Preliminary Studies Of Genetic Divergence By Molecular Markers Between Indica And Japonica Subspecies Of Oryza Sativa

The cultivated rice is one of the most important crop in the world and has been differentiated two distinctly discriminative subspecies,i.e.Oryza sativa ssp.indica and Oryza sativa ssp.japonica.The two subspecies showed separate characters in agronomic traits,yield and quality.So,by crossing between indica and japonica subspecies,selecting and breeding new cultivars or stronger Combining ability hybrid rice parents became important technique methods.But the research we have done was unsubstantial and lacked enough theoretical research about genetic diversity between subspecies of oryza sativa.In the present study,the sequence diversity of nuclear rDNA nontranscribed spacer, sequence-related amplified polymorphism(SRAP)and target region amplification polymorphism(TRAP)markers and then simple sequence repeat(SSR)and inter simple sequence repeat (ISSR)markers were used to analyze genetic diversity of indica and japonica subspecies,aiming to supply further theory about genetic characters of the subspecies of oryza sativa.The main results were as follows:1.The comparative analysis of the rDNA internal transcribed spacers(ITS)entire sequences of 18 cultivars in Oryza sativa L.was carried out with O.rufipogon as outgroup. In the 18 cultivars,there were 9 Oryza sativa ssp.indica varieties,6 typical Oryza sativa ssp.japonica,2 cultivars of Oryza sativa ssp.javanica and 1 cultivars of upland rice.The result indicated that there were 23 variational sites which were 5.41%of entire bases and 8 informative sites which were 1.82%of entire bases in Oryza sativa.The length range of ITS sequence in Oryza sativa ssp.indica was ranging from 425 to 430 bp;the percentage of G+C content varied from 74.25 to 75.59%.The length range of ITS sequence in Oryza sativa ssp. japonica was 425 bp and the percentage of G+C content varied from 74.25 to 75.59%.The phylogenetic tree constructed on the basis of the ITS sequences showed that indica and japonica subspecies of Oryza sativa species formed separate clades clearly,meanwhile,Oryza sativa ssp.javanica have closer relationships with Oryza sativa ssp.indica.The study shows that the sequences analysis of the internal transcribed spacer was a good tool to discriminate between subspecies of Oryza sativa and was significant in the research of Oryza sativa evolution.2.The analysis of the rDNA intergenic spacer entire sequences of 13 cultivars in Oryza sativa.L was carried out with O.rufipogon as outgroup.In the 13 cultivars,there were 6 typical Oryza sativa ssp.japonica,5 typical Oryza sativa ssp.indica,2 Oryza sativa ssp.javanica.The result indicated that the length range of IGS sequences was from 2130 to 2145 bp,the percentage of G + C content varied from 74.59%to 75.29%, there were 229 variational loci which were 10.70%of entire bases and 76 informative loci which were 3.51%of entire bases in Oryza sativa.38 indica and japonica specific loci were existed in the IGS sequences of Oryza sativa,in which about 34 ones were covered a about 400 bp segment located in the upstream of IGS sequence.The indica and japonica subspecies of Oryza sativa,as well as some closely related cultivars,can be divided clearly based on the phylogenetic tree constructed by IGS sequences.Oryza sativa ssp.javanica had a closed relationship with japonica subspecies.3.In order to exploit the utilization of heterosis between rice subspecies,SSR,ISSR,SRAP,TRAP markers were used to analyze the relationship of inter-subspecies and intra-subspecies in oryza sativa.The result indicated that all 4 markers were highly correlated,and 90 indica-japonica specific markers that could help differentiate between typical indica and japonica subspecies were identified which 17 with SSR,18 with ISSR,25 with SRAP,30 with TRAP.Comparison of genome database in GenBank,the indica-japonica specific markers all distributed in coding region or no-ncoding region which located with quantitative character(QTL).Japonica consanguinity was detected in hybrid rice sterile lines breeding from indica and japonica crossing, particularly in Peiai64S(the female parent of super hybrid rice),this helped us to build a bridge between the indica and japonica rice.Much indica-specific and japonica-specific markers were detected in oryza sativa ssp.javanica by 4 marker systems.It indicated oryza sativa ssp.javanica was a middle type between inidca and japonica subspecies.Considered the ITS sequences of javanica rice had closer relationship with indica rice, while had closer relationships with japonica with IGS sequences, our research supported the opinion that oryza sativa ssp. javanica was a independent subspecies of oryza sativa.To the common wild rice,Some accessions showed different indicaclinous or japonicaclinous characters in different marker systems.As a whole,the common wild rice included not only much indica,but much japonica specific markers,indicated the aboriginality of undefined indica-japonica differentiation.This supported the double origin theory of Asian common wild furtherly:indica and japonica rice originated from common wild rice,separately.These result showed that the four marker systems all can used in analysis of indica and japonica specific markers.SRAP and TRAP had more potential application.4.The indica,japonica,indicaclinous and japonicaclinous type were choosed from the F₂ hybrid crossing from the indica rice Guangluai4 and japonica rice akihikari according to chen' index methods.Reproduced offspring to F₇,and then determined their main agronomic traits and Chen' s index methods. Meanwhile,the ITS of rDNA of the indica and japonica type in their offspring was also comparatively analyzed.The result showed that the ITS both from the male parent and female parent were existed in F₂ greatly because of the intense separation of chromosome.At the same time,some base mutations were found in some sequences.With the continuous reproduction,the ITS fragments evolved into convergence through sequence elimination.As a result,the obvious predominant ITS appeared in F₄,and it nearly homogenized in F₇,although some single base differences still existed in some repeat sequences.This study holds that the agronomic traits of the hybrid offspring F₆-F₇ were generally constant by means of direct screening,and the genetic materials among F₇ were also stable,yet the real homozygosis in base level were not achieved.