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Marker-aided selection in hybrid rice breeding

Screening for Rf-3 and tms-2 genes
We have successfully incorporated DNA marker-assisted selection (MAS) in IRRI’s hybrid rice breeding program. Three traits were initially targeted—restoration ability for WA cytoplasm and thermosensitive genetic male sterility.

For WA fertility restoration, the major dominant QTL on chromosome 1 designated as Rf-3 underwent MAS using the sequence-tagged site (STS) marker RG140 with PvuII digestion. Restorers were identified by a 1.25-kb band, whereas nonrestorers possess a 1.15-kb band when resolved in an agarose gel (Fig. 1). Using this method, about 50% of the lines were identified as suspected restorers, which were then testcrossed with CMS lines. This method therefore reduced the number of lines to be testcrossed by 50%. Up to 80% of these testcrossed lines were confirmed as WA-CMS restorers. This demonstrated that MAS could be performed on the parental lines to increase our screening efficiency for restorers.

Fig. 1. PCR amplification products using the STS primers RG140L (F) and RG140B (R) and digestion with PvuII. Restorers such as IR24 and IR36 exhibit a 1.25-kb band, whereas a 1.15-kb band is produced in nonrestorers such as IR58025A.

For thermosensitive genetic male sterility (TGMS), the major recessive gene tms-2 located on chromosome 7 was targeted. Heterozygous plants that are to be used for generation advance were selected from fertile F2, F3, and F4 plants using the simple sequence repeat (SSR) marker RM11 (Fig. 2). This method reduced the number of materials to be handled by about 33% and eliminated the homozygous fertile plants.

Fig. 2. PCR amplification products using the SSR primer RM11. Fertile plants exhibit homozygous (upper band) and heterozygous bands. Heterozygous plants are selected based on the banding pattern for generation advance.
 

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Marker-aided selection in hybrid rice breeding