QTL mapping for the paste viscosity characteristics in rice (Oryza sativa L.)

In order to understand the genetic basis of the paste viscosity characteristics (RVA profile, which is tested on the Rapid Visco Analyser) of the rice grain, we mapped QTLs for RVA profile parameters using a DH population derived from a cross between an indica variety, Zai-Ye-Qing 8 (ZYQ8), and a japonica variety, Jing-Xi 17 (JX17). Evidence of genotype-by-environment interaction was found by comparing the mapped QTLs between two locations, Hainan (HN) and Hangzhou (HZ). A total of 20 QTLs for six parameters of the RVA profiles were identified at least one location. Only the waxy locus (wx) located on chromosome 6 was detected significantly at both environments for five traits, i.e. hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), consistency viscosity (CSV) and setback viscosity (SBV). This locus explained 19.5%–63.7% of the total variations at both environments, suggesting that the RVA profiles were mainly controlled by the wx gene. HPV, CPV, BDV, CSV and SBV were also controlled by other QTLs whose effects on the respective parameter were detected only in one environment, while for the peak viscosity (PKV), only 2 QTLs, 1 at HN,the other at HZ, were identified. These results indicate that RVA profiles are obviously affected by environment. Received: 18 July 1999 / Accepted: 27 August 1999     

Molecular mapping of genes conferring aluminum tolerance in rice (Oryza sativa L.)

Crop productivity on acid soil is restricted by multiple abiotic stress factors. Aluminum (Al) tolerance seems to be a key to productivity on soil with a pH below 5.0, but other factors such as Mn toxicity and the deficiency of P, Ca and Mg also play a role. The development of Al-tolerant genotypes of rice is an urgent necessity for improving crop productivity in developing countries. Inhibition of root growth is a primary and early symptom of Al toxicity. The present study was conducted to identify genetic factors controlling the aluminum tolerance of rice. Several parameters related to Al tolerance, most importantly the relative root growth under Al stress versus non-stress conditions, were scored in 188 F₃ selfed families from a cross between an Al-tolerant Vietnamese local variety, Chiembau, and an Al-susceptible improved variety, Omon269–65. The two varieties are both Oryza sativa ssp. indica, but showed a relatively high level of DNA polymorphism, permitting the assembly of an RFLP map consisting of 164 loci spanning 1,715.8 cM, and covering most of the rice genome. A total of nine different genomic regions on eight chromosomes have been implicated in the genetic control of root and shoot growth under aluminum stress. By far the greatest effects on aluminum tolerance were associated with the region near WG110 on chromosome 1. This region does not seem to correspond to most of the genes that have been mapped for aluminum tolerance in other species, nor do they correspond closely to one another. Most results, both from physiological studies and from molecular mapping studies, tend to suggest that aluminum tolerance is a complex multi-genic trait. The identification of DNA markers (such as WG110) that are diagnostic for aluminum tolerance in particular gene pools provides an important starting point for transferring and pyramiding genes that may contribute to the sustainable improvement of crop productivity in aluminum-rich soils. The isolation of genes responsible for aluminum tolerance is likely to be necessary to gain a comprehensive understanding of this complex trait. Received: 29 March 2000 / Accepted: 16 August 2000     

Fine mapping of the grain chalkiness QTL qPGWC-7 in rice (Oryza sativa L.)

Chalkiness of rice grain is an important quality component of rice, as it has a profound influence on eating and milling qualities. We has determined the inheritance of percentage of grain with chalkiness (PGWC) using a set of chromosome segment substitution lines, made from a cross between cv. PA64s and cv. 9311. Two loci controlling PGWC, designated as qPGWC-6 and qPGWC-7, were located on, respectively, chromosomes 6 and 7. Comparisons were made between C-51 (a CSSL harbouring qPGWC-7 and having a chalky endosperm) and the recurrent parent 9311 (translucent endosperm) to characterize the physical and chemical differences between translucent and chalky endosperm. Unlike the translucent endosperm, the chalky endosperm contains loosely packed starch granules, and there were significant difference between C-51 and 9311 for amylopectin structure and degree of crystallinity, but not for either amylose content or starch viscosity. Segregation analysis of the F₂ population from the cross between C-51 and 9311 showed PGWC is a semi-dominant trait, controlled by single nuclear gene. A large F₂ population was constructed from the cross C51 × 9311, and used for the fine mapping of qPGWC-7, which was located to a 44-kb DNA fragment, containing thirteen predicted genes. This result provides a springboard for the map-based cloning of qPGWC-7 and allowed for marker-assisted selection for endosperm texture.     

基于CSSL的水稻穗颈长度QTL的代换作图

水稻穗颈长度是影响杂交水稻制种产量提高的重要农艺性状之一。文章利用94个以籼稻品种9311为遗传背景、粳稻品种日本晴为染色体片段供体的覆盖全基因组的染色体片段置换系(Chromosome segment substi-tution lines, CSSL)为材料, 调查和分析CSSL群体及双亲的穗颈长度。结果表明: 在17个置换系中检测到8个控制水稻穗颈长度的数量性状位点(Quantitative trait loci, QTL), 分别位于第2、3、7、8、9和第11染色体; 利用代换作图法, 定位了其中的7个穗颈长度QTL; 其加性效应值介于0.10~3.20之间, 其中qPE-9和qPE-11的加性效应值较大, 平均效应值分别为3.15和2.95, 表现为主效基因特征; qPE-2-2、qPE-3-1、qPE-3-2、qPE-7和qPE-8等5个QTL被定位在小于10.0 cM的区段内。利用CSSL可以有效地鉴定水稻穗颈长度QTL, 这些QTL为分子标记辅助选育穗颈长度适中的水稻品系及其进一步的精细定位奠定了基础。     

Low irradiance stress tolerance in rice (Oryza sativa L.)

The effect of low irradiance on three rice cultivars (shade tolerant cvs. Swarnaprabha and CO 43 and shade susceptible cv. IR 20) was studied. The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase with molecular mass of 55 kDa was reduced in cv. IR 20 grown under low irradiance (LI). Native protein profile studied showed, under LI, reduction in the contents of proteins with RF values 0.03, 0.11 and 0.37. Analysis of chloroplast polypeptides revealed an induction of light-harvesting chlorphyll-protein 2 (LHCP2) under shade. The induction was more expressed in cv. CO 43 than in cv. IR 20. Under LI, in vivo labelled protein bands in the molecular range of 26 - 27 kDa were induced. These proteins were highly turned over in the LI-grown plants of cv. CO 43 than in cv. IR 20. A signal for rbcL gene sequences in EcoRI digested lanes was also found. Isozyme analysis of peroxidase showed an induction of a new band with RF 0.43 in cv. IR 20 subjected to LI. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis and QTL mapping for grain chalkiness characteristics of brown rice (Oryza sativa L.)

Grain chalkiness is one of the important appearance qualities in rice marketing. But it is a complex trait, controlled by polygenes and easily influenced by the environment. Genetic analysis and QTL detection was carried out on six characteristics of grain chalkiness consisting of the percentage of chalkiness (PGC), white belly (PWB) and white core grains (PWC), and the area of chalkiness (CA), white belly (WBA) and white core (WCA) in brown rice. A total of 16 main-effect QTLs associated with chalkiness characteristics of brown rice were mapped on seven chromosomes over two years. Among them,qPGC7.1 andqPWB7.2 were simultaneously located on chromosome 7 flanked by 7038 and 7042 at LOD scores 4.34 and 3.76, whileqPWC2.1 andqWCA2.1 were simultaneously located on chromosome 2 flanked by RM492 and RM324 with LOD scores of 2.50 and 3.39. Twelve epistatic combinations were detected for five chalkiness characteristics except for CA. Results indicated that WBA was mainly influenced by the additive effects of main-effect QTLs. PGC and PWC were affected by the effects of epistatic QTLs and the interactions between additive-by-additive effects and the environment. The effects of epistatic QTLs and the main-effect QTLs played important roles on CA, PWB and WCA. For the genetic improvement of grain chalkiness in breeding system, more attention should be paid to epistatic effects and the additive effects of main-effect QTLs.     

QTLs and epistasis for aluminum tolerance in rice (Oryza sativa L.) at different seedling stages

To investigate the genetic background for aluminum (Al) tolerance in rice, a recombinant inbred (RI) population, derived from a cross between an Al-sensitive lowland indica rice variety IR1552 and an Al-tolerant upland japonica rice variety Azucena, was used in culture solution. A molecular linkage map, together with 104 amplified fragment length polymorphism (AFLP) markers and 103 restriction fragment length polymorphism (RFLP) markers, was constructed to map quantitative trait loci (QTLs) and epistatic loci for Al tolerance based on the segregation for relative root length (RRL) in the population. RRL was measured after stress for 2 and 4 weeks at a concentration of 1mM of Al³⁺ and a control with a pH 4.0, respectively. Two QTLs were detected at both the 2nd and the 4th weeks on chromosomes 1 and 12 from unconditional mapping, while the QTL on chromosome 1 was only detected at the 2nd stress week from conditional mapping. The effect of the QTL on chromosome 12 was increased with an increase of the stress period from 2 to 4 weeks. The QTL on chromosome 1 was expressed only at the earlier stress, but its contribution to tolerance was prolonged during growth. At least one different QTL was detected at the different stress periods. Mean comparisons between marker genotypic classes indicated that the positive alleles at the QTLs were from the Al-tolerant upland rice Azucena. An important heterozygous non-allelic interaction on Al tolerance was found. The results indicated that tolerance in the younger seedlings was predominantly controlled by an additive effect, while an epistatic effect was more important to the tolerance in older seedlings; additionally the detected QTLs may be multiple allelic loci for Al tolerance and phosphorus-uptake efficiency, or for Al and Fe²⁺ tolerance. Received: 29 July 1999 / Accepted: 13 October 1999     

QTL mapping of grain length in rice (Oryza sativa L.) using chromosome segment substitution lines

Chromosome segment substitution (CSS) lines have the potential for use in QTL fine mapping and map-based cloning. The standard t-test used in the idealized case that each CSS line has a single segment from the donor parent is not suitable for non-idealized CSS lines carrying several substituted segments from the donor parent. In this study, we present a likelihood ratio test based on stepwise regression (RSTEP-LRT) that can be used for QTL mapping in a population consisting of non-idealized CSS lines. Stepwise regression is used to select the most important segments for the trait of interest, and the likelihood ratio test is used to calculate the LOD score of each chromosome segment. This method is statistically equivalent to the standard t-test with idealized CSS lines. To further improve the power of QTL mapping, a method is proposed to decrease multicollinearity among markers (or chromosome segments). QTL mapping with an example CSS population in rice consisting of 65 non-idealized CSS lines and 82 chromosome segments indicated that a total of 18 segments on eight of the 12 rice chromosomes harboured QTLs affecting grain length under the LOD threshold of 2.5. Three major stable QTLs were detected in all eight environments. Some minor QTLs were not detected in all environments, but they could increase or decrease the grain length constantly. These minor genes are also useful in marker-assisted gene pyramiding.     

Drought-induced site-specific DNA methylation and its association with drought tolerance in rice (Oryza sativa L.)

An indica pyramiding line, DK151, and its recurrent parent, IR64, were evaluated under drought stress and non-stress conditions for three consecutive seasons. DK151 showed significantly improved tolerance to drought. The DNA methylation changes in DK151 and IR64 under drought stress and subsequent recovery were assessed using methylation-sensitive amplified polymorphism analysis. Our results indicate that drought-induced genome-wide DNA methylation changes accounted for ～12.1% of the total site-specific methylation differences in the rice genome. This drought-induced DNA methylation pattern showed three interesting properties. The most important one was its genotypic specificity reflected by large differences in the detected DNA methylation/demethylation sites between DK151 and IR64, which result from introgressed genomic fragments in DK151. Second, most drought-induced methylation/demethylation sites were of two major types distinguished by their reversibility, including 70% of the sites at which drought-induced epigenetic changes were reversed to their original status after recovery, and 29% of sites at which the drought-induced DNA demethylation/methylation changes remain even after recovery. Third, the drought-induced DNA methylation alteration showed a significant level of developmental and tissue specificity. Together, these properties are expected to have contributed greatly to rice response and adaptation to drought stress. Thus, induced epigenetic changes in rice genome can be considered as a very important regulatory mechanism for rice plants to adapt to drought and possibly other environmental stresses.     

Tagging and mapping the thermo-sensitive genic male-sterile gene in rice (Oryza sativa L.) with molecular markers

The thermo-sensititve genic male-sterile (TGMS) gene in rice can alter fertility in response to temperature and is useful in the two-line system of hybrid rice production. However, little is known about the TGMS gene at the molecular level. The objective of this study was to identify molecular markers tightly linked with the TGMS gene and to map the gene onto a specific rice chromosome. Bulked segregant analysis of an F₂ population from 5460s (a TGMS mutant line) x Hong Wan 52 was used to identify RAPD markers linked to the rice TGMS gene. Four hundred RAPD primers were screened for polymorphisms between the parents and between two bulks representing fertile and sterile plants; of these, 4 primers produced polymorphic products. Most of the polymorphic fragments contained repetitive sequences. Only one singlecopy sequence fragment was found, a 1.2-kb fragment amplified by primer OPB-19 and subsequently named TGMS1.2. TGMS1.2 was mapped on chromosome 8 with a RIL population and confirmed by remapping with a DHL population. Segregation analysis using TGMS1.2 as a probe indicated that TGMS1.2 both consegregated and was lined with the TGMS gene in this population. It is located about 6.7 cM from the TGMS gene. As TGMS1.2 is linked to the TGMS gene, the TGMS gene must be located on chromosome 8.This research was supported by the Rockefeller Foundation and China National High-Tech Research and Development Program. The first author is a Rockefeller Career Fellow at Texas Tech University     

Genetic analysis of salinity tolerance in rice (Oryza sativa L.)

Summary The genetics of salinity tolerance in rice was investigated by a nine-parent complete diallel including reciprocals. Test materials involved susceptible (IR28, IR29, and MI-48), moderately tolerant (IR4595-4-1-13, IR9884-54-3-1E-P1, and IR10206-29-2-1), and tolerant (Nona Bokra, Pokkali, and SR26B) parents. Twoweek-old seedlings were grown in a salinized (EC = 12 dS/m) culture solution for 19 days under controlled conditions in the IRRI phytotron. Typical characteristics of salinity tolerance in rice were found to be Na⁺ exclusion and an increased absorption of K⁺ to maintain a good Na-K balance in the shoot. Genetic component analysis (GCA) revealed that a low Na-K ratio is governed by both additive and dominance gene effects. The trait exhibited overdominance, and two groups of genes were detected. Environmental effects were large, and the heritability of the trait was low. Our findings suggest that when breeding for salt tolerance, selection must be done in a later generation and under controlled conditions in order to minimize environmental effects. Modified bulk and single-seed descent would be the suitable breeding methods. Combining ability analysis revealed that both GCA and specific combining ability (SCA) effects were important in the genetics of salt tolerance. Moderately tolerant parents — e.g., IR4595-4-1-13 and IR9884-54-3-1E-P1 — were the best general combiners. Most of the best combinations had susceptible parents crossed either to moderate or tolerant parents. The presence of reciprocal effects among crosses necessitates the use of susceptible parents as males in hybridization programs. Large heterotic effects suggest the potential of hybrid rice for salt-affected lands.     

Phosphorylation upon cold stress in rice (Oryza sativa L.) seedlings

The response of plants to cold stress is not well understood at the biochemical level, although it has been studied extensively at the ecological level. To investigate whether protein phosphorylation may play an important role in cold stress, we exposed rice seedlings to low temperatures, prepared protein extracts from the leaves and incubated these in the presence of [γ-³²P]ATP. The proteins were then separated by two-dimensional polyacrylamide gel electrophoresis. While several proteins were found to be phosphorylated upon cold stress one protein, pp35, which has an isoelectric point of 8.0, was more phosphorylated than the others. The pp35 protein was found to be phosphorylated when rice seedlings were incubated for 6 h at 5°C before the leaf protein extract was prepared and radioactive labeling was performed. The pp35 was, however, significantly more phosphorylated in cold-tolerant rice varieties. Antibodies were raised against purified pp35 in adult rabbits. Using this pp35 antibody, which can recognize the RuBisCO large-chain subunit (LSU), and from amino acid sequencing of pp35, we were able to identify and confirm the pp35 protein as the fragment of RuBisCO LSU (EC 4.1.1.39). Phosphorylation of the RuBisCO LSU may be important in cold tolerance. Received: 7 July 1998 / Accepted: 19 December 1998     

Biochemical and molecular mechanisms associated with Zn deficiency tolerance and signaling in rice (Oryza sativa L.)

In this study, zinc (Zn) deficiency caused a significant reduction in growth parameters and tissue Zn concentrations in BRRI 33 (sensitive) but not in Pokkali (tolerant). The increase of proton extrusion in both genotypes under high pH suggests that it gets triggered as a common consequence of reducing pH and solubilization of Zn. Real-time PCR showed pronounced upregulation of OsZIP4, OsDMAS1, OsNAS2 and OsPCS1 in Zn-deficient roots of Pokkali, and to a lesser extent in BRRI 33 only for OsZIP4 and OsPCS1. This suggests that OsDMAS1, OsNAS2 and OsPCS1 functions as secondary consequences leading to higher chelation and uptake of Zn under Zn deficiency in Pokkali. Further, a major increase in CAT, POD, SOD, GR and key metabolites suggests that high antioxidant defense plays a critical role in Zn deficiency tolerance in Pokkali. Further, Pokkali self-grafts and plants having Pokkali rootstock combined with BRRI 33 scion showed no significant decline in plant height, root dry matter and Zn concentration along with upregulation of Zn transporters (OsZIP4 and OsIRT1) under Zn deficiency, suggesting that signal driving mechanisms for Zn deficiency tolerance mechanisms are generated in the root and Zn-inefficient BRRI 33 is not capable of producing signals or sensing them.     

水稻微卫星标记的发展和应用

微卫星又称简单序列重复。它是由几个核苷酸（一般２～４个）为重复单位组成的串联重复序列。相同座位上的重复序列由于重复次数的不同而造成序列长度的多态性。微卫星标记是一种共显性标记,具有等位基因丰富、检测技术简单等优点。微卫星标记在基因组作图、品种鉴定、种质保存、分子标记辅助选择等方面有着广泛的应用。目前水稻中已发展了３００多个微卫星标记。     

Mapping of QTLs for phosphorus-deficiency tolerance in rice (Oryza sativa L.)

 Phosphorus (P) deficiency of soils is a major yield-limiting factor in rice production. Increasing the P-deficiency tolerance of rice cultivars may represent a more cost-effective solution than relying on fertilizer application. The objective of this study was to identify putative QTLs for P-deficiency tolerance in rice, using 98 backcross inbred lines derived from a japonica×indica cross and genotyped at 245 RFLP marker loci. Lines were grown on P-deficient soil and P uptake, internal P-use efficiency, dry weight, and tiller number were determined. Three QTLs were identified for dry weight and four QTLs for P uptake, together explaining 45.4% and 54.5% of the variation for the respective traits. Peaks for both traits were in good agreement which was to be expected considering the tight correlation of r=0.96 between dry weight and P uptake. For both traits the QTL linked to marker C443 on chromosome 12 had a major effect. Two of the three QTLs detected for internal P-use efficiency, including the major one on chromosome 12, coincided with QTLs for P uptake; however, whereas indica alleles increased P uptake they reduced P-use efficiency. We concluded that this was not due to the tight linkage of two genes in repulsion but rather due to an indirect effect of P uptake on P-use efficiency. Most lines with high use efficiency were characterized by very low P uptake and dry weight and apparently experienced extreme P-deficiency stress. Their higher P-use efficiency was thus the result of highly sub-optimal tissue-P concentrations and did not represent a positive adaptation to low P availability. The number of tillers produced under P deficiency is viewed as an indirect indicator of P-deficiency tolerance in rice. In addition to the major QTL on chromosome 12 already identified for all other traits, two QTLs on chromosome 4 and 12 were identified for tiller number. Their position, however, coincided with QTLs for tiller number reported elsewhere under P-sufficient conditions and therefore appear to be not related to P-deficiency tolerance. In this study P-deficiency tolerance was mainly caused by differences in P uptake and not in P-use efficiency. Using a trait indirectly related to P-deficiency tolerance such as tiller number, we detected a major QTL but none of the minor QTLs detected for P uptake or dry weight. Received: 9 February 1998 / Accepted: 29 April 1998     

Genetic mapping of hypervariable minisatellite sequences in rice (Oryza sativa L.)

Minisatellites, or DNA fingerprinting sequences, have been utilized in animal linkage studies for several years but have not been used as markers for plant genome mapping. In animal genome mapping they have resulted in limited success because they are evenly dispersed in some species but are often clustered near telomeric regions, as observed on human chromosomes. The purpose of the present study was to generate DNA fingerprints utilizing several rice-derived minisatellites containing different core sequences and numbers of repeat units, followed by assessing their potential for use as genetic markers when mapped to a rice recombinant inbred line (RIL) population. Sites of segregating minisatellite loci were mapped onto 11 of the 12 rice RIL linkage maps. The implications for the use of rice minisatellite core sequences as genetic markers on linkage maps in rice are discussed. Received: 1 March 1999 / Accepted: 22 June 1999     

Association mapping of stigma and spikelet characteristics in rice (Oryza sativa L.)

Stigma and spikelet characteristics play an essential role in hybrid seed production. A mini-core of 90 accessions developed from USDA rice core collection was phenotyped in field grown for nine traits of stigma and spikelet and genotyped with 109 DNA markers, 108 SSRs plus an indel. Three major clusters were built upon Rogers’ genetic distance, indicative of indicas, and temperate and tropical japonicas. A mixed linear model combining PC-matrix and K-matrix was adapted for mapping marker-trait associations. Resulting associations were adjusted using false discovery rate technique. We identified 34 marker-trait associations involving 22 SSR markers for eight traits. Four markers were associated with single stigma exsertion (SStgE), six with dual exsertion (DStgE) and five with total exsertion. RM5_Chr1 played major role indicative of high regression with not only DStgE but also SStgE. Four markers were associated with spikelet length, three with width and seven with L/W ratio. Numerous markers were co-associated with multiple traits that were phenotypically correlated, i.e. RM12521_Chr2 associated with all three correlated spikelet traits. The co-association should improve breeding efficiency because single marker could be used to assist breeding for multiple traits. Indica entry 1032 (cultivar 50638) and japonica entry 671 (cultivar Linia 84 Icar) with 80.65 and 75.17% of TStgE, respectively are recommended to breeder for improving stigma exsertion.     

Mapping QTLs for phosphorus deficiency tolerance in rice (Oryza sativa L.)

 The amplified fragment length polymorphism (AFLP) technique combined with selective genotyping was used to map quantitative trait loci (QTLs) associated with tolerance for phosphorus (P) deficiency in rice. P deficiency tolerant cultivar IR20 was crossed to IR55178-3B-9-3 (sensitive to P-deficiency) and 285 recombinant inbred lines (RILs) were produced by single-seed descent. The RILs were phenotyped for the trait by growing them in P-sufficient (10.0 mg/l) and P-deficient (0.5 mg/l) nutrient solution and determining their relative tillering ability at 28 days after seeding, and relative shoot dry weight and relative root dry weight at 42 days after seeding. Forty two of each of the extreme RILs (sensitive and tolerant) and the parents were subjected to AFLP analysis. A map consisting of 217 AFLP markers was constructed. Its length was 1371.8 cM with an average interval size of 7.62 cM. To assign linkage groups to chromosomes, 30 AFLP and 26 RFLP markers distributed over the 12 chromosomes were employed as anchor markers. Based on the constructed map, a major QTL for P-deficiency tolerance, designated PHO, was located on chromosome 12 and confirmed by RFLP markers RG9 and RG241 on the same chromosome. Several minor QTLs were mapped on chromosomes 1, 6, and 9. Received: 21 April 1998 / Accepted: 9 June 1998