A Physical Map Encompassing a Major Root QTL and Candidate Gene for Root Penetration Ability on Rice Chromosome 4

Abstract

A major quantitative trait loci (QTL) associated with basal root thickness (BRT) on chromosome 4 was used as the target for physical mapping. Two parental rice lines contrasting in their root characteristics, CT9993-5-10-1-M (CT) and IR62266-42-6-2 (IR), were used in this study. The objectives of this study were to fine map the major BRT QTL on chromosome 4 in rice and to isolate candidate genes related to root penetration index (RPI) trait which located in this region. The BAC library available for screening in this study was constructed from a rice cultivar Nipponbare (japonica) with average insert sizes of 130 to 150 kb and with a 10X genome equivalent. The RFLP markers located near the target BRT QTL on rice chromosome 4 were used for screening BAC filters. All candidate BAC clones identified by colony hybridization were confirmed by Southern hybridization and by searching the database available at the Clemson University Genomics Institute (CUGI) website. After confirming and comparing clones with the CUGI database, 24 BAC clones were identified. Three BAC islands were established at the positions where the markers RG939, RZ905 and S15892 were located. Additional BAC clones selected from the CUGI database were added to the BAC islands in order to assemble the physical map encompassing the BRT QTL region. Results from sequence analysis indicated that the physical distance between RG939 and RZ905 was approximately 1.12 mb. The BAC clones in the BRT QTL region were digested with the restriction enzyme Hind lll and were used for Southern hybridization. The Differential Display (DD) technique was employed in order to isolate genes responsible for root growth in rice. The DD fragments CR17G1, CR19C1, CR23A1, CR39C2, CR39C7, and CR56A1 were used as probes to hybridize with these BAC clones. It was found that the DD fragment CR19C1 hybridized with the BAC clones 87D24 and 17B10, whereas the DD fragment CR23A1 hybridized with the BAC clone 17B10. The BAC clone 87D24 was located at the same position as the BAC clone AL606647sd1, which has a known nucleotide sequence. The clone 17B10, which hybridized with both the DD fragment CR19C1 and CR23A1, was located at the same position as the BAC clone AL606628sd1. In order to predict the functions of genes involved in root growth, the DD fragments CR19C1 and CR23A1 were submitted to the BLAST search against the Arabidopsis database at the TAIR website. It was interesting to find that the DD fragment CR19C1 has some degree of similarity to the locus AT1G72960 on chromosome 1 of the Arabidopsis genome. This locus was similar to RHD3, a putative gene conferring root hair defects in Arabidopsis thaliana. The DD fragment CR23A1 was similar to the locus AT1G76490 on chromosome 1 of Arabidopsis thaliana. This locus was a putative gene coding for 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the enzyme involved in cell division in many species. These findings give us some ideas about the functions of candidate genes involved in root growth mechanisms in rice.