Studies On Gene Expression Profiles Between Rice Hyrid And Parents And Molecular Basis Of Heterosis Using CDNA Microarray

Utilization of heterosis has become a major strategy for increasing crop productivityof plants and animals and preserving land source, it has been contributed greatlyworldwide to the production of many crop species including maize and rice. Though aconsiderable amount of efforts has been invested in unraveling the genetic basis ofheterosis in crop plants, the biological mechanisms of heterosis remained largelyuncharacterized. It's necessary to explore the biological mechanisms of heterosis inpromoting crop genetic improvement and breeding practice. As heterosis is the result ofgene expression regulation in heterozygous state, studies have been confirmed that it's animportant strategy to explore relationships between gene expression and heterosis at thelevel of gene expression. Microarray technique is a high throughput tool for surveyinggenome-scale gene expression, it has been widely applied in gene expression profiling ofimportant organisms. As seedling growth is the basis for canopy development and panicledifferentiation is critical for panicle size of rice, investigation of gene expression amonghybrid and its parents in these stages may provide information for understanding heterosisat the gene expression level.In this study, we attempt to investigate the gene expression differences in two eliterice hybrids, Shanyou 63 and Liangyoupei 9, in comparison with the parents at specificdevelopment stages of seedling and panicle, using a self-customized cDNA microarrayconsisting of 9198 unique expressed sequence tags (EST). The objectives were to revealpossible patterns of gene expression during seedling and young panicle development,discern processes likely to be associated with heterosis in the rice hybrid, and identifypotential candidate genes that are involved in seedling and young panicle developmentand heterosis by comparing with data accumulated in previous studies.Global gene expression profiles were surveyed in shoots and roots of the threegenotypes of both hybrid combinations at 72 hours after germination and 4-leaf stages,and in young panicles of Shanyou 63 and its parents at three panicle development stages(secondary branch primordium, pistil/stamen primordium, and pollen-mother cellformation stages). The raw data were normalized through Quantile and Loess methodsusing R program, hybridization signals were detected for 9048 ESTs in all threegenotypes in at least one seedling tissue in the combination of Liangyoupei 9 and itsparents. In the survey of Shanyou 63 and its parents, 8652 ESTs showed hybridizationsignals in all three genotypes in at least one of the seedling tissues and 8422 ESTs had hybridization signals at three panicle development stages. Differences in expression levelamong the three genotypes of each combination were assessed statistically using ANOVA,and validated with 100 random permutations (p＜0.05). At seedling stage, significantdifferences were detected for 1139 and 1283 ESTs among the three genotypes of thecombinations Liangyoupei 9 and Shanyou 63. And 438 showed significant expressiondifferences among Shanyou 63 and its parents in at least one of the three stages of youngpanicle development. About 77% to 94% of the ESTs showing significant expressionpolymorphisms among the three genotypes also revealed significant expressiondifferences between the hybrid and one or both parents. 174 ESTs in shoots or roots ofboth hybrid combinations, 75 ESTs in seedling and young panicle of Shanyou 63 wereshared. Severn ESTs showing expression polymorphisms at seedling stages of both hybridcombinations were selected as probes for Northern blot analysis and the results wereconsiderable consistent with those from cDNA microarray hybridization.Significant heterotic expression as evaluated by mid-parent heterosis was detectedfor ESTs showing expression polymorphisms using an h-statistic with 100 randompermutations at p＜0.05 level. It was shown that 78 and 51 ESTs respectively showedheterotic expression in Liangyoupei 9 and Shanyou 63 at the seedling stage, and 141ESTs showed significant heterosis in young panicle of Shanyou 63. In both Liangyoupei 9and Shanyou 63, the number of ESTs showing positive heterotic expression was largerthan those showing negative heterotic expression in shoots at the seedling stage, whereasthe reverse was the case in roots. In the young panicle of Shanyou 63, a much largernumber of sequences showed negative heterosis than ones showing positive heterosis. Byhomology search, 436 out of the 438 sequences that showed significant expressionpolymorphisms could be mapped to the 12 chromosomes. Of them, 64 sequences werelocalized to 26 intervals spanning 282.0 cM of the genome where QTLs were previouslyidentified for panicle traits, including number of primary branches, number of secondarybranches, number of spikelets on primary branches, number of spikelets on secondarybranches and number of spikelets per panicle.About 50% sequences on the microarray had known functions, a largest number ofdifferentially expressed sequences showing homology to genes of known functions inshoots and roots of both hybrid combinations was in the category of metabolism,followed by genetic information processing, and environment information processing. Butthe largest number of polymorphic expressed sequences showing known functions inyoung panicle of Shanyou 63 was in the category of genetic information processing, followed by metabolism and environment information processing. The category ofcellular processing had the smallest number of sequences in both rice development stages.The results of x~2 test showed that overall there was no significant discrepancy between theexpected and the observed numbers of polymorphic expressed sequences in the variousfunctional categories in seedling and young panicle of Shanyou 63 combination. Thereverse was the case in seedling of Liangyoupei 9 combination, the observed numbers ofpolymorphic expressed sequences in the category of metabolism and environmentinformation processing were larger than the expected, whereas the numbers of sequencesrelevant to secondary metabolism and transcription regulation were smaller than theexpected, indicating significant differential occurrence of sequences in these functionalcategories.