Identification of maize inbred lines and validation of genetic relationship among maize inbred lines using RAPD markers
--Zhang, CL; Sun, ZL; Jin, DM; Sun, SM; Guo, BT; Wang, B

The objective of our study was to identify twelve maize inbred lines and validate their genetic relationship. RAPD analysis of these inbred lines' genomic DNAs was used to generate genetic fingerprintings. Two hundred and twenty arbitrary, ten-mer primers were screened to amplify all the genotypes. Thirteen of those were selected for the study because they gave reproducible, polymorphic DNA amplification patterns. The experimental results were as follows:

1. Inbred line identification: Nine primers were used to identify all the inbred lines. Four of them gave unique banding patterns for five inbred lines (Table 1). OPA08 differentiates Wenhuang-31413 (Fig. 1), OPA10 distinguishes 488, OPC06 characterizes 8112. OPH12 is the most useful primer because it can identify Mo17 and H21 simultaneously (Fig. 2).

Table 1. Molecular markers for the identification of five maize inbred lines.
 

Inbred line	Primer	Primer sequence	Difference	M. W. of marker
Wenhuang-31413	OPA08	GTGACGTAGG	absent	1.2kb
488	OPA10	GTGATCGCAG	present	0.8kb
8112	OPC06	GAACGGACTC	present	0.9kb
Mo17	OPH12	ACGCGCATGT	absent	0.7kb, 0.8kb
H21	OPH12	ACGCGCATGT	absent	0.7kb, 1.0kb

Five combinations of 2-3 primers produced unique banding patterns for other inbred lines when data from each primer were analyzed in groups. OPC06/OPH12, OPF05/OPH12 and OPE12/OPH12 differentiate Huangzao-4, 515 and 340, respectively; both OPC06/OPN11 and OPA10/OPC07/OPG12 can distinguish two inbred lines, the former 512-196 and 512, the latter 478 and 107 (Table 2).

Table 2. Combinations of primers and their identified inbred lines.
 

Combination of primers	Inbred line	Combination of primers	Inbred line
OPC06/OPH12	Huangzao-4	OPN11/OPC06	502, 502-196
OPF05/OPH12	515	OPA10/OPC07/OPG12	478, 107
OPE12/OPH12	340

2. Genetic relationship: Twelve primers were selected to validate the potential of RAPDs for detecting genetic variability among maize inbred lines. In order to quantify the level of polymorphism observed, Nei's estimates of similarity were used to generate a similarity matrix. All of the inbred lines fell in the range of 0.65 to 1.00 like the case in tomato and wheat. This indicates that only little polymorphism exists at their DNA level and it may be due to self-pollinating.

A dendrogram was constructed from the similarity index values (Fig. 3). Three clusters can be observed, which are further divided into subgroups. The first cluster consists of Huangzao-4 and four inbred lies whose parent is Huangzao-4; 502-196 and 502 form the first subgroup at 0.98 of similarity since 502-196 is an improved material of 502; Huangzao-4 and Wenhuang-31413 form the second subgroup; H21 forms the third subgroup because its other parent is an American inbred line. The second cluster includes two inbred lines, 478 and 488, which came from an F2 plant. The five remaining inbred lines form a separate cluster for their different origin; Mo17, a well-known American inbred line, forms the first subgroup; 107 and 8112, which also came from the United States, form the second subgroup; 340 and 515 form the third subgroup, 340 is a native inbred line whose parents are Chinese local varieties and 515 has American and Chinese genealogy. This showed that genetic relationship established in the study conforms to the pedigree of the twelve maize inbred lines.

In summary, our study provides information on the molecular basis of the polymorphism detected by RAPD markers in maize. It demonstrates that RAPD assay can identify maize inbred lines and validate genetic relationship among maize inbred lines.

Figure 1. Amplification products generated from genomic DNAs of twelve maize inbred lines using arbitrary primer OPA08. The arrow indicates Wenhuang-31413's unique RAPD marker. Template DNAs in different lanes were as follows: 1) 502-196; 2) Huangzao-4; 3) 478; 4) Mo17; 5) H21; 6) 107; 7) 488; 8) Wenhuang-31413; 9) 340; 10) 515; 11) 8112; 12) 502. M represents DNA molecular weight marker VI.

Figure 2. Amplification products generated from genomic DNAs of twelve maize inbred lines using arbitrary primer OPH12. The pair of arrows on the left indicate Mo17's unique RAPD markers and the pair of arrows on the right indicate H21's unique RAPD markers. Template DNAs in different lanes were as follows: 1) 502-196; 2) Huangzao-4; 3) 478; 4) Mo17; 5) H21; 6) 107; 7) 488; 8) Wenhuang-31413; 9) 340; 10) 515; 11) 8112; 12) 502. M represents DNA molecular weight marker VI.

Figure 3. Dendrogram constructed from the similarity index values for different maize inbred lines with average linkage cluster analysis (UPGMA). 

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