genetic diversity in wild and cultivated peanut_khanal_2008

Genetic Diversity in Wild and Cultivated Peanut

Sameer KhanalB.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

Topical Breakdown

1. Introduction

3. Discovery and characterization of SSRs from tetraploid EST Database4. 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 Importance1.3. Production Constraints1.4. Improvement Gridlocks1.5. Genomics and Molecular Breeding

1.6. Research ObjectivesObjectivesMaterials and MethodsResults and DiscussionConclusion

1.1. Arachis spp.

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

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

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

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

Bertioli1. 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

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

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. stenosperma170 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

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

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

2.1. Objectives

To estimate enrichment for genic DNA sequences using methylation-filtration

To develop and characterize genome survey sequence-derived SSRs (GSS-SSRs)

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

Arachisduranensis

Arachisbatizocoi

Arachishypogaea

Num

ber o

f GSS

sUFMF

9,517 unique methylation filtered (MF) and unfiltered (UF) GSSs (GenBank Acc. No. DX505875-DX517373)

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.

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.

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


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


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


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

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.

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

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

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


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 HexanucleotideSSR 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


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

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


Figure 3.2.1. Arachis germplasm screened for EST-SSR marker amplification and length polymorphisms among 28 tetraploids and 4 diploid peanut accessions


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

394952%

317643%

521%

491%187

3%

DiTriTetraPentaHexa

Figure 3.3.3. Distribution of di- and trinucleotide repeats


GSSs ESTsSeqs. With SSRs (%) 10% 7.3%SSR Freq. 1/4.7 kb 1/5 kbPolymorphic (n, among diploids) 93% 81%Polymorphic (n, among tetraploids) 40% 32%Heterozygosity (H, among diploids) 0.57 0.50Heterozygosity (H, among tetraploids) 0.24 0.11

Table 3.3.1. Comparing ESTs with GSSs

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.


Figure 3.3.4. Inference on number of populations and population structure of tetraploid peanut.


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.


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.

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

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

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


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


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

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..


Figure 4.3.1. Inference on number of populations and population structure of diploid peanut.


Figure 4.3.2. Population structure of diploid peanut accessions.


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


D1D2D3 D4

D5

D6

D7

D9

D10

D11

D12D13 D14

D15D16

D17

D18

D19

D20

D21

D22

D23D24

D25

D26

D27D28

D29

D30 D31

D32

D33

D34

D35

D36 D37

B1

B2

B5

B7

B8B9B10B11

S1

S2S4S5

S7 S8

S9

S10S11

S12

S13

S14S15

S16

Coord. 1

Coo

rd. 2

DURBATSTP

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.


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).

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

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

AcknowledgementGraduate committee members

Institute of Plant Breeding, Genetics and Genomics

Project supported by:

USDA

NRI Competitive Grant

Grant # 2006-35604-17242

genetic diversity in wild and cultivated peanut_khanal_2008

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