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Linkage Map with SNP Dosage Data in Tetraploid Urochloa decumbens
Rebecca Caroline Ulbricht FerreiraUniversity of Campinas – UNICAMP, Brazil
Pasturelands in Brazil2
• 100 M ha cultivated pastures (247 M ac)
• 209 M cattle heads
• Brazil: main beef and tropical forage seeds exporter in the world
Main species• Urochloa decumbens• U. humidicola• U. ruziziensis• U. brizantha
each one with a specific purpose or niche
Figure. Pasturelands areas in Brazil.
African forage grasses ABIEC, 2018
Evolution of the Pasture Area x Productivity3
Pasture area- M/ha
Productivity Ton/ha/year
ABIEC, 2018
Last 30 years....
PROGRESS OF CLASSIC BREEDING PROGRAMS
CHALLENGES:
• Reduction of losses due to biotic stresses: spittlebugs attacks
• Increase of adaptation, based on forecasted climate changes: heat, drought, and flood tolerance
• Improvement of nutritive value: + beef and milk / kg of pasture
Support of genomic breeding↓ Pasture areas ↑ cattle herds
Current Genomic Tools in Urochloa Genus
Molecular markers: SSR and SNP
Transcriptome (leaf and root)
Genetic mapping : Single-dose markers
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Challenges: Apomixis and Polyploid
Autopolypoids
Allopolypoids
Segmental allopolypoids
Bourke et al., 2018
More genomic information to reach thenext-generation breeding
Current Method for Polyploid Genotyping
o Genetic studies aim to identify SNP markers based on NGSo Generation of accurate genotypic data is crucial
Assignment of marker dosage “Dosage” is a very important concept in genetic analysis of polyploids
Example os dosage classes in autotetraploid species:
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Bourke et al., 2018
Allele dosage estimation: more genetic information
imparted by each SNP locus
aaaa Aaaa AAaa AAAa AAAA
The Aim of the Study6
To build a GBS-based integrated genetic map and identify QTLs
related to spittlebug resistance using autotetraploid allele dosage
information in a bi-parental progeny of U. decumbens
Species of Study
Urochloa decumbens (Signalgrass)
Syn. Brachiaria decumbens
Adaptation to poor and acidic soils
Susceptible to pastures spittlebugs
Segmental allopolyploid
2n= 4x= 36
Genome size ≈ 1,600 Mbp
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No sequencedgenome
ORPHAN SPECIES
(Notozulia entreriana) Urochloa decumbens
Intraspecific Mapping Population8
Sexual(Tetraploidized)
xApomictic
=217 F1 hybrids
Base Population of the U. decumbens breedingprogram of Embrapa Beef Cattle
D24/27 cv. Basilisk
Figure. Mapping population of Urochloa decumbens
GBS Library Construction and Sequencing 9
DNeasy Plant Kit (QUIAGEN)
NextSeq 500Platform
Restriction enzyme NsiI(Elshire et al., 2011)
GBS LibraryDNA Extraction Sequencing
SNP Calling and Genetic Mapping10
Tassel-GBS for polyploid
SNP Calling
Alignment
• Setaria viridis• S. italica• Panicum virgatum• P. hallii• U. decumbens
transcriptome
Allele Dosage
SuperMASSASoftware
Genetic Mapping
TetraploidSNPMapSoftware
Pereira et al., 2018 Hackett et al., 2017
• Interval Mapping• Phenotypic Trait:
resistance to spittlebug(Notozulia entreriana)
QTLUse of the total amount
of reads
~1 billion reads with high quality
Setaria spp. genomes: good assembly
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0
10
20
30
40
50
60
70
Tassel forpolyploid
SuperMassa FiltrationsN
umb
erof
SNP
ma
rker
s(x1
000)
Amount of SNP markers
P. virgatumP. hallii
S. viridisS. italicaTranscriptome
Sequencing and SNP Calling
GeneticMap
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Simplex
DuplexDouble-simplexX-double-simplexDuplex-simplexDouble-duplex
SNP Configuration
QTL to spittlebug resistance
1,000 SNP markers1,335.09 cM
U. decumbens Genotyping13
↑ information imparted by loci0
100
200
300
400
500
600
700
Mapped SNPs Configuration
Aaaax
aaaa
AAaax
aaaa
Aaaax
Aaaa
Aaaax
AAaa
AAaax
AAaa
AAAax
Aaaa
Molecular data fitting atetrasomic segregation model
• Mode of inheritance• Parental tetraploidized
autotetraploid
U. decumbens genetic map
The majority of markers mapped were within 1 cM of each other.
Two largest distances between adjacentmarkers: 11.2 cM and 12.4 cM
• Centromeric regions• Regions without recombination• Distorted portions of genome
Possible method to fill these distances:• Two restriction enzyme• Alignment with Urochloa decumbens genome
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Num
ber
ofm
ark
ers
Distance between adjacente markers (cM)
QTL to Spittlebug Resistance
3 QTL
4.66% to 6.24% of the phenotypic variation
Resistance alleles from male parent
Small number of QTL with small effects U. decumbens presents low genetic variability for spittlebug resistance
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Segregation of different alleles in each parental: polymorphism for the trait Notozulia entreriana
Conclusions
Building of the first genetic map of U. decumbens with SNPs with allele dosage information
Allele dosage estimation: important evolution for polyploid studies
Identification of genomic regions related to spittlebug (N. entreriana) resistance in U. decumbens
Opened new molecular perspectives for forage grasses of Urochloa genus
First step towards genome assembly, MAS and GS
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Related Work....17
Collaborations
EMBRAPA BEEF CATTLE
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UNICAMP/CBMEG/LAGM USP/ESALQ
Aline Moraes
Felipe Martins
Thamiris Deo Lucimara ChiariCacilda do Valle
Sanzio BarriosAntônio Garcia
Letícia Lara
Fernanda de Oliveira
Anete de Souza
José Valério
