidy, with a diploid subpopulation closely related to U.
401 ruziziensis, and a tetraploid subpopulation closely related to U. brizantha. We observed clearly
402 differentiated gene pools in U. brizantha, which were not related with origin or ploidy. One of
403 these gene pools, named “brizantha-1”, clustered relatively distant to the rest of the agamic
404 accessions despite having significant shared ancestry with tetraploid U. decumbens. Among
405 the possible evolutive scenarios to explain this observation, it would support a single
406 polyploidization event taking place to establish both the tetraploid U. brizantha and U.
407 decumbens. The “brizantha-1” gene pool should be further explored for prospective founders
408 in the agamic group since U. brizantha is particularly tolerant to neotropic insects, which is
409 one of the main traits under selection. We also identified a well-supported subpopulation
410 containing both polyploid U. decumbens and U. brizantha accessions that likely constitutes a
411 third independent gene pool for both species. We observed two gene pools in U. humidicola.
412 One subpopulation, “humidicola-2”, was significantly smaller but likely includes the only known
413 sexual accession. We also observed one case of natural hybridization between both U.
414 humidicola groups. Our results offer a definitive picture of the available diversity and resolve
415 questions raised by previous studies. They provide an insight into the diversity available for
15 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
416 improvement through crossing, and a platform to identify target genes for forage grass
417 improvement, also providing gene sequences to allow for genome editing (CRISPR/Cas9)
418 approaches. Furthermore, as performance data become available, the data could be further
419 leveraged for GWAS (Genome Wide Association Studies), genotyping array construction, and
420 development of genetic markers for selection in breeding programmes.
421
422 AUTHOR CONTRIBUTIONS
423 JJDV, RACM, JT, TS and JSHH conceived and managed the project. JH and JJDV completed
424 the bioinformatics analysis. PT analysed and provided ploidy level and accessions
425 information. TKP, VC and JA selected, validated and collected the samples. TKP carried out
426 RNA extraction. JH and JJDV wrote the manuscript with contributions from all the authors.
427
428 ACKNOWLEDGEMENTS
429 This work was supported under the RCUK-CIAT Newton-Caldas Initiative “Exploiting
430 biodiversity in Brachiaria and Panicum tropical forage grasses using genetics to improve
431 livelihoods and sustainability”, with funding from UK’s Official Development Assistance
432 Newton Fund awarded by UK Biotechnology and Biological Sciences Research Council
433 (BB/R022828/1). Additional funding for this study was received from the CGIAR Research
434 Programmes on Livestock; and Climate Change, Agriculture and Food Security (CCAFS). JH
435 and JJDV received additional funding from the Biotechnology and Biology Sciences Research
436 Council (BBSRC)’s Global Challenge Research Fund (Project BB/P028098/1) and core
437 strategic funding (Project BBS/E/T/000PR9818).
438 We are grateful to CIAT’s Genebank and USDA’s Germplasm Resources Information Network
439 (GRIN) for their generous provision of germplasm. Germplasm held in the CIAT and USDA
440 collections is available on request.
441
442 DATA AVAILABILITY
443 All the raw reads were deposited in SRA under Bioproject PRJNA513453.
16 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
444
445 CONFLICT OF INTEREST
446 The authors declare no conflict of interest.
447
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556
557
21 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
558 Table 1: Summary of the species (rows) and ploidy level (columns) of the 111 accessions
559 used in this study. Details about each accession were included in Suppl. Table 1.
2n 4n 5n 6n 7n 8n/9n ? Total
U. ruziziensis 10 1 11
U. decumbens 8 17 1 26
U. brizantha 17 9 2 1 29
U. humidicola 10 16 2 28
U. maxima 12 1* 13
U. spp. 2 1 1 4**
560 *Accession 26438 was received as U. humidicola but it was reassigned to U. maxima based
561 on the results. **Accession 1752 (U. spp.) clustered within the agamic group.
562
22 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
563 Figure 1. Geographical origin of 92 accessions with collection coordinates (74 accessions) or
564 country of origin (18 accessions). Accessions were coloured by subpopulation. (A) Origin in
565 Sub-Sahara Africa. (B) Zoom into East Africa. (C) Zoom into the Great Lakes region.
A B
C
ag-admixed brizantha-1 brizantha-2 decumbens-P2 decumbens-P4 decumbens/brizantha humidicola-1 humidicola-2 humidicola-admix maxima ruziziensis 566
23 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
567 Figure 2. Percentage of reads aligning in either uniquely or in multiple positions in the genome.
568 The 111 accessions were coloured by species (A) or ploidy (B).
A B
U. decumbens U. brizantha U. humidicola U. maxima U. ruziziensis U. spp.
569
24 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.21.453213; this version posted July 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
570 Figure 3. Admixture analysis of the genetic ancestry inferred in the complete set of 111
571 accessions (A), the subset of 67 accessions in the agamic group (B), and the subset of 28 U.
572 humidicola accessions (C). Ploidy level is included at the foot of each column. Each accession
573 is represented by a stack column partitioned by proportion of ancestral genetic component,
574 where each identified ancestral genetic component is represented with a different colour.
575 Accessions with a single colour are “pure”. A minimum threshold of 50 % (A) or 70 % (B and
576 C) genetic composition was used to assign accessions to groups. In panel C, “x” for
577 “humidicola-admix”.
A agamic group 1 agamic gr. 2 U. humidicola U. maxima Ad. 1
0.75
0.5
0.25
0 6944 16060 16061 26438 26944 6906 PI284156 6890 16017 26723 6171 16031 16004 PI657653 PI508570 PI508571 67 accessions (Panel B) 28 accessions (Panel C) 13 acc. 3 a. B ruziziensis decum.-P2 decumbens-P4 decumb./brizan. brizantha-2 brizantha-1 agamic-adm. 1.001
0.750.75
variable
ag67_ruzi ag67_decP2 0.500.5 ag67_decP4 ag67_dec_bri Genotype ag67_bri2 ag67_bri1
0.250.25
2 2 2 2 2 2 2 2 2 2 2 2 2 ? 2 2 2 2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 6 5 5 4 5 5 5 4 6 4 4 4 4 ? 4 4 4 4 4 4 5 5 5 4 4 ? 2 5 ? 0.000 4 6 7 9 5 8 2 10 31 18 23 28 29 33 35 11 19 22 34 27 30 32 26 16 20 21 25 48 55 15 24 44 36 40 41 43 45 47 42 46 54 37 51 57 59 52 39 38 56 49 50 58 105 104 106 108 109 113 114 111 112 101 110 127
PI226049 PI210724PI292187
606 665 664 Taxa 6419 6132 6702 6370 6298 6693 1752 26295 26170 26163 16102 26162 26348 26174 26171 26172 26308 26301 26306 26300 26305 26185 26569 16503 16501 16498 26182 16497 16490 26183 16472 26032 26745 26112 16173 16122 16149 16152 16170 16290 16296 16348 16153 16292 16133 16842 16125 16794 26109 26115 16809 16147 26117 16141 26106 16480 16505 26175 16494 26110 6133A C humidicola-1 x. hum.-2 PI226049 PI210724 PI292187 1.001
0.750.75
variable 0.50.50 Urochloa.humidicola1 Urochloa.humidicola2 Genotype
0.250.25
7 6 6 6 7 7 6 7 8 7 7 7 6 7 7 9 7 6 7 7 6 6 6 7 7 6 7 7 0.000
62 66 68 71 72 74 76 79 80 84 85 88 89 90 64 65 63 61 75 69 70 77 81 82 83 119 117 118
Taxa 682 679 6133 6709 6013 578 26407 16350 26145 26151 26159 26375 26413 36083 16867 16180 16882 26430 16887 26573 16871 16880 16894 16876 26425 26154 26155 16878 26570
579
25 580 Figure 4. Population structure by Principal Component Analysis (PCA) using the top two
581 components to separate the complete set of 111 accessions (A and C) or the components 1
200 582 and 3 to separate the subset of 67 accessions in the agamic group (B and D). Accessions
583 were coloured by species (A and B) or subpopulation (C and D).
A Species of 111 accessions B Species of 67 accessions (agamic group) 100 species_finalSpecies
U. maxima U. brizantha U. brizantha U. decumbens U. humidicola
PC2 U. decumbens 4n U. maxima agamic U. ruziziensis group 0 U. spp. hybrids U. brizantha U. brizantha PC3 (3.2%) PC3 PC2 (15.9%) PC2 U. decumbens U. humidicola U. ruziziensis U. decumbens 2n
U. ruziziensis −100
200 PC1 (37.3%) PC1 (46.9%) −200 −100 Subpopulations0 of 111 accessions100 Subpopulations of 67 accessions (agamic group) C PC1 D maxima Subpopulationssubpopulation_final brizantha-1 admixed decumbens/brizantha ag−admixed 100 brizantha−1 decumbens-P4 brizantha−2 decumbens−P2 brizantha-2 decumbens−P4 agamic-admixed PC2 admixed decumbens/brizantha humidicola−1 brizantha-2 humidicola-2
PC2 (15.9%) PC2 humidicola−2 0 brizantha-1 humidicola−admix humidicola-1 ruziziensis maxima decumbens-P2 ruziziensis humidicola-admix unknown ruziziensis
PC1 (37.3%) PC1 (46.9%) 584 −100 585 −200 −100 0 100 PC1
26 586 Figure 5: Distribution of accessions in subpopulations according to the species and ploidy
587 annotations. The number on each stream represents the number of accessions in that division.
588 Streams without a number represent a single accession. .
ruzi 10 ruziziensis U. 1 11 decumbens-P2 7 2n 6 10 decumbens-P4
decumbens 10
U. 6 decum./brizanth. 2 5 10 brizantha-1 10 4n 13 2 brizantha brizantha-2 2
U. 8 13 3 agamic-adm. 2 5 5n 13 maxima 2 2 U. maxima U. spp. 3 admixed 6n 9
humidicola-1 23 11 7n
humidicola 3 8n U. 4 9n humidicola-2 2 1 humi.-admix ? 589
590
27 591 Supplementary Table 1. Sample number, accession number, species, ploidy, subpopulation,
592 architecture, collection location, and PCA position for each of the 111 accessions used in this
593 study.
594
595 Supplementary figure 1: Cross-validation (CV) error and chosen value for number of groups
596 (K) for the complete dataset of 111 accessions (A), the subset of 67 accessions in the agamic
597 group (B), and the subset of 28 U. humidicola accessions (C). Cross-validation error is shown
598 on the Y-axis (vertical) and the number of hypothetical populations on the X-axis (horizontal).
599
600 Supplementary figure 2: Admixture analysis for alternative values for number of groups (K =
601 3, 4 -selected-, and 5) in the complete set of 111 accessions. Numbered by “sample id”.
602
603 Supplementary figure 3: Admixture analysis for alternative values for number of groups (K =
604 5, 6 -selected-, and 7) in the subset of 67 accessions in the agamic group. Numbered by
605 “sample id”.
606
607 Supplementary figure 4: Admixture analysis for alternative values for number of groups (K =
608 2 -selected, 3 and 4) in the subset of 28 U. humidicola accessions. Numbered by “sample id”.
28