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

2. 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 (F IS, F IT, F ST ), 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 EventExpected effect on diversity LoggingDecrease Land-clearingDecrease FireIncrease – if it increases regeneration GlaciersDecrease – bottlenecks FloodIncrease 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. Y L D C A F E H K PopulationCountySample Size ASullivan18 CMorgan30 DParke29 EJennings30 FPosey29 HHarrison29 KGrant30 LCarroll30 YPulaski29 Materials and Methods 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. LocusnAllele Size# AllelesHeHo AAG 141150-18050.6810.659 WAG 6187134-172150.5620.567 WAG 27131199-245190.8680.863 WAG 32173163-217290.9250.855 WAG 69175165-185160.7140.509 WAG 72168130-180110.5990.542 WAG 7648230-26080.7000.688 WAG 82178140-250400.9670.916 WAG 89149180-230260.9220.893 WAG 9049135-185160.9050.918 WAG 9731150-185130.8960.871  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

10. Population# Unique AllelesFrequency IN_A90.049 IN_C100.051 IN_D90.045 IN_E0N/A IN_F10.029 IN_H30.023 IN_K70.039 IN_L40.039 IN_Y20.029  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_AIN_CIN_DIN_EIN_FIN_HIN_KIN_LIN_Y IN_A--- IN_C0.01--- IN_D0.0340.009--- IN_E0.0860.0680.09--- IN_F0.0660.0580.0750.068--- IN_H0.0710.0590.0760.0780.007--- IN_K0.0560.0520.0560.0660.0120.011--- IN_L0.0640.0570.0630.0770.0080.010.003--- IN_Y0.0670.0620.0710.0790.010.0050.0060.008---  Pairwise F ST 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.