QTL analysis on rice grain appearance quality,as exemplifying the typical events of transgenic or backcrossing breeding

Rice grain shape and yield are usually controlled by multiple quantitative trait loci (QTL). This study used a set of F_9–10 recombinant inbred lines (RILs) derived from a cross of Huahui 3 (Bt/Xa21) and Zhongguoxiangdao, and detected 27 QTLs on ten rice chromosomes. Among them, twelve QTLs responsive for grain shape/ or yield were mostly reproducibly detected and had not yet been reported before. Interestingly, the two known genes involved in the materials, with one insect-resistant Bt gene, and the other disease-resistant Xa21 gene, were found to closely link the QTLs responsive for grain shape and weight. The Bt fragment insertion was firstly mapped on the chromosome 10 in Huahui 3 and may disrupt grain-related QTLs resulting in weaker yield performance in transgenic plants. The introgression of Xa21 gene by backcrossing from donor material into receptor Minghui 63 may also contain a donor linkage drag which included minor-effect QTL alleles positively affecting grain shape and yield. The QTL analysis on rice grain appearance quality exemplified the typical events of transgenic or backcrossing breeding. The QTL findings in this study will in the future facilitate the gene isolation and breeding application for improvement of rice grain shape and yield.     

QTL pyramiding for improving of cold tolerance at fertilization stage in rice

Vigorous cold tolerance at the fertilization stage (CTF) is a very important characteristic for stable rice production in cold temperature conditions. Because CTF is a quantitatively inherited trait, pyramiding quantitative trait loci (QTLs) using marker-assisted selection (MAS) is effective for improving CTF levels in rice breeding programs. We previously identified three QTLs controlling CTF, qCTF7, qCTF8 and qCTF12, using backcrossed inbred lines derived from a cross between rice cultivar Eikei88223 (vigorous CTF) and Suisei (very weak CTF). However, pyramiding of these QTLs for the application of MAS in practical rice breeding programs have not yet been elucidated. In this study, we examined the effect of pyramiding QTLs for improvement of CTF level using eight possible genotype classes from the 152 F₃ population derived from a cross between Eikei88223 and Suisei. Increasing of CTF levels in combinations between qCTF7 and qCTF12 and between qCTF8 and qCTF12 were detected. Furthermore, we compared the haplotype pattern around the QTLs for CTF among the rice cultivars from Hokkaido. These results are useful for improvement of new cultivars with high CTF levels using MAS and identification of genetic resources with the novel QTL(s) for CTF.     

Rice tungro spherical virus resistance into photoperiod-insensitive japonica rice by marker-assisted selection

Rice tungro disease (RTD) is one of the destructive and prevalent diseases in the tropical region. RTD is caused by Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. Cultivation of japonica rice (Oryza sativa L. ssp japonica) in tropical Asia has often been restricted because most japonica cultivars are sensitive to short photoperiod, which is characteristic of tropical conditions. Japonica1, a rice variety bred for tropical conditions, is photoperiod-insensitive, has a high yield potential, but is susceptible to RTD and has poor grain quality. To transfer RTD resistance into Japonica1, we made two backcrosses (BC) and 8 three-way crosses (3-WC) among Japonica1 and RTSV-resistant cultivars. Among 8,876 BC₁F₂ and 3-WCF₂ plants, 342 were selected for photoperiod-insensitivity and good grain quality. Photoperiod-insensitive progenies were evaluated for RTSV resistance by a bioassay and marker-assisted selection (MAS), and 22 BC₁F₇ and 3-WCF₇ lines were selected based on the results of an observational yield trial. The results demonstrated that conventional selection for photoperiod-insensitivity and MAS for RTSV resistance can greatly facilitate the development of japonica rice that is suitable for cultivation in tropical Asia.     

Genetical and morphological characterization of cold tolerance at fertilization stage in rice

Cold temperature during the reproductive phase leads to seed sterility, which reduces yield and decreases the grain quality of rice. The fertilization stage, ranging from pollen maturation to the completion of fertilization, is sensitive to unsuitable temperature. Improving cold tolerance at the fertilization stage (CTF) is an important objective of rice breeding program in cold temperature areas. In this study, we characterized fertilization behavior under cold temperature to define the phenotype of CTF and identified quantitative trait loci (QTLs) for CTF. A wide variation in CTF levels has been identified among local cultivars in Hokkaido, which is one of the most northern regions for rice cultivation in the world. Clear varietal differences in pollen germination, and pollen tube elongation due to cold temperature have been observed. These differences may confer a degree of CTF among this population. We conducted QTL analysis for CTF using 120 backcrossed inbred lines derived from a cross between Eikei88223 (vigorous CTF) and Suisei (very weak CTF). Three QTLs for CTF were identified. A clear effect by QTL, qCTF7, for increasing the level of CTF was validated using advanced progeny. These results will facilitate marker-assist selection for desirable QTLs for CTF in rice breeding program.     

Confirming a major QTL and finding additional loci responsible for field resistance to brown spot (Bipolaris oryzae) in rice

Brown spot is a devastating rice disease. Quantitative resistance has been observed in local varieties (e.g., ‘Tadukan’), but no economically useful resistant variety has been bred. Using quantitative trait locus (QTL) analysis of recombinant inbred lines (RILs) from ‘Tadukan’ (resistant) × ‘Hinohikari’ (susceptible), we previously found three QTLs (qBS2, qBS9, and qBS11) that conferred resistance in seedlings in a greenhouse. To confirm their effect, the parents and later generations of RILs were transplanted into paddy fields where brown spot severely occurred. Three new resistance QTLs (qBSfR1, qBSfR4, and qBSfR11) were detected on chromosomes 1, 4, and 11, respectively. The ‘Tadukan’ alleles at qBSfR1 and qBSfR11 and the ‘Hinohikari’ allele at qBSfR4 increased resistance. The major QTL qBSfR11 coincided with qBS11 from the previous study, whereas qBSfR1 and qBSfR4 were new but neither qBS2 nor qBS9 were detected. To verify the qBSfR1 and qBSfR11 ‘Tadukan’ resistance alleles, near-isogenic lines (NILs) with one or both QTLs in a susceptible background (‘Koshihikari’) were evaluated under field conditions. NILs with qBSfR11 acquired significant field resistance; those with qBSfR1 did not. This confirms the effectiveness of qBSfR11. Genetic markers flanking qBSfR11 will be powerful tools for marker-assisted selection to improve brown spot resistance.     

Quantitative trait loci for whiteness of cooked rice detected in improved rice cultivars in Hokkaido

Improving the eating quality of cooked rice has been one of the most important objectives in rice breeding programs. Eating quality of cooked rice is a complex trait including several components, such as external appearance, taste, aroma, and texture. Therefore, dissection of these components followed by marker-assisted selection of detected QTL(s) may be a useful approach for achieving desirable eating quality in rice breeding. Whiteness of cooked rice (WCR) is an important factor related to the external appearance of cooked rice. WCR is known to be associated with the amylose and protein contents of the endosperm. However, the genetic basis of WCR remains unclear. In this study, we evaluated phenotypic variation in WCR among recently developed rice cultivars from Hokkaido, Japan. Then, we developed doubled haploid lines (DHLs) derived from a cross between two cultivars from Hokkaido, Joiku No. 462 (high WCR) and Jokei06214 (low WCR). Using the DHLs, we detected two QTLs for WCR, qWCR3 and qWCR11, on chromosomes 3 and 11, respectively. We also examined the dosage effect of the two QTLs based on both the categorized segregants in the DHLs and the relationship between the WCR phenotype and inheritance around the QTL regions in cultivars from Hokkaido.     

Variation in heading date conceals quantitative trait loci for other traits of importance in breeding selection of rice

To identify quantitative trait loci (QTLs) associated with the primary target traits for selection in practical rice breeding programs, backcross inbred lines (BILs) derived from crosses between temperate japonica rice cultivars Nipponbare and Koshihikari were evaluated for 50 agronomic traits at six experimental fields located throughout Japan. Thirty-three of the 50 traits were significantly correlated with heading date. Using a linkage map including 647 single-nucleotide polymorphisms (SNPs), a total of 122 QTLs for 38 traits were mapped on all rice chromosomes except chromosomes 5 and 9. Fifty-eight of the 122 QTLs were detected near the heading date QTLs Hd16 and Hd17 and the remaining 64 QTLs were found in other chromosome regions. QTL analysis of 51 BILs having homozygous for the Koshihikari chromosome segments around Hd16 and Hd17 allowed us to detect 40 QTLs associated with 27 traits; 23 of these QTLs had not been detected in the original analysis. Among the 97 QTLs for the 30 traits measured in multiple environments, the genotype-by-environment interaction was significant for 44 QTLs and not significant for 53 QTLs. These results led us to propose a new selection strategy to improve agronomic performance in temperate japonica rice cultivars.     

Genome-wide association mapping focusing on a rice population derived from rice breeding programs in a region

Plant breeding programs in local regions may generate genetic variations that are desirable to local populations and shape adaptability during the establishment of local populations. To elucidate genetic bases for this process, we proposed a new approach for identifying the genetic bases for the traits improved during rice breeding programs; association mapping focusing on a local population. In the present study, we performed association mapping focusing on a local rice population, consisting of 63 varieties, in Hokkaido, the northernmost region of Japan and one of the northern limits of rice cultivation worldwide. Six and seventeen QTLs were identified for heading date and low temperature germinability, respectively. Of these, 13 were novel QTLs in this population and 10 corresponded to the QTLs previously reported based on QTL mapping. The identification of QTLs for traits in local populations including elite varieties may lead to a better understanding of the genetic bases of elite traits. This is of direct relevance for plant breeding programs in local regions.     

Localization of QTL for basal root thickness in <Emphasis Type="Italic">japonica</Emphasis> rice and effect of marker-assisted selection for a major QTL

Root traits are key components of plant adaptation to drought environment. By using a 120 recombined inbred lines (RILs) rice population derived from a cross between IRAT109, a japonica upland rice cultivar and Yuefu, a japonica lowland rice cultivar, a complete genetic linkage map with 201 molecular markers covering 1,833.8 cM was constructed and quantitative trait loci (QTLs) associated with basal root thickness (BRT) were identified. A major QTL, conferring thicker BRT, located on chromosome 4, designated brt4, explained phenotypic variance of 20.6%, was selected as target QTL to study the effects of marker-assisted selection (MAS) using two early segregating populations derived from crosses between IRAT109 and two lowland rice cultivars. The results showed that the flanking markers of brt4 were genetically stable in populations with different genetic backgrounds. In the two populations under upland conditions, the difference between the means of BRT of plants carrying positive and negative favorable alleles at brt4 flanking markers loci was significant. Phenotypic effects of BRT QTL brt4 were 5.05–8.16%. When selected plants for two generations were planted at Beijing and Hainan locations under upland conditions, MAS effects for BRT QTL brt4 were 4.56–18.56% and 15.46–26.52% respectively. The means of BRT for the homozygous plants were greater than that of heterozygous plants. This major QTL might be useful for rice drought tolerance breeding. L. Liu and P. Mu are contributed equally to this work.     

Variation in cooking and eating quality traits in Japanese rice germplasm accessions

The eating quality of cooked rice is important and determines its market price and consumer acceptance. To comprehensively describe the variation of eating quality in 183 rice germplasm accessions, we evaluated 33 eating-quality traits including amylose and protein contents, pasting properties of rice flour, and texture of cooked rice grains. All eating-quality traits varied widely in the germplasm accessions. Principal-components analysis (PCA) revealed that allelic differences in the Wx gene explained the largest proportion of phenotypic variation of the eating-quality traits. In 146 accessions of non-glutinous temperate japonica rice, PCA revealed that protein content and surface texture of the cooked rice grains significantly explained phenotypic variations of the eating-quality traits. An allelic difference based on simple sequence repeats, which was located near a quantitative trait locus (QTL) on the short arm of chromosome 3, was associated with differences in the eating quality of non-glutinous temperate japonica rice. These results suggest that eating quality is controlled by genetic factors, including the Wx gene and the QTL on chromosome 3, in Japanese rice accessions. These genetic factors have been consciously selected for eating quality during rice breeding programs in Japan.     

Mapping and validation of QTLs for rice sheath blight resistance

Sheath blight, caused by Rhizoctonia solani, is one of the most serious diseases of rice. Among 33 rice accessions, mainly from National Institute of Agrobiological Sciences (NIAS) Core Collection, we found three landraces from the Himalayas—Jarjan, Nepal 555 and Nepal 8—with resistance to sheath blight in 3 years’ field testing. Backcrossed inbred lines (BILs) derived from a cross between Jarjan and the leading Japanese cultivar Koshihikari were used in QTL analyses. Since later-heading lines show fewer lesions, we used only earlier-heading BILs to avoid association with heading date. We detected eight QTLs; the Jarjan allele of three of these increased resistance. Only one QTL, on chromosome 9 (between markers Nag08KK18184 and Nag08KK18871), was detected in all 3 years. Chromosome segment substitution lines (CSSLs) carrying it showed resistance in field tests. Thirty F₂ lines derived from a cross between Koshihikari and one CSSL supported the QTL.     

Characterization and identification of cold tolerant near-isogenic lines in rice

To exploit the genetic mechanism of cold tolerance in rice, cold tolerant near-isogenic lines (NILs) were developed by backcrossing Kunmingxiaobaigu (KMXBG), reported to be the most cold-tolerant variety at the booting stage, as donor, with the cold sensitive Japanese commercial japonica variety, Towada. Comparisons of cold tolerance-related traits between five BC₆F₅ NILs and recurrent parent Towada under cold treatment and normal temperatures at the booting stage showed that the differences between the NILs and Towada were significant only for spikelet fertility-related traits. Analyses of cold tolerance in the NILs at the budding (germination), seedling and booting stages indicated both correlated effects and differences. Lines 1913-4 and 1916-1 showed strong and stable tolerance at all three stages. Whole genome marker screening showed that the proportion of genetic background recovery was more than 98%. Seventeen markers from KMXBG were introgressed in two or more NILs, and cold tolerance genes were possibly present in these marker regions. The NILs should be excellent materials for both rice improvement and map-based cloning of cold tolerance QTLs.     

Development and validation of a PCR-based functional marker system for the brown planthopper resistance gene Bph14 in rice

Brown planthopper (BPH) is the most damaging rice pest globally. Resistant varieties are the most effective and environmental strategy for protecting the rice crop from BPH. Functional markers (FMs) designed from polymorphic sites within gene sequences affecting phenotypic variation are highly efficient when used for marker assisted selection (MAS). Bph14 is the first and only cloned insect resistance gene so far in rice. Compared to the sequences of its non-effective alleles there are a number SNP differences. In this study, the method of allele-specific amplification (ASA) was adopted to design a simple, co-dominant, functional marker Bph14P/N for Bph14. Bph14P/N was combined with two specific dominant markers: one, named Bph14P, targets the promoter region of Bph14 and amplifies 566 bp fragments; and the other, Bph14N, targets the LRR region of bph14 and amplifies 345 bp fragments. Specificity and applicability of the functional marker system were verified in two breeding populations and a Chinese mini core collection of Oryza sativa. We recommend the use of this simple, low-cost marker system in routine genotyping for Bph14 in breeding populations.     

Characterization and fine mapping of thermo-sensitive chlorophyll deficit mutant1 in rice (Oryza sativa L.)

Chlorophyll content is one of the most important traits controlling crop biomass and economic yield in rice. Here, we isolated a spontaneous rice mutant named thermo-sensitive chlorophyll deficit 1 (tscd1) derived from a backcross recombinant inbred line population. tscd1 plants grown normally from the seedling to tiller stages showed yellow leaves with reduced chlorophyll content, but showed no significant differences after the booting stage. At temperatures below 22°C, the tscd1 mutant showed the most obvious yellowish phenotype. With increasing temperature, the yellowish leaves gradually turned green and approached a normal wild type color. Wild type and tscd1 mutant plants had obviously different chloroplast structures and photosynthetic pigment precursor contents, which resulted in underdevelopment of chloroplasts and a yellowish phenotype in tscd1. Genetic analysis indicated that the mutant character was controlled by a recessive nuclear gene. Through map-based cloning, we located the tscd1 gene in a 34.95 kb region on the long arm of chromosome 2, containing two BAC clones and eight predicted candidate genes. Further characterization of the tscd1 gene is underway. Because it has a chlorophyll deficit phenotype before the tiller stage and little influence on growth vigor, it may play a role in ensuring the purity of hybrids.     

Combination of stem elongation ability with submergence tolerance in rice

Summary A study was conducted with six F₂ populations to test the possibility of combining submergence tolerance and stem elongation ability into a single genotype of rice (Oryza sativa L.). Submergence tolerance and stem elongation ability could be combined in the same genotype if strongly submergence tolerant genes are present in submergence tolerant parents.     

Development of a new selection method and quality improvement of sugary-1 rice mutants

Brown rice of sugary-1 mutants has a wrinkled character because of the presence of phytoglycogen instead of starch in the inner part of the endosperm. Because the wrinkled phenotype was used as a sole selection marker for progeny of the sugary-1 strain, identification of mutant seeds with improved appearance is very difficult. We found that sugary-1 varieties contained not only phytoglycogen but also free glucose in the endosperm, and these were positively correlated. In the segregated F₂ seeds that resulted from crossing Hokurikutou237 (sugary-1) and Koshihikari strains, glucose and phytoglycogen were also significantly correlated. Thus, we identified new sugary types with improved appearance from these progeny using glucose measurements. The F₄ seeds of the improved strain had moderate phytoglycogen contents and seed germination characteristics. Native-PAGE showed that pullulanase activity in the improved strain increased in developing seeds compared with Hokurikutou237, although isoamylase activity was extremely low and similar to that in sugary-1 types. The new selection method in this study efficiently aids the development of improved sugary rice types that lack the wrinkled phenotype.     

Integrating marker assisted background analysis with foreground selection for identification of superior bacterial blight resistant recombinants in Basmati rice

Basmati rice is highly susceptible to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae. Transfer of BB resistance genes from non‐Basmati sources to Basmati through cross‐hybridization requires strict monitoring for recovery of the desirable Basmati quality traits in the recombinants, which show complex inheritance pattern. We integrated background analysis using mapped microsatellite markers with foreground selection to identify superior lines that combine useful genes from a non‐Basmati BB resistance donor line IRBB55 with grain and cooking quality characteristics of the popular Basmati rice variety ‘Pusa Basmati 1’ (PB 1) employing backcross pedigree strategy. Foreground selection using linked markers ensured presence of two genes, xa13 and Xa21 for BB resistance from IRBB55, and the recurrent parent PB 1 allele for the waxy locus giving intermediate amylose content and maintainer allele at fertility restorer locus in the BC₁F₅ recombinants. Background analysis enabled selection of recombinants with recurrent parent genome to the extent of 86.3% along with the quality traits. The extent of introgression of non‐Basmati donor chromosome segments in the superior selections was estimated to be < 7.8 Mb and < 6.7 Mb in the xa13 and Xa21 linked genomic regions, respectively. Association mapping identified three quantitative trait loci, one each for 1000‐grain weight, fertile grains/panicle and cooked kernel length. The backcross‐pedigree breeding strategy facilitated recovery of additional desirable characteristics from the donor in some of the selections. The elite selection Pusa 1460‐01‐32‐6‐7‐67 with maximum genomic background and quality characteristics of the recurrent Basmati parent gave resistance reaction against BB, similar to that of the non‐Basmati resistant check variety and recorded an yield advantage of 11.9% over the best check in the multiplication agronomic trial in the Basmati growing region of India. This line, which has been released as a new variety in the name of ‘Improved Pusa Basmati 1’ for commercial cultivation in India, is an example of successful application of marker assisted selection to variety development.     

Genome-wide indel markers shared by diverse Asian rice cultivars compared to Japanese rice cultivar ‘Koshihikari’

Insertion-deletion (indel) polymorphisms, such as simple sequence repeats, have been widely used as DNA markers to identify QTLs and genes and to facilitate rice breeding. Recently, next-generation sequencing has produced deep sequences that allow genome-wide detection of indels. These polymorphisms can potentially be used to develop high-accuracy polymerase chain reaction (PCR)-based markers. Here, re-sequencing of 5 indica, 2 aus, and 3 tropical japonica cultivars and Japanese elite cultivar ‘Koshihikari’ was performed to extract regions containing large indels (10–51 bp) shared by diverse cultivars. To design indel markers for the discrimination of genomic regions between ‘Koshihikari’ and other diverse cultivars, we subtracted the indel regions detected in ‘Koshihikari’ from those shared in other cultivars. Two sets of indel markers, KNJ8-indel (shared in eight or more cultivars, including ‘Khao Nam Jen’ as a representative tropical japonica cultivar) and C5-indel (shared in five to eight cultivars), were established, with 915 and 9,899 indel regions, respectively. Validation of the two marker sets by using 23 diverse cultivars showed a high PCR success rate (≥95%) for 83.3% of the KNJ8-indel markers and 73.9% of the C5-indel markers. The marker sets will therefore be useful for the effective breeding of Japanese rice cultivars.     

Feasibility of selection for traits associated with cold tolerance in rice under rapid generation advance method

Summary Variability of the hybrid population can be preserved through generations during the process of rapid generation advance (RGA). Effective selection can be done in an early generation for those traits having high heritability value like growth duration and plant height. But selection is not advantageous for traits having low heritability. Thus, selection for panicle exsertion, cold tolerance at seedling stage and panicle length would not be wise during the early generations. However, in the low temperature areas growth duration and plant height are two important factors which determine varietal suitability. As selection of these two characters are possible through RGA, it should be a standard parctice during RGA to eliminate the plants with long growth duration and select plants with optimum plant height.     

QTL analysis for panicle characteristics in temperate japonica rice

To understand the genetic background of panicle characteristics in temperate japonica rice (Oryza sativa L.), we genetically analyzed DH lines derived from a cross between two temperate japonica rice cultivars, ‘Akihikari’ and ‘Koshihikari’,in 1996 and 1997. Four traits of panicle characteristics, number of primary branches per panicle (NPB), number of secondary branches per panicle (NSB), average number of spikelets on one primary branch (NSP)and average number of spikelets on one secondary branch (NSS), in 212 DH lines were measured, and the interval mapping of QTLs for these traits was carried out using169 DNA markers with an LOD threshold of2.5. Five, three and one putative QTLs for NPB, NSB and NSS were identified,respectively, and no QTLs relating to NSP appeared. The percentages in total phenotypic variation explained by all putative QTLs for NPB were 35.5%: and43.8% in 1996 and 1997, respectively. All putative QTLs for NSB accounted for 35.5%and 27.5% of total phenotypic variation in1996 and 1997, respectively. The QTLs identified in this study will be useful intemperate japonica rice breeding for improved spikelet yield. This revised version was published online in August 2006 with corrections to the Cover Date.