Microsatellites as Indicators of Genetic Diversity in Natural Populations of Black Walnut (Juglans nigra L.)

Across Indiana

1Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN2USDA Forest Service, North Central Research Station, Hardwood Tree Improvement and Regeneration Center, Purdue University, West Lafayette, IN

Erin Victory1, Keith Woeste2, Olin E. Rhodes, Jr.1

IntroductionIntroduction

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.

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?)

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

Y

LDCAF

E

H

K

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

Materials and Methods

Figure 1. Sampling locations where black walnut leaves were collected throughout Indiana.

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)

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.

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

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

Results and Discussion

Results and DiscussionPopulation # 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

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

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.