1. Genetic Diversity in Wild and
Cultivated Peanut
Sameer Khanal
B.S. Tribhuvan University, Nepal
Advisors: Dr. Steven J. Knapp
Committee: Dr. Albert K. Culbreath
Dr. E. Charles Brummer
In Partial Fulfillment of the Requirements for the Degree Master of Science in Agronomy

2. Topical Breakdown
1. Introduction
3. Discovery and characterization of
SSRs from tetraploid EST Database
4. SSR Diversity in A- and B-Genome
Diploid Peanut Species
2. Mining A- and B-Genome Diploid
and AB-Genome Tetraploid GSSs for
SSRs
5. Summary
1.2. Economic Importance
1.3. Production Constraints
1.4. Improvement Gridlocks
1.5. Genomics and Molecular
Breeding
1.6. Research Objectives
Objectives
Materials and Methods
Results and Discussion
Conclusion
1.1. Arachis spp.

3. 1.1. Arachis spp.
 Belongs to the family of legumes (Fabaceae)
 Gene pool includes 80 accepted species assembled
into 9 sections1,2
 Cultivated peanut (Arachis hypogaea L.) belongs to
section Arachis together with 32 other wild diploids
and a wild tetraploid species, A. monticola
1. Krapovickas and Gregory 1994 2. Valls and Simson 2005

4. 1.2. Economic Importance
 Cultivated in tropical, sub-tropical, and warm-
temperate regions of more than 100 countries
 Second most important legume after soybean
 Third most important source of vegetable protein
 Fourth most important source of vegetable oil
 Twelfth most important food crop
Source: FOASTAT (http://faostat.fao.org

5. 1.3. Production Constraints
Source: Compiled from plantpathology.tamu.edu
edis.ifas.ufl.edu/IN176
Biotic
Constraints
Diseases
Insect-Pests
Abiotic
Constraints
Drought
High Temp.
Abiotic
Constraints
Drought
High Temp.
Source: environmentalgraffiti.com

6. 1.4. Improvement Constraints
2n Gametes
Or
Spontaneous
Chromosome
Doubling
AA (n=10) BB (n=10)
AABB (n=20)
Parents
Allotetraploid peanut
Source: Figure Modified from Bertioli
1. Kochert et al. 1991 2. Burow et al. 2001 3. Halward et al. 1991 4. Knauft and Gorbet 1989 5. Stalker 1997
Low genetic variation
and breeding
bottlenecks
Reasons:
• Reproductive isolation
of an amphidiploid1
• Monophyletic origin2
• Autogamy3
• Narrow genetic base4
• Rearing susceptible
genotypes under
chemical intensive
systems5

7. 1.4. Improvement Constraints
A. cardinasii (AA) A. diogoi (AA)X
AA
TXAG-6
Parents
INTERSPECIFIC HYBRIDIZATION
F1 hybrid
CHROMOSOME DOUBLING
Tetraploid
X
Florunner
AB
INTERSPECIFIC HYBRIDIZATION
X
A. batizocoi (BB)
BC5F3 COAN1
Development of COAN and NemaTAM
NemaTAM2
BC7F3
 Introgression of traits
of interest from exotic
spp. is desirable
 Large number of
introgression lines
 Very few introgression
cultivars have been
released
Reasons:
1. Complex crosses
2. Resource intensive
3. Linkage drag
4. Lack of molecular tools
1. Simpson and Starr 2. Simpson et al. 2003

8. 1.5. Genomics and Molecular
Breeding
 Lagging in genomic resources to address pertinent problems with
molecular breeding solutions.
Linkage maps
1. Diploid RFLP map produced from an interspecific hybrid1
Mapping population: A. stenosperma x A. cardinasii
117 markers mapped along 11 linkage groups
2. Microsatellite-based map published in Arachis2
Mapping population A. duranensis x A. stenosperma
170 SSRs mapped along 11 linkage groups
3. Tetraploid RFLP map produced from a complex hybrid3
A. hypogaea x ‘synthetic’ amphidiploid [A. batizocoi x A.
diogoi] 370 RFLP loci mapped on 23 linkage
groups
1. Halward et al. 1993 2. Burow et al. 2002 3. Moretzsohn et al. 2005

9. 1.6. Research Objectives
 To access the frequency of polymorphic SSRs in the genic and non-
genic DNA sequences of Arachis
 To access genetic diversity in wild and cultivated peanut
 To develop and report additional DNA markers and genomics
resources

10. 2. Mining A- and B-Genome
Diploid and AB-Genome Tetraploid
GSSs for SSRs

11. 2.1. Objectives
 To estimate enrichment for genic DNA sequences using
methylation-filtration
 To develop and characterize genome survey sequence-derived
SSRs (GSS-SSRs)

12. 2.2. Materials and Methods
Figure 2.2.1. Number of
genome survey
sequences (GSSs) from
methylation-filtered (MF)
and unfiltered (UF)
genomic libraries of
peanut.
0
500
1000
1500
2000
2500
3000
3500
4000
Arachis
duranensis
Arachis
batizocoi
Arachis
hypogaea
NumberofGSSs UF
MF
 9,517 unique methylation filtered (MF) and unfiltered (UF) GSSs
(GenBank Acc. No. DX505875-DX517373)

13. 2.2. Materials and Methods
 Mining tool and criteria: SSRIT1
; minimum no. of repeats (n) = 5
 Primer design tool: Primer32
 PCR amplicons resolved in SSCP gels and haplotypes were scored
1. Temnykh et al. 2001 2. Rozen and Skaletsky 2000
Table 2.2.1. Arachis germplasm screened for SSR marker
amplification and length polymorphisms.

14. 2.3. Results and Discussion
 Methylation filtration
produced enrichment in
the range of 4.4 to 14.6 for
genic DNA
 Gene enrichment
suggested a reduction of:
- 1,240 Mb A. duranensis to
279 Mb gene space
- 951 Mb A. batizocoi to
65.14 Mb gene space
- 2,813 Mb A. hypogaea to
478.40 Mb space
 500,000 to 10,000,000 MF
sequence reads for 1x raw
coverage of Arachis
genome
Table 2.3.1. Example: Calculation of filter
power for A. duranensis.

15. 2.3. Results and Discussion
 Table 2.3.2. Annotation statistics of methylation-filtered (MF) and
unfiltered (UF) GSSs from Arachis.
 MF showed reduced representation of repetitive fraction of the genome
in the sequence database

16. 2.3. Results and Discussion
 A total of 1,168 SSRs were interspersed in 960 GSSs
Figure 2.3.1. Abundance of di-, tri-, and tetranucleotide repeats among
9,517 genomic survey sequences of Arachis.
 Dinucleotide repeats were the
predominant repeat type in the
GSSs

17. 2.3. Results and Discussion
 97 out of 153 SSR markers amplified alleles across species and
accessions and produced high-quality genotypes
 A. duranensis and A. batizocoi contributed 21 markers each and A.
hypogaea contributed 55 markers
 93% of the markers were polymorphic among diploids with an
average heterozygosity (H) of 0.57
 40% of the markers were polymorphic among tetraploids (H = 0.24)
 70-80% of A. hypogaea based markers were transferable to the
diploids

18. 2.3. Results and Discussion
 56.7% of the markers were in genic regions and 43.3% were in non-
genic regions
 Among tetraploids, frequency of polymorphic markers was higher for
the genic (41.8%) than the non-genic ones (38.1%)
 Average heterozygosity (H) among tetraploids was equal for the genic
and the non-genic SSRs
 However, among diploids, H was slightly higher for the non-genic
(0.59) than for the genic (0.55) SSRs

19. 2.3. Results and Discussion
 Dinucleotide repeats were more polymorphic (H=0.62) than
trinucleotide (H=0.55) and tetranucleotide (H=0.51) repeats
 Among tetraploids, SSRs longer than 26 bp were nearly fourfold more
polymorphic (H=0.44) than SSRs shorter than 26 bp (H=0.12)
Figure 2.3.2.
Relationship between
the simple sequence
repeat length (bp) and
heterozygosity for 97
SSR markers among
eight peanut
germplasm
accessions.

20. 2.4. Conclusions
 Methylation filtration effectively enriched for the genic DNA sequences
 GSSs were abundant in SSRs
 93 polymorphic SSRs were developed
 Tetraploids showed narrow allelic variation, while diploids were highly
polymorphic

21. 3. Discovery and Characterization
of SSRs from Tetraploid Peanut
EST Database

22. 3.1. Objectives
 To describe the abundance and characteristics of simple sequence
repeats (SSRs) in peanut expressed sequence tags (ESTs)
 To assess polymorphisms offered by a broad spectrum of SSR repeat
motifs, repeat lengths, and repeat locations
 To decipher population structure of tetraploid peanut

23. 3.2. Materials and Methods
 Source of ESTs: PeanutDB; 71,448 long-read (Sanger) ESTs and
304,215 short-read (454) ESTs assembled into 101,132 unigenes
 SSR Mining Tool: SSRIT1
with minimum no. of repeats = 5
 Representative SSR-EST panel selected based on:
SSR Repeat Motif (M): Di-, Tri-, Tetra-, Penta-, and Hexanucleotide
SSR Length (K) = Motif (M) x Repeat Number (N)
SSR Location: Exon and UTRs
 Primer Design: Primer32
online tool
 Genotypes determined using ABI3730 DNA analyzer and
GeneMapper Software Version 4 (Applied Biosystems, Foster City,
CA)
1. Temnykh et al. 2001 2. Rozen and Skaletsky 2000

24. 3.2. Materials and Methods
 Power Marker V3.251
was used to estimate average heterozygosity (H)
 Microsat2
was used to generate pair wise genetic distance matrix
based on the proportion of shared bands (D = 1 – ps)
 Phylip v3.673
was used for the construction of neighbor-joining tree
and TreeDyn 198.34
was used for editing the tree
 Principle coordinate analysis (PCoA) was performed using Microsoft
excel based software GenAlexEx 6.15
 Software package structure 2.26
was used for deciphering population
structure
1. Liu and Muse 2005 2. Minch et al. 1997 3. Felsenstein et al. 1989 4. Chevenet et al. 5. Peakall and Smoush 2006

25. Panel (32 Genotypes)
Tetraploids (28 AABB Genotypes) Diploids (4 Genotypes)
Runner
Virginia
Valencia
Spanish
Botanical
Varieties
Historical
Germplasm
A. duranensis (A-
Genome)
A. batizocoi (B-Genome)
Exotic
Germplasm
3.2. Materials and Methods
Figure 3.2.1. Arachis germplasm screened for EST-SSR marker
amplification and length polymorphisms among 28 tetraploids and 4
diploid peanut accessions

26. 3.3. Results and Discussion
 A total of 7,413 SSRs were interspersed in 6,371 uniscripts
Figure 3.3.2. Abundance of di-, tri-,
and tetranucleotide repeats ESTs
 Dinucleotide repeats were the
predominant repeat type in the
ESTs
3949
52%
3176
43%
52
1%
49
1%187
3%
Di
Tri
Tetra
Penta
Hexa
Figure 3.3.3. Distribution of di-
and trinucleotide repeats

27. 3.3. Results and Discussion
GSSs ESTs
Seqs. With SSRs (%) 10% 7.3%
SSR Freq. 1/4.7 kb 1/5 kb
Polymorphic (n, among diploids) 93% 81%
Polymorphic (n, among tetraploids) 40% 32%
Heterozygosity (H, among diploids) 0.57 0.50
Heterozygosity (H, among tetraploids) 0.24 0.11
Table 3.3.1. Comparing ESTs with GSSs

28. 3.3. Results and Discussion
 In tetraploids, dinucleotide and trinucleotide SSRs were equally
polymorphic (H=0.14)
 Polymorphisms of SSRs in exons and UTRs were also equal
 Among tetraploids, SSRs longer than 26 bp were nearly three-fold
more polymorphic (H=0.18) than SSRs shorter than 26 bp (H=0.08)
Figure 2.3.2.
Relationship between
the simple sequence
repeat length (bp) and
heterozygosity for 59
SSR markers among
28 tetraploid peanut
accessions.

29. 3.3. Results and Discussion
Figure 3.3.4. Inference on
number of populations and
population structure of
tetraploid peanut.

30. 3.3. Results and Discussion
Figure 3.3.5.
Neighbor-joining tree
produced from
genetic distances
estimated from 59
EST-SSR markers
among 28 tetraploid
peanuts. Runners are
shown in red,
valencia in blue and
Spanish in Green.

31. 3.3. Results and Discussion
Figure 3.3.6.
Principal coordinate
analysis of mean
genetic distance
matrix. The first two
principle coordinates
explained 36.39% and
20.23% of the total
variance.

32. 3.4. Conclusion
 Expressed sequence tags were developed, mined, and characterized
for SSRs
 19 polymorphic markers were developed
 Polymorphic EST-SSRs were shown to be sufficient for developing a
critical mass of DNA markers for genetic mapping and downstream
application

33. 4. SSR Diversity in A- and B-
Genome Diploid Peanut Species

34. 4.1. Objectives
 To estimate SSR diversity in A- and B-genome diploid peanut species
 To decipher population structure of A- and B-genome diploid peanut
species
 To develop a large number of polymorphic SSR markers in A- and B-
genome mapping populations

35. 4.2. Materials and Methods
 32 previously mapped SSR markers from Moretzsohn et al.
(2005) were used for the analysis
 A total of 60 genotypes belonging to A. duranensis, A. batizocoi
and A. stenosperma were used
 Two of the genotypes (designated as BAT3 and DUR38) were
not included in the statistical analysis
 Final panel of 58 genotypes included 36 A. duranensis, 8 A.
batizocoi, and 14 A. stenosperma

36. 4.2. Materials and Methods
 Power Marker V3.25 was used to estimate average heterozygosity (H)
 Microsat2
was used to generate pair wise genetic distance matrix
based on the proportion of shared bands (D = 1 – ps)
 Phylip v3.67 was used for the construction of neighbor-joining tree
 Principle coordinate analysis (PCoA) was performed using Microsoft
excel based software GenAlexEx 6.1
 Software package structure 2.2 was used for deciphering population
structure

37. 4.3. Results and Discussion
 27 out of 32 markers showed amplification
 Almost all the markers amplified single band in all the
accessions
 Average H was 0.72
Table 4.3.1. Polymorphisms of the 27 SSR markers screened among
36 A. duranensis, 8 A. batizocoi, and 14 A. stenosperma accessions..

38. 4.3. Results and Discussion
Figure 4.3.1. Inference on
number of populations and
population structure of
diploid peanut.

39. 4.3. Results and Discussion
Figure 4.3.2. Population structure of diploid peanut accessions.

40. 4.3. Results and Discussion
 For accessions with
location information,
sites were tagged in
google map
 Most of the
accessions shown to
form a separate
population group were
collected at altitudes
above 900 masl
 Most other accessions
(26) were collected at
altitudes below 600
masl
Figure 4.3.3. Addressing sub-Population
structure in A. duranensis accessions

41. 4.3. Results and Discussion
D1
D2D3
D4
D5
D6
D7
D9
D10
D11
D12
D13 D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30 D31
D32
D33
D34
D35
D36
D37
B1
B2
B5
B7
B8
B9
B10B11
S1
S2
S4
S5
S7
S8
S9
S10
S11
S12
S13
S14
S15
S16
Coord. 1
Coord.2
DUR
BAT
STP
Figure 4.3.4.
Principal coordinate
analysis of mean
genetic distance
matrix. The first two
principle coordinates
explained 33.71% and
21.14% of the total
variance.

42. 4.3. Results and Discussion
Figure 4.3.5.
Neighbor-joining tree
produced from the
genetic distances
estimated from 27
SSR markers
screened among 58
diploid Arachis
accessions including
36 A. duranensis
(DUR), 8 A. batizocoi
(BAT), and 14 A.
stenosperma (STP).

43. 4.3. Results and Discussion
 A total of 612 previously reported and 97 GSS-SSRs were screened
among 12 diploids.
Table 4.3.2. Polymorphisms of 556 SSR markers among A. duranensis,
A. batizocoi, A. kuhlmanii, and A. diogoi germplasm accessions

44. 4.4. Conclusion
 We observed large genetic diversity among the diploid accessions
 It is feasible to develop a critical mass of polymorphic SSR markers
for the construction of high-density A- and B-genome intraspecific
maps of Arachis species

45. Acknowledgement
Graduate committee members
Institute of Plant Breeding, Genetics
and Genomics
Project supported by:
USDA
NRI Competitive Grant
Grant # 2006-35604-17242