Using Simple Sequence Repeats to Identify the Presence of Ab10 in Maize

Alexandra Volker1, Von Mark Cruz2, Candice Gardner2,3, Carolyn Lawrence2,3,4

1Iowa State University, Ames, IA 50011; 2Dept. of Agronomy, Iowa State University, Ames, IA 50011;

3 USDA-ARS; 4 Dept. of Genetics, Developmental, and Cellular Biology, Iowa State University, Ames, IA 50011

Abstract

Conservation of genetic diversity is important to both modern breeders and Native American tribes. The presence of Abnormal Chromosome 10 (Ab10) potentially threatens the genetic diversity of maize due to preferential segregation of all chromosome knobs and linked loci. To identify the presence of Ab10, SSR markers were developed to diagnose chromosome constitution. Thus far, no SSRs have been found that conclusively distinguish Ab10 from the normal chromosome 10 (N10). Further work to develop molecular markers is required to determine whether Ab10 reduces the genetic diversity of seed stocks.

About The AuthorI am a junior at Iowa State University double majoring in Biology and History with a minor in French. I come from the Eastern band of the Cherokee nation. I am interested in infectious diseases and genetics. Of particular interest to me is Sacred Eagle Corn, which originated with the Cherokee, and the genetic events that may have caused the Cherokee corn to lose its purple colour.

IntroductionThe chromosome variant Abnormal 10 (Ab10) was first described by Albert Longley in 1937.1 Ab10 possess extra heterochromatin near the end of the long arm of the chromosome. Marcus Rhoades continued to study Ab10, and discovered the phenomenon of preferential segregation.2 This refers to the ability of Ab10 to ensure its presence in the basal megaspore, resulting in a non-Mendelian ratio in the progeny (Fig.1). In the presence of Ab10, all chromosomes with knobs are preferentially drawn into the basal megaspore3, a process known as meiotic drive. It is necessary to determine whether Ab10 is threatening genetic diversity so a conservation program can be implemented if needed. The maintenance of maize diversity is crucial to both commercial breeders and Native American tribes. Breeders require diversity to strengthen their maize lines via cross-breeding. Many of the traditional uses of maize by Native Americans mandate using certain types of maize.

In order to preserve maize diversity, there must be a way to diagnose chromosome constitution. The development of simple sequence repeat markers (SSRs) that could distinguish between Ab10 and the normal chromosome (N10) would be an important part of protecting maize diversity. Once a diagnostic method has been developed, maize accessions can be screened for the presence of Ab10. These accessions could also be screened over generations to determine if the percentage of Ab10 is increasing and whether its presence is threatening the diversity of the accessions. If that is the case, conservation efforts may be implemented.

Materials and MethodsSeveral accessions of known genotype were planted in the field. Samples of these plants were taken by using a hole punch on the second leaf of the plant. The maize DNA was then extracted using extraction and dilution solutions from a kit. The DNA was stored in a 4ºC refrigerator.

Thirteen SSR markers were available for testing. Each SSR was run with two samples of each of the ten known accessions, as well as five inbred controls. PCR was performed for each SSR using the REDExtract-N-Amp PCR kit. After PCR, the amplified DNA was run in a 4% agarose gel via electrophoresis.

The bands in a gel correspond to DNA fragments of different length. To diagnose chromosome constitution, a fragment that was unique to either the plants of N10 or Ab10 origin needed to be present. Gel banding patterns were analyzed for unique fragments that corresponded to the genotypes of the maize. If a gel did not exhibit a band pattern based on genotype, that SSR was considered unable to diagnose chromosome constitution.

Conclusions•None of the thirteen SSRs analyzed were able to diagnose chromosome constitution.

•Further development on a larger scale should be done to find the appropriate SSR markers.

Field Work at the Plant Introduction Station

Field work was also undertaken at the North Central Regional Plant Introduction Station to gain an understanding of how and why germplasm is conserved and maintained. During the five weeks I spent at the PI Station, I rotated through various plant crews to learn how the station worked. For the first two weeks I worked with Matt Lively and the maize crew and learned how to propagate corn and prepare seed for germination tests. I worked with Joe-Ann McCoy and the medicinal crew on Hypericum spp., specifically on preparing the plants for pollination. Dave Brenner shared his research on amaranth and possible contamination of crops by weedy amaranths. He also showed me his attempts to breed dwarf ornamental amaranths. Charlie Block and Bill Van Roekel of plant pathology gave me the opportunity to study some plant diseases. We spent most of our time investigating Stewart’s wilt, a bacterial infection of maize. They also taught me how to run an ELISA test for the presence of Stewart’s wilt.

AcknowledgementsI would like to thank the following people and organizations for making this program possible:

•National Science Foundation

•George Washington Carver Internship Program

•The curators and staff of NCR-PIS

•USDA-ARS

References1, 2Birchler, James. “Perspectives.” Genetics July 2003:835-841.

3Dawe, R. Kelly. “Plant neocentromeres: fast, focused, and driven.” Chromosome Research 2004: 655-669.

Fig. 4: The ten accessions and five controls (as shown in Table 1) run with the p-umc1084 SSR primers.

Fig.5: The ten accessions and five controls run with p-umc2351 SSR primers.

Accession Number Genotype Presence of Ab10?

1 homozygous A1 C1 r1 N10

No

2 R1 Ab10-I/r1 N10 Yes

3 R1-nj N10/r1 N10 No

4 R1-st N10/r1 N10 No

5 homozygous C1 sh1 wx1 R1 K9s-1 Ab10-I

Yes

6 homozygous r1 Ab10-I Yes

7 R1 Ab10-II/r1 N10 Yes

8 r1 Ab10-II/r1 N10 Yes

9 homozygous R1 N10 No

10 R1 N10/r1-x1 No

Table 1: Accessions and their genotypes.

Rr Rr

rr rr

R

r

rr

Fig.1: A Punnett square showing typical 50:50 genotypic ratio of the R allele in maize for a testcross.

Fig. 2: Normally segregating maize ear (top) and maize affected by Ab10 (bottom). In the bottom ear, the genetic constitution is r-Ab10/R-N10.

Fig. 3: The ten accessions and five controls (as shown in Table 1) run with p-bnlg1028 (top) and p-umc1045 (bottom) SSR primers.

11 H99 No

12 B73 No

13 Mo17 No

14 Tx303 No

15 Co159 No