Next-generation sequencing of African yam bean (Sphenostylis stenocarpa) using Restriction-site associated DNA Sequencing

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Next-generation sequencing of African yam bean
(Sphenostylis stenocarpa) using Restriction-site associated
DNA Sequencing
AINA, Ademola Ibukun 1,2,3, POTTER Daniel 3, ABBERTON Michael 2, ILORI Christopher
Olumuyiwa1, ALLEN Van-Deynze 3, CARRASQUILLA Garcia-Noelia 3, OYATOMI Olaniyi 2
1 Department of Crop Protection and Environmental Biology, Faculty of Agriculture, Ibadan,
Ibadan, Nigeria.
2 Genetic Resources Centre, International Institute of Tropical Agriculture, Ibadan, Nigeria.
3 Department of Plant Sciences, University of California, Davis, USA.
21ST Annual Symposium of IARSAF

INTRODUCTION
• African Yam bean (AYB) is a tuberous legume of tropical
origin
• AYB is the most economically important among the
seven species of Sphenostylis
• It is a neglected and under-utilized species (NUS)
• AYB has immense potential as food and nutrition
security crop in sub-Saharan Africa
2

• 15-29 % high protein content in seeds and tubers
• Higher amino acids content than pigeon pea, cowpea and
Bambara groundnut (Adewale and Dumet, 2010; Uguru
and Madukaife, 2001)
• Seeds and tubers contains more than twice the protein in
sweet or Irish potatoes and ten times more than in
Cassava roots (NRC, 2006)
3

USES
• Food and feed
• Socio-cultural uses
• Biological control
(Potential)
• Fixes soil nitrogen
Plate 1: AYB Plant
4

Production constraints
• Continual loss of genetic diversity
• Long maturity period
• Hard seed coat
• Need for stakes and training of vines
• Human perception
5

• Prior to this study, some molecular markers have been
used in diversity studies for a few selected AYB
accessions (RAPD and AFLP)
• Transferability of cowpea derived SSR markers in AYB
have been reported by Shitta et al., 2015
6

• NGS are high-throughput sequencing technologies
• New methods or approaches to NGS
 Reduced- representation libraries (RRLs) or Complexity
reduction of polymorphic sequences (CRoPs)
 Restriction-site associated DNA sequencing (RADSeq)
 Low coverage genotyping
NEXT- GENERATION SEQUENCING (NGS)
7

• Reduced labor, saves time and cost
• High resolution population genomic data
• Thousands of sequenced markers across many
individuals
• Large depth coverage of sequence
• Can be used for crops without a reference genome
Advantages of RADSeq
8

• Persistent neglect of the crop has resulted in continual
loss of essential AYB germplasm with implications on its
variability
• Research on diversity and yield evaluation of this crop is
germane to its improvement
• Using more recent genomic tools to understand the
extent of genetic variability present in AYB will be
beneficial to breeders
JUSTIFICATION
9

OBJECTIVE
• To our knowledge, RADSeq has not been reported in
AYB therefore the objective of this study is to employ
RADSeq in unraveling and understanding the genetic
diversity in AYB germplasm
10

• Ninety three (93) accessions of AYB collected from the
Genetic Resources Centre, IITA, were used for this study
• Five seeds from each accession was planted in plastic
pots and grown in the vegetable crops field and
greenhouses, UC Davis, USA
• Two weeks old leaf seedlings were collected and bulked
into single samples
• Genomic DNA was extracted from lyophilised leaf
tissues using the Qiagen DNeasy plant mini kit
MATERIALS AND METHODS
11

• 96 multiplex GBS library was prepared using the Double
- Digest GBS protocol (modified version using
streptavidin dynabeads - Michelmore lab)
• GBS library was validated on Bioanalyzer and
sequencing was performed on Illumina Hiseq 4000 at the
DNA Technologies Core of the UC Davis, USA
12

PLATE 2 : BIOANALYZER PLATE 3: ILLUMINA HISEQ 4000
13

• Tassels UNEAK pipeline was used to identify RAD tags
with SNPs
• SNPs called was used for admixture analysis for K1 = 2,
K2 = 3 and K3 = 4
• Distance matrixes was used to construct the phylogenetic
tree using the ape library in R software
• STRUCTURE 2.3.1 was used to infer the population
structure
ANALYSIS
14

Fig 1: Classification of 93 AYB accessions into populations
RESULTS
15

Fig 2: Phylogenetic tree
1
2
3
4
5
16

TABLE 1: Mean Genetic Distances
CLUSTERS MEAN GENETIC DISTANCE NUMBER OF ACCESSIONS
C 1 0.17 13
C 2 0.16 8
C 3 0.21 37
C 4 0.19 31
C 5 0.10 4
17

• Four structured populations were postulated from this
study in the 93 accessions of African yam bean based
on SNP discovery
• Genetic variability was highest in cluster 3, with a
mean genetic distance of 0.21 while cluster 5 with the
least number of accession was the least variable
• TSs 47 in cluster 3 and TSs 4 in cluster 5 were the
most distantly related accessions in this study
DISCUSSION
18

CONCLUSION
• Phenotypic characterization
• Cowpea derived SSR markers
• Conservation and breeding
19

• Prof. Michael Abberton - IITA
Prof. Dan Potter - UC Davis
Prof. Douglas Cook - UC Davis
Dr. C.O. Ilori - UI
Mr. Olaniyi Oyatomi - IITA
ACKNOWLEDGEMENTS
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Next-generation sequencing of African yam bean (Sphenostylis stenocarpa) using Restriction-site associated DNA Sequencing

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Next-generation sequencing of African yam bean (Sphenostylis stenocarpa) using Restriction-site associated DNA Sequencing