Detection of high levels of polymorphism among dent and popcorn inbred lines using Inter-Simple Sequence Repeat (ISSR) amplification technique
--R.V. Kantety, X. Zeng, J.L. Bennetzen, B.E. Zehr

Nineteen elite popcorn inbred lines and eight dent corn inbred lines were used in a study to investigate the degree of polymorphism resulting from a novel DNA marker technique, Inter-Simple Sequence Repeat (ISSR) amplification (Zietkiewicz et al., Genomics 20:176-183, 1994). ISSR involves the use of designed primers to anchor a subset of tandem di-, tri-, or tetra-nucleotide repeats in the genome, resulting in amplification of the DNA sequence between two repeat units. This technique combines the advantages of RAPDs (Random Amplified Polymorphic DNA) (Welsh and McClelland, Nucleic Acids Res. 18:7213-7218, 1990; Williams et al., Nucleic Acids Res. 18:6531-6535, 1990), i.e. not requiring prior knowledge of specific genomic sequences, with that of SINE-morphs (Short Interspersed repetitive Elements) (Kaukinen and Varvio, Nucleic Acids Res. 20:2955-2958, 1992), i.e. the ability to target multiple genomic loci in a single polymerase chain reaction (PCR).

Each PCR contained a total reaction volume of 20 µL; with 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% Gelatin, 0.01% Triton-X-100, 125 µM dNTPs, 1.25 µM primer DNA, 1 µCi 32P dCTP (3000 Ci/mmol; 10 mCi/ml), 2.5 U Taq DNA polymerase, 0.1 mM cresol red dye, and 25 ng template DNA. PCR conditions were ordered as follows: first step denaturation at 94C for 90 s; then 35 cycles of i) 30 s at 94C (denaturation), ii) 45 s at 45C (annealing), iii) 90 s at 72C (extension); and a final extension for 5 min. at 72C. Six-percent polyacrylamide gels (acrylamide:N,N'-methylenebisacrylamide, 30:1) were used to separate PCR products under non-denaturing conditions, with 3 M urea, in 1X TBE buffer, cast in 0.04 x 38 x 50 cm dimension, and electrophoresed at 1000 V for 2 to 3 h. Gels were dried at 80C under vacuum and exposed to X-ray film for 8 to 12 h at room temperature.

Ten primers were designed based on di- [(CA)n, (GT)n] and tri- [(AGC)n, (GCT)n] nucleotide tandem repeats (Table 1). Primers with the same tandem repeat were differentiated by one or two bases at their 3' end, in order to amplify sequences between tandem repeats and to achieve a degree of specificity for these unique regions. Primers were obtained from both Integrated DNA Technologies Inc. and the Purdue DNA synthesis laboratory.

For each inbred line, two DNA samples derived from separate extractions were used in each PCR run (see Figure 1). Only bands which were consistent across separate DNA extractions were considered as reproducible, and thus scored. Repeated PCR runs involving separate DNA extractions indicated that > 98% of ISSR bands were reproducible. A total of 455 scoreable bands were identified among all primers, with fragment sizes ranging from 100 to 3000 bp. The number of bands scored per primer ranged from 10 to 63, giving an average of ~ 45 markers (band positive or null)/primer/inbred line (Table 1). An average of 95% polymorphism was detected among all pop- and dent corn lines analyzed. Approximately 80% polymorphism was detected among 19 popcorn lines, compared to 50% polymorphism using over 100 probes with three restriction enzymes each in RFLP analysis (data not shown). Approximately 95% polymorphism was detected among the eight dent corn inbred lines used in this study (data not shown).

Table 1. Summary of the primer structure and polymorphism obtained using Inter-Simple Sequence Repeat (ISSR) amplification.*
 

Primer Sequence	Total Number Bands	Number Bands Polymorphic	Percentage Polymorphism
(CA)6 R	58	57	98
(CA)6RY	42	40	95
(CA)6RG	47	46	98
(GT)6YR	63	59	94
(GT)6AY	10	9	90
(GCT)4Y	47	45	96
(AGC)4Y	63	58	92
(AGC)4GR	37	36	97
(AGC)4GY	46	44	96
(AGC)4AY	42	40	95
Average	45	43	95

*R = purine and Y = pyrimidine in primer sequence notation. Percentage polymorphism indicates at least
one difference in band expression across the combined set of pop- and dentcorn inbred lines.

In conclusion, ISSR was successful in generating a high degree of polymorphic markers, and appeared to be more effective in this regard when compared to RFLP analysis using popcorn germplasm. ISSR provided a high number of scoreable and reproducible markers per primer. Also, the ISSR technique was simple to perform and cost-time efficient when compared to RFLP technique. Therefore, ISSR appears to hold promise for application in studies of genetic diversity, DNA marker-assisted breeding and genomic mapping.

Figure 1. Example of ISSR autoradiograph using primer (CA)6 RY. Lanes represent (left to right) nineteen popcorn and eight dent corn inbred lines. For each inbred, separate DNA samples were run in adjacent lanes. Two blank lanes were included after samples from the second dent corn line. Exposure of X-ray film was for 12 h without an intensifying screen.

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