Molecular Mapping of QTLs for Yield and Yield-Related Traits in Oryza sativa cv Swarna × O. nivara (IRGC81848) Backcross Population

Advanced backcross QTL analysis was used to identify QTLs for seven yield and yield-related traits in a BC2F2 population from the cross between a popular Oryza sativa cv Swarna and O. nivara IRGC81848. Transgressive segregants with more than 15% increased effect over Swarna were observed for all the traits except days to heading and days to 50% flowering. Thirty QTLs were detected for seven yield and yield-related traits using interval and composite interval mapping. Enhancing alleles at 13 (45%) of these Q...     

Development and Identification of Introgression Lines from Cross of Oryza sativa and Oryza minuta

Introgression line population is effectively used in mapping quantitative trait loci(QTLs),identifying favorable genes,discovering hidden genetic variation,evaluating the action or interaction of QTLs in multiple conditions and providing the favorable experimental materials for plant breeding and genetic research.In this study,an advanced backcross and consecutive selfing strategy was used to develop introgression lines(ILs),which derived from an accession of Oryza minuta(accession No.101133) with BBCC genome,as the donor,and an elite indica cultivar IR24(O.sativa),as the recipient.Introgression segments from O.minuta were screened using 164 polymorphic simple sequence repeat(SSR) markers in the genome of each IL.Introgressed segments carried by 131 ILs covered the whole O.sativa genome.The average number of homozygous O.minuta segments per introgression line was about 9.99.The average length of introgressed segments was approximate 14.78 cM,and about 79.64% of these segments had sizes less than 20 cM.In the genome of each introgression line,the O.minuta chromosomal segments harbored chromosomal fragments of O.sativa ranging from 1.15% to 27.6%,with an overall average of 8.57%.At each locus,the ratio of substitution of O.minuta alleles had a range of 1.5% 25.2%,with an average of 8.3%.Based on the evaluation of the phenotype of these ILs,a wide range of alterations in morphological and yield-related traits were found.After inoculation,ILs 41,11 and 7 showed high resistance to bacterial blight,brown planthopper and whitebacked planthopper,respectively.These O.minuta-O.sativa ILs will serve as genetic materials for identifying and using favorable genes from O.minuta.     

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.     

New Stably Expressed Loci Responsible for Panicle Angle Trait in Japonica Rice in Four Environments

Panicle angle (PA) of 254 recombinant inbred lines derived from a cross between two japonica varieties Xiushui 79 and C Bao was investigated under four environments,and a genetic linkage map including 111 SSR markers was constructed.Genetic analysis was conducted by mixed major gene plus polygene inheritance models,and quantitative trait loci (QTLs) identification by the QTLNetwork 2.0 and the composite interval mapping approach of WinQTLCart 2.5 software.Results showed that the PA trait was controlled by two major genes plus polygenes,mainly by major genes.Eight QTLs for PA were detected by the QTLNetwork 2.0 software,and each locus explained 0.01% to 39.89% of the phenotypic variation.Twelve QTLs for PA were detected by the WinQTLCart 2.5 software,with each locus explaining 2.83% to 30.60% of the phenotypic variation.Two major QTLs (qPA9.2 and qPA9.5) distributed between RM3700 and RM3600 and between RM5652 and RM410,respectively,and a moderate QTL (qPA9.7) distributed between RM257 and OSR28,were both detected by the two methods in all of the four environments.The negative effect alleles of the three QTLs were from Xiushui 79.In addition,eight pairs of epistatic QTLs with minor effects were also detected.QTL × environment interactions were not significant for additive QTLs and epistatic QTL pairs.     

Dissection of Genetic Mechanism of Abnormal Heading in Hybrid Rice

Abnormal heading in hybrid rice production has caused great economic loss in recent years,but the genetic basis of this phenomenon remains elusive.In this study,we developed four testcross populations using 38 introgression lines(ILs)from Shuhui 527(SH527)/Fuhui 838(FH838)//SH527population as male parents and four male sterile lines(MSLs;namely II-32A,Xieqingzao A,Gang 46A and Jin 23A)as female parents.Progeny testing allowed us to identify 55 abnormal heading combinations in Hefei,but had late heading date in Hangzhou and Guangzhou of China.By one-and two-way analysis of variance,a total of 21 QTLs and 31 pairs of epistatic QTLs associated with photosensitivity were identified in the four populations,respectively.Genotypic analysis showed that the IL parent of most abnormal heading combinations showed some introgressions at markers RM331 and RM3395 on chromosome 8(strongly associated with the known genes OsHAP3H/DTH8/Ghd8/LHD1)of donor FH838alleles,and these two markers were also identified as affecting photosensitivity.The observation that the recipient parent(SH527),donor parent(FH838),their testcross combinations with four MSLs,and the IL parents of abnormal heading combinations had normal heading date in Hefei suggested that OsHAP3H/DTH8/Ghd8/LHD1 showed no independent regulation on abnormal heading in the abnormal heading combinations.It is noteworthy that complex epistasis among RM331 or RM3395 with other loci,including dominant×additive,additive×dominant,and dominant×dominant epistases,were identified only in the four testcross populations of the current study,but not in the SH527/FH838//SH527 population,suggesting the cause of abnormal heading in abnormal heading combinations in Hefei and delayed heading in Hangzhou and Guangzhou.     

Identification of QTLs and Validation of qCd-2 Associated with Grain Cadmium Concentrations in Rice

Cadmium(Cd) is one of heavy metals harmful to human health. As rice is the main staple food in Asia and Cd is easily contaminated in rice, the molecular regulation of Cd accumulation should be explored. In this study, a recombinant inbred population derived from Xiang 743/Katy was grown in Cd-polluted fields and used to map the quantitative trait loci(QTLs) for Cd accumulation in rice grains. We identified seven QTLs distributed on chromosomes 2, 3, 6, 7, 8 and 10. These QTLs displayed phenotypic variances of 58.50% and 40.59% in 2014 and 2015, respectively. Two QTLs, qCd-2 and qCd-7, were identified in both the two years. qCd-2 was detected on the interval of RM250–RM207 on chromosome 2, with an LOD of 2.51 and a phenotypic contribution of 13.75% in 2014, and an LOD of 3.35 and a phenotypic contribution of 14.16% in 2015. qCd-7 co-localized with the cloned qCdT7 on chromosome 7 and may represent the correct candidate. The other five QTLs were detected only in one year. To further confirm the effects of qCd-2, a residual heterozygous line designated as RHL945, with a heterozygous interval of RM263–RM207 on chromosome 2, was selected from the recombinant inbred population and used to develop an F2 population consisting of 155 individual plants. By incorporating further simple sequence repeat markers into the segmental linkage map of the target region, qCd-2 was delimited in the interval of RM5404–RM3774, with an LOD value of 4.38 and a phenotypic contribution of 15.52%. These results reflected the genetic regulation of grain Cd in rice and paved the way for the future cloning of qCd-2.     

Genetic Analysis and QTL Mapping of Large Flag Leaf Angle Trait in Japonica Rice

Genetic segregation analysis for flag leaf angle was conducted using six generations of P1,P2,F1,B1,B2 and F2 derived from a cross of 863B(a maintainer line of japonica rice) and A7444(a germplasm with large flag leaf angle).Genotypes and phenotypes of flag leaf angle were investigated in 863B(P1),A7444(P2) and 141 plants in BC1F1(863B/A7444$$$$863B) population.An SSR genetic linkage map was constructed and QTLs for flag leaf angle were detected.The genetic map containing 79 information loci was constructed,which covers a total distance of 441.6 cM,averaging 5.6 cM between two neighboring loci.Results showed that the trait was controlled by two major genes plus polygene and the major genes were more important.Fifteen markers showed highly significant correlations with flag leaf angle based on single marker regression analysis.Two QTLs(qFLA2 and qFLA8) for flag leaf angle were detected by both composite interval method in software WinQTLCart 2.5 and composite interval method based on mixed linear model in QTL Network 2.0.The qFLA2 explained 10.50% and 13.28% of phenotypic variation,respectively,and was located at the interval of RM300 and RM145 on the short arm of chromosome 2.The qFLA8 explained 9.59% and 7.64% of phenotypic variation,respectively,and was located at the interval flanking RM6215 and RM8265 on the long arm of chromosome 8.The positive alleles at the two QTLs were both contributed from A7444.     

Inheritance and QTL Mapping of Salt Tolerance in Rice

An F2 population derived from the cross between Jiucaiqing (japonica) and IR36 (indica) was used to analyze the inheritance of salt tolerance in rice by genetic model of major-genes plus polygenes, and to map the corresponding QTLs by SSR molecular markers. Rice plants of P1, P2, F1 and F2 at 5- to 6- leaf stage were treated under 140 mmol/L NaCI for 10 days. Three indices representing the ability of salt tolerance of rice seedlings were measured, including salt tolerance rating (STR), Na^ /K^ ratio in roots and dry matter weight of shoots (DWS). STR, Na^ /K^ and DWS were all controlled by two major genes with modification by polygenes. Heritability of these traits from major genes was 17.8, 53.3 and 52.3%, respectively. The linkage map constructed by 62 SSR molecular markers covered a total length of about 1 142 cM. There were three QTLs detected for STR located on chromosome 1, 5 and 9, two QTLs for DWS on chromosomes 8 and 9, and two QTLs for Na^ /K^ on chromosomes 2 and 6, one on each chromosome respectively. Single QTL accounted for 6.7 to 19.3% of phenotypic variation. Identification method of salt tolerance in rice and breeding of rice varieties with salt tolerance based on molecular markers assisted selection had been discussed.     

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.     

Mapping of qTGW1.1,a Quantitative Trait Locus for 1000-Grain Weight in Rice (Oryza sativa L.)

1000-grain weight(TGW) is one of the three component traits of the grain yield in rice(Oryza sativa L). This study was conducted to validate and fine-map qT GW1.1, a minor QTL for TGW which was previously located in a 3.7-Mb region on the long arm of rice chromosome 1. Five sets of near isogenic lines(NILs) were developed from two BC2F4 populations of the indica rice cross Zhenshan 973/Milyang 46.The NIL sets consisted of two homozygous genotypic groups differing in the regions RM11448-RM11522,RM11448- RM11549, RM1232- RM11615, RM11543-RM11554 and RM11569-RM11621, respectively. Four traits, including TGW, grain length, grain width and heading date, were measured. Phenotypic difference between the two genotypic groups in each NIL population was analyzed using SAS procedure GLM.Significant QTL effects were detected on TGW with the Zhenshan 97 allele increasing grain weight by0.12 g to 0.14 g and explaining 8.30% to 15.19% of the phenotypic variance. Significant effects were also observed for grain length and width, whereas no significant effect was found for heading date. Based on comparison among the five NILs on the segregating regions and the results of QTL analysis, qT GW1.1was delimited to a 376.9-kb region flanked by DNA markers Wn28382 and RM11554. Our results indicate that the effects of minor QTLs could be steadily detected in a highly isogenic background and suggest that such QTLs could be utilized in the breeding of high-yielding rice varieties.     

Breeding of R8012,a Rice Restorer Line Resistant to Blast and Bacterial Blight Through Marker-Assisted Selection

Genetic improvement is one of the most effective strategies to prevent rice from blast and bacterial blight(BB) diseases,the two most prevalent diseases jeopardizing rice production.Rice hybrids with dural resistance to blast and BB are needed for sustainable production of food.An incomplete diallele design resulted in 25 crosses between five blast and five BB resistant germplasm accessions.Only one pair of parents,DH146 × TM487,showed polymorphism for all the markers to identify one blast resistance gene Pi25 and three BB resistance genes,Xa21,xa13 and xa5,thus it was used in the marker-assisted selection(MAS).F2 individuals of DH146 × TM487 were genotyped using flanking markers of RM3330 and sequence tagged site(STS) marker SA7 for Pi25.The resistant F2 plants with Pi25 were used for pyramiding BB resistance genes Xa21,xa13 and xa5 identified by the markers pTA248,RM264 and RM153,respectively in subsequent generations.Finally,after selection for agronomic traits and restoration ability among 12 pyramided lines,we acquired an elite restorer line,R8012 including all four target genes(Pi25+Xa21+xa13+xa5).Hybrid Zhong 9A/R8012 derived from the selected line showed stronger resistance to blast and BB,and higher grain yield than the commercial checks uniformally in experimental plots,2007 state-wide yield trial and 2008 nation-wide yield trial.This study provides a paradigmatic example to show that MAS is a practically feasible tool in effectively pyramiding multiple resistance genes.The resultant restoring line and its hybrid would play an important role in securing rice production in China.     

Genetic Analysis and Preliminary Mapping of Two Recessive Resistance Genes to Brown Planthopper,Nilaparvata lugens Stl in Rice

An F2 population derived from the cross of WB01, an introgression line resistant to brown planthopper (BPH) originated from Oryza rufipogon Griff. and a susceptible indica variety 9311, was developed for genetic analysis and gene mapping. The population with 303 F2:3 families was genotyped by 141 simple sequence repeat (SSR) markers and used for gene mapping. Two softwares, Mapmaker/Exp 3.0 and Windows QTL Cartographer V2.0 were applied to detect QTLs. Totally, two QTLs resistant to BPH, named temporarily a...     

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.     

Identification and Genetic Mapping of a Lesion Mimic Mutant in Rice

A lesion mimic stripe mutant,designated as lms1(lesion mimic stripe 1),was obtained from the M2 progeny of a 60Co γ-radiation treated japonica rice variety Jiahua 1.The lms1 mutant displayed propagation type lesions across the whole growth and developmental stages.Physiology and histochemistry analysis showed that the mutant exhibited a phenotype of white stripe when grown under high temperature(30 ℃),and the lesion mimic caused by programmed cell death under low temperature(20 ℃).The genetic analysis indicated that this lesion-mimic phenotype is controlled by a single locus recessive nuclear gene.Furthermore,by using simple sequence repeat markers and an F2 segregating population derived from two crosses of lms1 × 93-11 and lms1 × Pei'ai 64S,the lms1 gene was mapped between markers Indel1 and MM0112-4 with a physical distance of 400 kb on chromosome 6 in rice.     

Agro-Morphological,Physico-Chemical and Molecular Characterization of Rice Germplasm with Similar Names of Bangladesh

Thirty-one duplicate and similar named rice germplasms of Bangladesh were studied to assess the genetic variation for the agro-morphological and physico-chemical traits and simple sequence repeat banding patterns during 2009–2012 at Bangladesh Rice Research Institute. The range of variations within the cultivar groups showed higher degree. The principal component analysis showed that the first five components with vector values 1 contributed 82.90% of the total variations. The cluster analysis grouped the genotypes into four clusters, where no duplicate germplasm was found. The highest number(11) of genotypes was constellated in cluster I and the lowest(3) in cluster II. The intra- and inter-cluster distances were the maximum in cluster I(0.93) and between clusters I and IV(24.61), respectively, and the minimum in cluster IV(0.62) and between clusters I and III(5.07), respectively. The cluster mean revealed that the crosses between the genotypes of cluster I with those of clusters II and IV would exhibit high heterosis for maximum good characters. A total of 350 alleles varied from 3(RM277) to 14(RM21) with an average of 7.8 per locus were detected at 45 microsatellite loci across the 31 rice accessions. The gene diversity ranged from 0.48 to 0.90 with an average of 0.77, and the polymorphism information content values from 0.44(RM133) to 0.89(RM206) with an average of 0.74. RM206, RM21, RM55, RM258 and RM433 were considered as the best markers on the basis of their higher polymorphism information content values. The dendrogram from unweighted pair-group method with arithmetic average clustering also classified the genotypes into four groups, where group IV comprised of 20 genotypes and group III of one genotype, but no duplicate was found. Finally, similar and duplicate named rice germplasms need to be conserved in gene bank as are distinct from each other.     

Seed Length Controlled by Same Locus in Four Different AA Genome Species of Genus Oryza

To broaden the genetic basis and overcome the yield plateau in Asian cultivated rice, the exploitation and utilization of favorable alleles from rice species with the AA genome has become important and urgent in modern breeding programs. Four different interspecific populations were used to detect quantitative trait locus （QTL） for seed length, including a BC4F2 population derived from Oryza glumaepatula crossed with Dianjingyou 1 （a japonica cultivar）, a BC4F2 population derived from O. nivara crossed with Dianjingyou 1, a BCzF1 population derived from a cross between O. Iongistaminata and RD23 （an indica cultivar）, and a BCsF1 population derived from a cross between O. glaberrima and Dianjingyou 1. The QTLs for seed length in four different populations were termed as SL-3a, SL-3b, SL-3c and SL-3d, respectively. They had good collinearity and accounted for 49% to 60% of the phenotypic variations. Sequencing data indicated that four QTLs were different alleles of GS3 which were responsible for the seed length variation between O. sativa and its four AA genome relatives. These results will be valuable for confirming the evolution of GS3 and also be helpful for rice breeding.     

Mapping of QTLs for Leaf Malondialdehyde Content Associated with Stress Tolerance in Rice

Malondialdehyde (MDA) is the final product of lipid peroxidation, and MDA content can reflect the stress tolerance of plants. To map QTLs conditioning the MDA content in rice leaves, a recombinant inbred line (RIL) population with 247 lines derived from an indica-indica cross Zhenshan 97B×Milyang 46, and a linkage map consisting of 207 DNA markers were used. The RIL population showed a transgressive segregation in the MDA content of rice leaves. Two QTLs for the MDA content in rice leaves were detected in t...     

Characterization and mapping of QTLs on chromosome 2D for grain size and yield traits using a mutant line induced by EMS in wheat

Production of mutants with altered phenotypes is a powerful approach for determining the biological functions of genes in an organism. In this study, a high-grain-weight mutant line M8008 was identified from a library of mutants of the common wheat cultivar YN15 treated with ethylmethane sulfonate(EMS). F2 and F2:3generations produced from crosses of M8008 × YN15(MY) and M8008 × SJZ54(MS) were used for genetic analysis. There were significant differences between M8008 and YN15 in plant height(PH), spike length(SL),fertile spikelet number per spike(FSS), grain width(GW), grain length(GL), GL/GW ratio(GLW), and thousand-grain weight(TGW). Most simple correlation coefficients were significant for the investigated traits, suggesting that the correlative mutations occurred in M8008. Approximately 21% of simple sequence repeat(SSR) markers showed polymorphisms between M8008 and YN15, indicating that EMS can induce a large number of mutated loci. Twelve quantitative trait loci(QTLs) forming QTL clusters(one in MY and two in MS) were detected. The QTL clusters coinciding with(MY population) or near(MS population) the marker wmc41 were associated mainly with grain-size traits, among which the M8008 locus led to decreases in GW, factor form density(FFD), and TGW and to increases in GLW. The cluster in the wmc25–barc168 interval in the MS population was associated with yield traits, for which the M8008 locus led to decreased PH, spike number per plant(SN), and SL.     

Identification of QTL Affecting Protein and Amino Acid Contents in Rice

The phenotypes of protein and amino acid contents were measured in an F9 recombinant inbred line population derived from a cross between Zhenshan 97B and Delong 208. A total of 48 and 64 QTLs were identified in 2004 and 2005, respectively. The contribution of each QTL to the phenotypic variation ranged from 4.0% to 43.7%. Most QTLs co-localized, forming 29 QTL clusters on the chromosomes with three major ones detected in both years, which were mapped on chromosomes 1, 7 and 9, respectively. The two QTL clus...     

Informative ISSR Markers Help Identify Genetically Distinct Accessions of Oryza rufipogon in Yield Improvement

Inter simple sequence repeat(ISSR) polymorphism was used to determine genetic diversity and phylogenetic relationships in 90 genotypes of wild and cultivated species of Oryza from different geographical regions of the world. In all the 17 primers used in ISSR-PCR, a total of 11 464 bands were amplified at 253 band positions/loci. The primer UBC-809 amplified the maximum bands(1 059) at 21 band positions. UBC-810 and UBC-835 amplified the minimum of 391 bands each at 7 and 14 band positions, respectively. The mean polymorphism information content ranged from 0.44 to 0.84 and resolving power ranged from 8.69 to 23.53. Un-weighted pair group method with arithmetic mean dendrogram and population structure based on the 17 primers separated all genotypes into 4 major clusters with a genetic similarity of 53%–100%. The first two clusters consisted of 30 O. rufipogon accessions each. In the third cluster, O. nivara and O. longistaminata grouped as one sub-cluster and all other O. nivara accessions and cultivars grouped as another sub-cluster. The fourth cluster had only five O. rufipogon accessions which can be a source of new genes. Four sub-populations were identified within O. rufipogon and two sub-populations within O. nivara at K = 7. A subset of six primers with high resolving power values were the most informative and grouped all genotypes almost similarly as the 17 primers did. Use of these six highly informative primers in ISSR-PCR is a cost effective and robust method for assessing genetic diversity in large germplasm collections of wild rice species.