Comparative Analysis of Genetic Diversity and Structure in Rice Using ILP and SSR Markers

Genetic diversity of 36 rice entries from the United States Department of Agriculture(USDA)rice collection was assessed using 103 ILP(intron length polymorphism)and 54 SSR(simple sequence repeats)markers.A total of 236 and 332 alleles were detected by the ILP and SSR markers,respectively.On average,the SSR markers produced higher polymorphism information content value and number of alleles than the ILP markers.Whereas the Nei's genetic distance measured using the SSR markers was much higher than that measured using the ILP markers.Mantel's test indicated that there was a statistically significant correlation(r=0.827,P0.001)between the two marker systems.UPGMA clustering based on the ILP and SSR markers resulted in consensus dendrograms.The cophenetic correlation coefficient(r=0.918,0.878 and 0.924,P0.001 for the ILP,SSR and combined markers,respectively)showed a highly accurate dendrogram represented the genetic distance among these entries.The 36 entries were divided into four groups.Four African Oryza glaberrima accessions were clustered within a distinct group(Ⅰ),and the remaining entries were separated into three groups(Ⅱ,Ⅲ and IV).All the entries could be also clustered into two main groups:One was composed of Ⅲ and IV,considered as indica group, and the other was composed of Ⅰ(O.glaberrima)and Ⅱ(japonica-like).Model-based cluster analysis revealed that the japonica-like group maintained very pure ancestry while the indica group shared mixed ancestry,especially for group Ⅲ, which had seven admixtures sharing from 19.5%to 30.0%of ancestry with group Ⅳ(based on SSR markers).It is suggested that ILP and SSR markers could be very useful for the genetic study and breeding in rice.     

Genetic Characterization of Indigenous Rice Varieties in Eastern Himalayan Region of Northeast India

The eastern Himalayan region of Northeast （NE） India is home to a large number of indigenous rice varieties, which are traditionally classified as Oryza sativa subspecies indica, japonica or intermediate types. The classification based on traditional Cheng’s index is often inconclusive due to phenotypic plasticity of morphological characters, which are influenced by environmental conditions. We used molecular markers specific for indica and japonica subspecies to assess the degree of genetic relatedness of indigenous rice varieties in NE India. The results revealed that majority of upland （jum） and glutinous rice varieties, traditionally considered as japonica, were genetically close to the subspecies indica. All varieties of boro ecotype were found to be indica type, and only a few varieties cultivated in lowland and upland areas were japonica type. Some of the lowland varieties of the sali ecotype were intermediate between indica and japonica, and they showed a closer genetic affinity to O. rufipogon.     

Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

The discovery of thermo-sensitive genic male sterility(TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F_2 populations derived from crosses between IR68301 S, an indica TGMS rice line, and IR14632(tropical japonica), Supanburi 91062(indica) and IR67966-188-2-2-1(tropical japonica), respectively.Approximately 1:3 ratio between sterile and normal pollen of F_2 plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat(SSR) and insertion-deletion(InDel) markers were used to identify markers linked to the tms gene. The linkage analysis based on the three populations indicated that the tms locus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F_2 population, the results showed that the tms locus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from the tms gene to these two flanking markers were 1.10 and 0.82 cM, respectively.InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to the tms gene using F_2 populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S.     

A novel approach for discovery and exploitation of new male sterile cytoplasm resources for hybrid rice

Using a simple sequence repeat molecular method (SSR), we found that cytoplasmic male sterile lines and their maintainer lines used widely in China could be completely identified by one of the special SSR markers in hybrid rice. This marker have been applied to screen a new male sterile cytoplasm resources from landrace and germplasm. The results showed that 2 of the 109 varieties screened possess different male sterile cytoplasm resources. When they were crossed with common maintainer tines, some male sterile plants produced from their offsprings. They could be     

Genetic Diversity of Main Inbred Indica Rice Varieties Applied in Guangdong Province as Revealed by Molecular Marker

Genetic diversity of 299 inbred indica rice varieties, including 33 introduced varieties, applied in Guangdong Province of China were assessed using 20 ILP(intron length polymorphism) and 34 SSR(simple sequence repeat) markers. Totally, 154 loci were screened for the 299 varieties, with the average number of alleles(Na), rare alleles(Nr), and polymorphism information content(PIC) scored at 3.4, 0.7and 0.32, respectively. The Nei’s genetic distance(GD) was estimated ranging from 0 to 0.7529 with an average of 0.4797. There was no significant difference of Na, Nr, PIC or GDs between the introduced and local varieties. Neighbor-joining(NJ) analysis showed that the 299 varieties failed into three main distinct groups, and the 33 introduced varieties were distributed over all the groups or subgroups. Model-based cluster analysis demonstrated that only 73(24.4%) of the 299 varieties and 7(21.2%) of the 33 introduced varieties could be distinctly classified into the three groups. Analysis of molecular variance showed that within the groups divided by NJ analysis, the genetic variations revealed by ILP, SSR and these two combined were 7.7%, 5.6% and 6.6%, and within the groups divided by region(Guangdong local and the introduced varieties), the genetic variables were 2.1%, 4.6%, 5.4%, respectively. These results suggested that the genetic diversity of the 299 inbred rice varieties in Guangdong Province was low, simultaneously relationship among varieties was poor and close in all kind of groups. Hence, it is very necessary to extend the genetic diversity during the breeding and selection practical procedure.     

Genetic Diversity of Rice Landraces from Lowland and Upland Accessions of China

Genetic diversity of rice landraces from lowland and upland accessions of China was investigated using 66 polymorphic simple sequence repeat (SSR) markers.The total number of alleles detected from all 324 tested accessions was 555 with an average allele number (Na) of 8.409 per locus,the average effective number of alleles (Ne) of 3.574 and the average Shannon’s information index (I) of 1.378.The genetic diversity was higher for the indica landraces compared to the japonica landraces,and the upland landraces were more genetically diverse than the lowland landraces.The SSR markers,RM72,RM232,RM219,RM241,RM224 and RM3 showed the highest rates of polymorphism and these SSR markers were suitable to assess the genetic diversity of rice germplasm resources.A dendrogram of 324 accessions of lowland and upland landraces showed that all rice accessions were mainly subdivided into two groups,japonica and indica,with some being intermediate.The distribution of lowland and upland landraces among the japonica and indica rice groups was distinct,with obvious differentiation between the lowland and upland landraces in japonica rice,but no such clear distinction in indica rice.     

Genetic Polymorphism of Wx Gene and Its Correlation with Main Grain Quality Characteristics in Rice

The allelic variation of the Wx gene in 50 non-glutinous rice varieties (lines) was analyzed by using the microsatellite marker RM190 [for (CT)n simple sequence repeat (SSR)] and cleaved amplified polymorphic sequence(CAPS) marker 484/W2R-ACCⅠ[for G/T single nucleotide polymorphism (SNP)]. Six homozygous (CT)n types, namely (CT)20, (CT)19, (CT)18, (CT)17, (CT)16, (CT)14, (CT)11 and (CT)10, and a heterozygous genotype (CT)11/(CT)18 were detected for RM190, of which (CT)11 and (CT)18 were predominant. Two homozygous Wx genotypes (G/G and T/T) and one heterozygous (G/T) were detected using 484/W2R-ACCⅠ . Most of the materials with a RM190 of (CT)11 were G/G for SNP of 484/W2R-ACC I, while T/T for SNP was predominantly appeared in materials with (CT)18. The materials tested could be grouped into 10 categories using the two markers together. Results indicated that 59.3% variance of amylose content was attributed to the polymorphism of Wx gene revealed by RM190, while 56.1% and 24.6% of the variances in amylose content and gel consistency were respectively to the polymorphism of Wx gene revealed by 484/W2R-ACC I. Furthermore, with both SSR and CAPS markers, 72.4% of the variance in amylose content could be explained. In addition, the application prospects of the two markers in breeding were also discussed.     

Genetic Diversity of Tropical Hybrid Rice Germplasm Measured by Molecular Markers

Investigation of genetic diversity and relationships among breeding lines is of great importance to facilitate parent selection in hybrid rice breeding programs.In this study,we characterized 168 hybrid rice parents from International Rice Research Institute with 207 simple sequence repeat (SSR) and 353 single nucleotide polymorphism (SNP) markers.A total of 1 267 SSR and 706 SNP alleles were detected with the averages of 6.1 (SSR) and 2.0 (SNP) alleles per locus respectively across all lines.Based on the genetic distances estimated from the SSR and SNP markers separately and combined,the unrooted neighbor-joining cluster and STRUCTURE analyses consistently separated the 168 hybrid rice parents into two major groups:B-line and R-line,which is consistent with known parent pedigree information.The genetic distance matrices derived from the SSR and SNP genotyping were highly correlated (r=0.81,P < 0.001),indicating that both of the SSR and SNP markers have distinguishable power to detect polymorphism and are appropriate for genetic diversity analysis among tropical hybrid rice parents.A subset of 60 SSR markers were also chosen by the Core Hunter with 368 alleles,and the cluster analysis based on the total and subset of SSR markers highly corresponded at r=0.91 (P < 0.001),suggesting that fewer SSR markers can be used to classify and evaluate genetic diversity among parental lines.     

贵州常用玉米自交系的SSR遗传分析

Genetic variation was analyzed of 35 maize inbred lines in Guizhou with SSR.Selected 10 core-primers which could test 75 allele sites in sample of 35 inbred lines.The average number of allele per SSR locus was 7.5 with arrange from 5 to 14.The average of polymorphic information content(PIC) was 0.739.Gene locus among the 35 inbred lines ranged from 0.154 to 0.838 with an average of 0.513.The clustering results showed that the inbred lines could be classified into 7 groups.The clustering results were consistent with the groups based on the available.The local germplasm lines could be divided into three heterotic groups,showed the local germplasm had wide genetic basis.     

SSR Analysis on Diversity of AA Genome Oryza Species in the Southeast and South Asia

To investigate genetic diversities among the AA genome Oryza species in the Southeast and South Asia, a total of 428 accessions of the AA genome Oryza species were genotyped using 36 simple sequence repeats (SSR) markers distributed throughout the rice genome. All of the 36 SSR markers generated polymorphic bands, revealing 100% polymorphism. The number of alleles per locus ranged from 3 to 17 with the mean of 8.6. The Nei’s genetic diversity index (He) ranged from 0.337 at RM455 to 0.865 at RM169 with an a...     

Genetic Diversity and Population Structure in Landraces and Improved Rice Varieties from India

A set of 50 rice genotypes comprising landraces, local selections, and improved varieties were characterized using simple sequence repeat(SSR) and inter simple sequence repeat(ISSR) markers to study genetic diversity and population structure. Following unweighted pair group method with arithmetic mean based clustering using binary data of polymorphic markers, the genotypes were grouped into 5 clusters and 11 sub-clusters, whereas population structure analysis separated 50 rice genotypes into 5 sub-populations. Grouping of rice genotypes showed better resemblance with the pedigree information of the genotypes. Both genetic diversity and population structure analysis separated majority of the improved varieties from landraces and local selections. Some of the SSR markers amplified unique alleles which were specific to a particular genotype and could distinguish them from the rest. The results indicate that these rice genotypes exhibit a higher genetic diversity and can be very useful in rice improvement program.     

Genetic Analysis and Mapping of Dominant Minute Grain Gene Mi3（t） in Rice

Grain size, determined chiefly by grain length, is one of the main factors affecting the grain yield in rice production. To study the trait of rice grain size, F1 and F2 populations were developed from crosses Shuhui 881/Y34 and Shuhui 527/Y34, and genetic analysis for minute grain was performed. The F1 populations showed minute grains, and grain size segregations in the two F2 populations were both in accordance with the ratio of 3：1, indicating that minute grain in Y34 was controlled by a completely dominant gene. By using the F2 population from Shuhui 881/Y34, this dominant gene, tentatively designated as Mi3（t）, was mapped based on SSR markers in the interval between RM282 （genetic distance of 5.1 cM） and RM6283 （genetic distance of 0.9 cM） on the short arm of chromosome 3.     

Genetic Structure and Indica/Japonica Component Changes in Major Inbred Rice Varieties in China

We used 39 SSR markers to analyze the genetic structure of 304 major Chinese inbred rice varieties, and to compare changes in the indica or japonica components in these varieties that have been widely cultivated from the 1950s to the 1990s in China. The genetic structure analysis showed that these rice varieties were distinctly divided into two populations, indica and japonica. The sub-structure of indica varieties was more complex than that of japonica ones. Among the various lines, late-season indica and early season japonica varieties had simpler genetic backgrounds. The seasonal ecotypes were not quite consistent with the subtypes of genetic structure. Twelve SSR loci with specific differentiation between indica and japonica were used to calculate the indica/japonica components. The differences in indica/japonica components among the five decades were not significant, except for late-season indica varieties in the 1990s, which had a significantly higher japonica component. These results will help to understand the genetic structure of the major Chinese inbred rice varieties and will be useful for indica-japonica hybrid breeding in China.     

Distribution of the Classification Traits in the F2 Progeny of Two Crosses of indica／japonica in Rice （Oryza sativa）

Comparison of seed setting rate, plant height and Cheng‘s morphological traits were made between the F2 progeny of indica-japonica hybrids, 02428/Pei‘ai 64 and Taichung 65/Teqing. There existed resemblance normal distribution in plant height, and the distribution had no relationship with the seed setting rate and plant height in F1 progeny, suggesting that there were no gametic selection for the plant height in the F2 progeny of indica-japonica hybrids. An average seed setting rate of the F2 was equal to the F1 populations in the recombination of 02428/Pei‘ai 64 in which there was an excellent cross compatibility, but the remarkable increase in the seed setting rate from the F1 to F2 in Taichung 65/Teqing, indicating that gametic selection only took place in the indica-japonica crosses with low cross compatibility while null or little in the hybrids with high compatibility. There were many intermediate types among the leaf pubescence, color of hull at heading, and length of the first and second panicle internode, and these Cheng‘s morphological traits was distributed normally; there were also a little intermediate types in hull hairness and phenol reaction with bi-peak distribution; while length to width ratio of grain was inclined to the japonica types obviously showing a skewed distribution in the Fz population. Although Cheng‘s morphological traits in F2 populations had inconsistent distribution types, Cheng‘s morphological traits index showed normal distribution, which suggested that intermediate types, i.e. indicalinous or japonicalinous, were primary in F2 population.     

Evaluation and Bulked Segregant Analysis of Major Yield QTL qtl12.1 Introgressed into Indigenous Elite Line for Low Water Availability under Water Stress

Near isogenic lines carrying large-effect QTL (qtl12.1), which has a consistent influence on grain yield under upland drought stress conditions in a wide range of environments, were evaluated under water stress in the fields. The line which gave higher yield under drought was crossed with a local elite line, PMK3, and forwarded to F2:3 generation. Significant variation was found among the F2:3 lines for agronomic traits under water stress in the fields. Low to high broad sense heritability (H) for investigated traits was also found. Water stress indicators such as leaf rolling and leaf drying were negatively correlated with plant height, biomass and grain yield under stress. Bulked segregant analysis (BSA) was performed with the markers in the vicinity of qtl12.1, and RM27933 was found to be segregated perfectly well in individual components of drought resistant and drought susceptible bulks which were bulked based on yield under water stress among F2:3 lines. Hence, this simple and breeder friendly marker, RM27933, may be useful as a potentially valuable candidate marker for the transfer of the QTL qtl12.1 in the regional breeding program. Bioinformatic analysis of the DNA sequence of the qtl12.1 region was also done to identify and analyze positional candidate genes associated with this QTL and to ascertain the putative molecular basis of qtl12.1.     

Development of New InDel Marker to Detect Genotypes of Rf-la Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility in Rice

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility （CMS） in japonica rice, a functional marker InDeI-Rf-la based on the difference of nucleotide sequence in Rf-la locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-larf-la showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-la locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F2 segregation population, and three genotypes in Rf-la locus could be distinguished distinctly. Therefore, the marker InDeI-Rf-la could be widely used for genetic id~ntifio.~tinn ~nd m~rkp.r-~.~.~i~fp.d .~.tAr.tinn （MA.~ in hr~=dinn i~nnnir~ r~fnr~=r lin==~     

A Comparative Study of SSR Diversity in Chinese Major Rice Varieties Planted in 1950s and in the Recent Ten Years （1995-2004）

Forty pairs of SSR markers were used to compare the genetic diversity changes in 151 Chinese major rice varieties planted in 1950s and in the recent ten years. Of 40 SSR loci, 39 were found to be polymorphic while one locus (RM479) monomorphic. A total of 213 alleles were identified from the 39 polymorphic loci. The average number of alleles per locus (Na) was of 5.5, ranging from 2 to 11. Nei’s gene diversity index (He) varied drastically among loci from 0.309 at RM174 to 0.869 at RM418, with an average value of 0.649. There existed significant difference in SSR allelic diversity between indica and japonica subspecies, and indica had more variation than japonica both in Na and He. By comparison with the genetic changes in Na and He, it was revealed that the varieties planted in 1950s had more alleles and higher He than those in the recent ten years both for indica and japonica rices. The difference between two subspecies for Na was significant in a tendency over time (indica: z = 2.677, P = 0.007; japonica: z = 3.441, P = 0.001), but not significant for He (indica: z = 1.471, P = 0.141; japonica: z = 1.932, P = 0.053). Analysis of molecular variance (AMOVA) indicated that there existed significant difference (P < 0.05) in genetic variation between the two periods, of which more genetic variation was contributed by indica (Fst = 0.050) and japonica (Fst = 0.082) subsets. Using locus-by-locus AMOVA procedure, significant genetic differentiations were observed in 13 loci (RM21, RM128, RM147, RM169, RM190, RM221, RM231, RM251, RM253, RM317, RM341, RM418, and RM478) for indica varieties and 11 loci (RM101, RM135, RM152, RM159, RM169, RM190, RM251, RM253, RM311, RM418, and RM478) for japonica ones between the two periods. It was found some alleles had been lost in current major rice varieties as comparing with those in 1950s. Therefore, it should be necessary to exploit more alien elite genetic resources for extension of genetic background in current rice breeding program.     

Mapping QTL for Heat-Tolerance at Grain Filling Stage in Rice

A mapping population of 98 lines (backcross inbred lines, BILs) derived from a backcross of Nipponbare/Kasalath// Nipponbare was planted at two experimental sites, Nanjing and Nanchang, and treated with high and optimal temperature during grain filling, respectively. The grain weight heat susceptibility index [GWHSI= (grain weight at optimum temperature-grain weight at high temperature) / grain weight at optimum temperature ×100] was employed to evaluate the tolerance of rice to heat stress. A genetic linkage map with 245 RFLP markers and a mixed linear-model approach was used to detect quantitative trait loci (QTLs) and their main effects, epistatic interactions and QTL×environment interactions (Q×E). The threshold of LOD score=2.0 was used to detect the significance of association between marker and trait. A total of 3 QTLs controlling heat tolerance during grain filling were detected, on chromosomes 1, 4 and 7, with LOD scores of 8.16, 11.08 and 12.86, respectively, and they explained the phenotypic variance of 8.94, 17.25 and 13.50 %, correspondingly. The QTL located in the C1100-R1783 region of chromosome 4 showed no QTL×environment interaction and epistatic effect, suggesting that it could be stably expressed in different environments and genetic backgrounds, and thus it would be valuable in rice breeding for heat tolerance improvement. This QTL allele, derived from Kasalath reduced 3.31% of the grain weight loss under heat stress. One located between R1613-C970 on chromosome 1 and the other between C1226-R1440 on chromosome 7, with additive effect 2.38 and 2.92%, respectively. The tolerance alleles of both these QTLs were derived from Nipponbare. Both of these QTLs had significant QTL×environment interactions, and the latter was involved in epistatic interaction also. Eight pairs of epistatic effect QTLs were detected, one pair each on chromosomes 1,2,3, 5, 7, 8, 10 and 12. The results could be useful for elucidating the genetic mechanism of heat-tolerance and the development of new rice varieties with heat tolerance during grain filling phase.     

A Set of SCAR Markers Efficiently Differentiating Hybrid Rice

Molecular markers have been widely used in crop genetic improvement,seed test and genetic mapping.Of which,sequence characterized amplified region(SCAR) markers are particularly popular for its diversity,stable reproducibility,and suitability for analyzing large number of samples.In this study,500 random amplified polymorphic DNA(RAPD) primers were tested,and a set of SCAR markers comprising 37 pairs of loci-specific primers were developed from the DNA fragments ranging from 300 to 1000 bp which correspond to the stable,distinctive RAPD banding patterns.Using these SCAR markers,59 hybrid rice combinations were assessed and distinguished into 58 subgroups at the similarity coefficient of 0.97 in a genetic clustering tree based on the allele diversities of the SCAR markers.Furthermore,13 hybrid rice combinations were reassayed with 40 randomly selected simple sequence repeat(SSR) markers to evaluate the effectiveness of these SCAR markers.SSR markers produced similar results to SCAR markers as the 13 hybrid rice combinations were completely separated at the similarity coefficient of 0.91 in the clustering tree established from SSR patterns.Taken together,SCAR markers prove to be effective tools for identifying and differentiating hybrid rice combinations.