Use of SSR Markers to Determine Reciprocal Outcrossing Rates Between Non-Herbicide-Resistant Rice and Red Rice

Outcrossing between rice and red rice has been a concern because of their morphological similarities and oftentimes synchronous flowering. A continuing study on reciprocal outcrossing between conventional rice and red rice was done in 2001. Pairs of rice and red rice with near synchronous flowering were planted in isolated field plots. In 2003, seedlings were produced from the pair ‘Kaybonnet’ and AR19948 black-hulled red rice planted in a greenhouse, and DNA was extracted from leaves of the 14-day-old seedlings. Outcrossing between Kaybonnet rice and AR1994-8 black-hulled red rice was estimated at 0.27% with AR1994-8 as the pollen donor, and about 0.20% with Kaybonnet as the pollen donor. Although these outcrossing rates were calculated from a total of only six hybrid plants, the results generally agree with an earlier report, which suggested that pollen movement from red rice to rice may be higher than from rice to red rice, and that outcrossing occurs even when the height difference between rice and red rice is as great as 60 cm. INTRODUCTION Conditions favorable for growing cultivated rice are also favorable for the growth, development, and reproduction of red rice, due to their morphological similarities (De Datta 1981; Gealy et al., 2002). These conditions create major concerns over hybridization between rice and its weedy relatives (Vaughan et al., 2001). These con1 This is a completed study. AAES Research Series 517 38 cerns have increased in recent years in the US due to the development of herbicide resistant cultivars. Gene flow between rice and red rice can proceed in both directions (Zhang et al., 2003). The objective of the study was to determine the reciprocal outcrossing rates between several adjacent pairs of conventional rice and red rice ecotypes under Arkansas field conditions using four SSR rice markers. MATERIALS AND METHODS Plant Materials Non-herbicide-resistant rice cultivars and red rice ecotypes were drill-seeded in isolated field plots at the University of Arkansas Rice Research and Extension Center on 2 May 2001 using seed sources and procedures similar to those described previously (Estorninos et al., 2003). Based on previous comparisons of numerous rice cultivars and red rice ecotypes under field conditions at Stuttgart, Ark., pairs of rice and red rice with overlapping flowering periods were selected for these experiments (Table D 1). The pairs studied in the present test included Kaybonnet rice and AR1994-8 (awned, black-hulled red rice) and ‘Starbonnet’ rice and AR1994-11D (awned, straw-hulled red rice). Due to time constraints, DNA analyses from only the first pair have been completed for both years. Heading notes were recorded three times per week throughout the reproductive periods of rice and red rice to obtain estimates of flowering dates within each plot. After flowering was completed, all red rice panicles in each plot (2 rows by 3 m) were bagged securely with perforated plastic bags to assure capture of all shattered and non shattered seeds. At maturity, heights of five rice and five red rice plants in each plot were measured to mid-panicle and to the tallest point on the plant. The bagged red rice panicles were harvested, threshed, cleaned, and dried to 7 to 10% moisture at room temperature, and 100-seed weights were determined. Rice panicles (not bagged) were harvested and processed similarly. The experimental design was a randomized complete block with three replications. The data were not analyzed statistically due to the extremely low number of hybrids detected. In 2003, seeds from the pairs of Kaybonnet (2060) and AR1994-8 RR (1460) grown in 2001 were selected and planted in soil in groups of 24 in greenhouse flats. Approximately 1500 and 1010 seedlings of Kaybonnet and AR1994-8 RR emerged, respectively. Leaves from 14-day old seedlings in each group were bulked and genomic DNA was extracted. Genomic DNA Analysis Genomic DNA (20 ng/ L) served as a template for amplification by polymerase chain reaction (PCR) using SSR rice markers. These markers, RM215, RM234, RM251, and RM 253, are known to differentiate between rice and red rice (Gealy et al., 2002). The reactions were carried out in 15-μL volumes and subjected to the following temperature profiles: one cycle at 94oC for 45 s, 35 cycles at 94oC for 45 s, 57oC for 45 s, and at 72oC for 60 s, and one cycle at 72oC for 7 min. DNA fragments were