Microsatellites as Indicators of Genetic Diversity in Natural Populations of Black Walnut (Juglans nigra L.) Across Indiana Erin Victory1, Keith Woeste2, Olin E. Rhodes, Jr.1 1Department of Forestry and Natural Resources, Purdue University, West

1

Microsatellites as Indicators of Genetic
Diversity in Natural Populations of Black Walnut
(Juglans nigra L.) Across Indiana
Erin Victory1, Keith Woeste2, Olin E. Rhodes, Jr.1
1Department of Forestry and Natural
Resources, Purdue University, West Lafayette,
IN 2USDA Forest Service, North Central Research
Station, Hardwood Tree Improvement and
Regeneration Center, Purdue University, West
Lafayette, IN
2
Introduction

• Introduction
• Black walnut is an important species
  ecologically, culturally, and economically
• It has been used throughout history for fuel,
  fencing, dyes, cooking, fine furniture,
  and gunstocks
• Despite its importance, not much is known about
  how it partitions its genetic variation in the
  wild
• It is not known how centuries of harvesting and
  anthropogenic use may have affected
  its genetic diversity
• Purpose
• To try to describe this, a study was conducted
  using 11 microsatellite loci on 9 natural Indiana
  populations to describe black walnut genetic
  diversity and how black walnut partitions its
  genetic variance
• Presented here are preliminary data describing
  genetic structure within and among Indiana
  populations as assessed by F statistics (FIS,
  FIT, FST), along with allele frequencies,
  expected and observed heterozygosities, and
  unique alleles.
• This is part of a larger project to elucidate
  patterns of variation within and among natural
  populations across the Central Hardwood Region.

3
Some events that have had probable influence on
black walnut gene flow and what that effect might
be
Black walnut has two modes of gene flow, pollen
and nut dispersal
Event Expected effect on diversity
Logging Decrease
Land-clearing Decrease
Fire Increase if it increases regeneration
Glaciers Decrease bottlenecks
Flood Increase by increasing gene flow (nut dispersal?)
4
Materials and Methods

• Sampling
• Leaves were collected from 40 trees in each of 9
  natural black walnut populations across Indiana
  (Figure 1)
• Natural populations chosen were at least 40 acres
  in size, and at least a mile from any walnut
  plantation
• Trees sampled were approximately 100 yards apart
  or more

5
Materials and Methods
Population County Sample Size
A Sullivan 18
C Morgan 30
D Parke 29
E Jennings 30
F Posey 29
H Harrison 29
K Grant 30
L Carroll 30
Y Pulaski 29
Figure 1. Sampling locations where black walnut
leaves were collected throughout Indiana.
6
Materials and Methods

• Laboratory
• DNA was isolated from freeze-dried leaf tissue
  using a modified CTAB protocol, and an automated
  nucleic acid extractor (Autogen NA 2000)
• Each Indiana sample was amplified at 11
  polymorphic nuclear microsatellite loci
• PCR products were amplified on an ABI 377
  Automated DNA sequencer (Figure 2)

7
Materials and Methods
Figure 2. Gel image of populations IN-K, IN-L,
and IN-Y at WAG 6, WAG 27, WAG 32, WAG 69.
8
Materials and Methods

• Analysis
• Genotypic data was extracted using Genotyper 2.5
• Genetic Data Analysis (GDA version 1.1) was used
  to perform all statistical analyses

9
Results and Discussion
Locus n Allele Size Alleles He Ho
AAG 1 41 150-180 5 0.681 0.659
WAG 6 187 134-172 15 0.562 0.567
WAG 27 131 199-245 19 0.868 0.863
WAG 32 173 163-217 29 0.925 0.855
WAG 69 175 165-185 16 0.714 0.509
WAG 72 168 130-180 11 0.599 0.542
WAG 76 48 230-260 8 0.700 0.688
WAG 82 178 140-250 40 0.967 0.916
WAG 89 149 180-230 26 0.922 0.893
WAG 90 49 135-185 16 0.905 0.918
WAG 97 31 150-185 13 0.896 0.871

• The number of alleles is high
• for all loci, and ranges from 5
• to 40
• Observed heterozygosities
• were high for all loci, and
• range from 0.509 to 0.918
• The notable deficiency of
• heterozygotes detected using
• WAG 69 suggests either a
• Wahlund effect or the
• presence of null alleles

10
Results and Discussion
Population Unique Alleles Frequency
IN_A 9 0.049
IN_C 10 0.051
IN_D 9 0.045
IN_E 0 N/A
IN_F 1 0.029
IN_H 3 0.023
IN_K 7 0.039
IN_L 4 0.039
IN_Y 2 0.029

• The number of alleles
• unique to a particular
• population ranges from 0
• to 10, and are all at
• relatively low frequency
• It appears that
• populations in southern
• Indiana (IN_E, IN_F, and
• IN_H) have fewer unique
• alleles than populations
• in other regions of the
• state however, data from
• fewer loci were collected
• from these populations

11
Results and Discussion
  IN_A IN_C IN_D IN_E IN_F IN_H IN_K IN_L IN_Y
IN_A ---                
IN_C 0.01 ---            
IN_D 0.034 0.009 ---            
IN_E 0.086 0.068 0.09 ---          
IN_F 0.066 0.058 0.075 0.068 ---        
IN_H 0.071 0.059 0.076 0.078 0.007 ---      
IN_K 0.056 0.052 0.056 0.066 0.012 0.011 ---    
IN_L 0.064 0.057 0.063 0.077 0.008 0.01 0.003 ---
IN_Y 0.067 0.062 0.071 0.079 0.01 0.005 0.006 0.008 ---

• Pairwise FST values range from 0.003 to 0.086
• Populations throughout Indiana are not very
  differentiated from one another
• Again, we see that southern Indiana populations
  show a greater degree of differentiation than
  other
• regions
• We also see a hierarchical effect populations
  within a region are somewhat more similar than
• populations from different regions

12
Future Research

• This research comprises the initial phase of
  a study that will elucidate patterns of genetic
  variation within and among populations on a
  regional scale.
• In total, 44 populations across 10 states across
  the northern portion of black walnut range will
  have 3 to10 natural populations genotyped at 12
  nuclear microsatellite loci.

Figure 3. Distribution map of black walnut. The
encircled portion highlights the 10 states that
comprise a regional study of genetic variation
Illinois, Indiana, Iowa, Kansas, Kentucky,
Missouri, Ohio, Pennsylvania, Tennessee, and West
Virginia.