A major QTL associated with cold tolerance at seedling stage in rice (Oryza sativa L.)

Chilling injury is one of the most important limiting factors affecting rice production in temperate and high-elevation areas. In this study, 146 microsatellite markers were employed to identify quantitative trait loci (QTL) conferring cold tolerance at seedling stage (CTS) .The mapping population consisted of 193 doubled haploid (DH) lines, which derived from a cross between a cold-tolerant japonica variety (AAV002863) and a cold-sensitive indica cultivar (Zhenshan97B). Tolerance to cold was assessed by the survival percentage of seedlings after cold treatment. In a climate chamber, after treatment at 6°C/10°C for 7 d, the measurement was taken on the sixth day of the recovery stage at room temperature. The phenotypic distribution of the DH population approximately fitted normality with skewness and kurtosis less than 0.3, and the difference among the three repetitions was not significant. Five main effect QTLs were identified with LOD > 4.0 on chromosomes 1, 2, 8 using a composite interval mapping approach. The accumulated contribution of the five QTLs was 62.28%, and a major QTL (LOD = 15.09) was identified on chromosome 2 flanked by RM561 and RM341, which explained 27.42% of the total phenotypic variation. Four significant epistatic interactions were also detected with a total contribution of 20.14%. Liang Chen and Qiaojun Lou had made the equal contribution for the research.     

基于高密度SNP遗传图谱的粳稻芽期耐低温QTL鉴定

水稻直播由于省时、省工和节约成本而备受农户关注。然而,芽期耐冷性不强致使现行推广的许多优良水稻品种不适于直播生产。因此,挖掘鉴定芽期耐冷位点,为后续的辅助育种提供基因资源就日益受到重视。本研究利用丽江新团黑谷和沈农265构建的重组自交系群体及其重测序构建的包含2818个bin标记的遗传图谱对水稻芽期的耐冷性进行QTL定位分析。共检测到5个芽期耐冷QTL,分布在水稻的1号、3号、9号和11号染色体上,增效等位基因均来自耐冷亲本丽江新团黑谷。这些QTL的LOD值的范围从3.05到24.01,表型贡献率为8.0%～53.5%。其中,表型贡献率最大的主效QTL是qCTB11b,位于11号染色体长臂端的21.24 Mb～22.03 Mb之间,物理图谱区间为790 kb。随后利用"选择作图"的策略进行了QTL验证和累加效应分析,明确了可以通过QTL的累加聚合实现芽期耐冷能力的遗传改良,聚合的增效QTL越多,耐冷能力提升越明显。上述研究结果不仅可以增强人们对芽期水稻耐冷能力遗传基础的认识和理解,也可以为后续直播品种的遗传改良提供理论依据和技术指导。     

Genetic divergence among cold tolerant rices (Oryza sativa L.)

Summary Genetic divergence was investigated among 17 rice varieties known to possess some degree of cold tolerance at different growth stages. The 17 varieties and their 102 F₁ hybrids with 6 male testers were studied for isozyme variation at 15 loci, spikelet fertility, and degree of cold tolerance at various stages. Multivariate analyses of the data provided several schemes of divergence based on various sources of evidence. All schemes gave similar results, and separated the varieties into japonica and indica groups. The japonica group displayed specific isozymes, a low F₁ fertility with indica testers, and a high degree of cold tolerance which was expressed in the F₁ progenies. The indica group displayed contrasting specific isozymes, a high F₁ fertility with indica testers, and a moderate to low degree of cold tolerance which was not expressed in the F₁ progenies. One variety, ARC 6000, displayed unique traits in most schemes and was classified into a distinct type based on the isozymes. The results emphasize that cold tolerance is a major trait for classification of rice into two varietal groups and that proper characterization of potential donors is essential in breeding. Isozyme studies are useful tools for this purpose.     

Marker‐assisted breeding for improvement of anaerobic germination in japonica rice (Oryza sativa)

Direct seeding is increasingly being practiced in rice cultivation areas because it saves labour and reduces cost of production and management. In this study, four japonica‐type breeding lines were developed from a cross of a japonica cultivar and an AG‐tolerant donor, KHO, using marker‐assisted selection. Validation of the target genes AG1 and AG2 in the selected lines was examined by PCR analysis and the background recovery rate by the recurrent parent was confirmed by genotyping using a 6K SNP chip. Compared to the recurrent, the survival rates of the selected lines were improved in a range from 33% to 115% in three conditions; in particular, the survival rate was increased in more than twice that of the recurrent parent under the hypoxic condition. The main agronomic trait, yield and quality‐related traits were similar to those of the recurrent parent. The use of these lines will contribute to the expansion of the cultivation area due to the proven economic benefits of direct seeding in Korea.     

Marker-assisted introgression of QTLs for yield under moisture stress into elite varieties of rice (Oryza sativa)

Rice production is severely constrained by the moisture stress. The present study was undertaken to transfer two important QTLs viz., (qDTY2.2 and qDTY4.1), which controls yield under moisture stress (DTY) into two elite varieties, that is, MTU1010 and NLR34449 through marker-assisted breeding. Foreground and background selections of backcross generations, that is, BC₁F₁, BC₂F₁ and BC₂F₂ identified several promising introgression lines (ILs) with two QTLs and single QTLs with an appreciable level of recovery of recipient parent genome. In ILs, the recovery of MTU1010 genome content was estimated to be 83%–93% while the recovery of NLR34449 was 84%–92%. The two-QTL ILs of MTU1010 and NLR34449 backgrounds (qDTY2.2 and qDTY4.1) have shown substantial yield advantage (32 to 84%) over the single-QTL ILs (either qDTY2.2 or qDTY4.1) under moisture stress conditions. Among all, two ILs, MBC-124 and NBC-127 are the best high yielding lines under moisture stress conditions. These outyielded ILs have the potential to be released as varieties in rainfed ecosystem and also can be used as donors in the existing breeding programme in rice.     

Genetic analysis of salt tolerance at seedling and reproductive stages in rice (Oryza sativa)

Salinity is a major abiotic stress that limits rice production across rice areas as high‐yielding modern rice varieties are generally sensitive to salt stress. The study was conducted to deduce heritability and combining ability estimates of rice for various morphological and physiological traits using a 7 × 7 full‐diallel‐cross analysis at seedling and reproductive stages. The salinity stress treatment was 12 dS m^?1 at the seedling stage and 8 dS m^?1 at the reproductive stage. Diallel analysis revealed high for salinity tolerance scores and shoot height, moderate for shoot dry weight and root dry weight and low for Na⁺ and K⁺ concentrations and K⁺/Na⁺ ratio. The low‐to‐moderate narrow‐sense heritability for number of panicles, number of fertile spikelets, grain weight, spikelet fertility and K⁺/Na⁺ ratio suggests a large breeding population and delayed selection for tolerance until later generations. Significant maternal effects indicate that selection of the female parent is very important for desired trait development. The results of this study confirmed that salinity tolerance at the seedling and reproductive stages is regulated by a different set of genes that could be pyramided using different donors to enhance the level of tolerance.     

Reinventing heterosis phenomenon through deployment of alien introgression lines in rice (Oryza sativa L.)

Introgression Lines (ILs) carrying alien genomic segments in the homozygous state may or may not be able to contribute positively to the phenotype on account of replacement of cultivated genome segment, however, the genetic elite in heterozygous condition can be attributed to the presence of both recipient and donor genome complements. Therefore, overdominance or pseudo‐overdominance effect at the heterozygous loci is anticipated. Set of 318 ILs, carrying several genomic segments from “A” genome donor wild species, were used for developing test hybrids with CMS line PMS17A. Of these, three hybrids observed significant yield advantage (>25%) over recurrent parents and checks. Parental ILs of these hybrids viz., IL326, IL901 and IL951 carried 5.62%, 2.52% introgression from Oryza rufipogon and 6.71% from Oryza nivara, respectively. These ILs were also crossed with their recurrent parent to develop introgression line hybrids (ILHs) to observe the contribution of the alien segment(s), in the homozygous and heterozygous state. The traits studied have higher mean value when genomic segments from wild species were in the heterozygous state as compared to the homozygous state.     

Wide compatibility in rice (Oryza sativa L.)

Summary Wide compatible varieties (WCVs) show normal spikelet fertility in crosses with Indica and Japonica rice varieties. Crosses of Indica and Japonica varieties frequently show high spikelet sterility which prevents exploitation of heterosis for grain yield. We screened 41 rice varieties for the wide compatibility trait by crossing each with three Indica and three Japonica testers. Varieties giving fertile F₁ hybrids with both groups of testers were classified as WCVs. Seven varieties viz., BPI-76 (Indica); N 22; Lambayeque-1 and Dular (Aus); Moroberekan, Palawan and Fossa HV (Japonicas), were identified as WCVs. The frequency of WCVs was higher among Aus and Japonicas. The wide compatibility trait in varieties: Dular and Moroberekan was controlled by a single dominant gene linked with the Est-2 and Amp-3 loci (mean recombination 32.0%). Est-2 and Amp-3 showed complete linkage. Pgi-2 was found to be linked with Est-2 and Amp-3 (mean recombination 16.1%). Est-2 and Amp-3, showed a tighter linkage with C ⁺ (mean recombination 4.1%). Pgi-2 showed a lower linkage with C ⁺ (mean recombination 17.3%). The recombination values between the WC gene in Dular and C ⁺ was much higher than those reported in Japan for the WC gene (S₅ ⁿ) from Ketan Nangka. It is possible that the WC gene from Dular is different from that in Ketan Nangka. Linkage intensities with the WC gene were not strong enough to be of use for indirect selection for the wide compatibility trait. A search for a more closely linked isozyme or DNA marker was proposed.     

水稻孕穗期耐热性QTLs分析

水稻籼粳亚种间杂种优势利用是提高水稻产量的重要途径。然而，异常高温或低温导致籼粳亚种间杂种育性下降是影响其优势利用的主要因素之一。本研究以USSR5（粳稻）/广解9号（籼稻）//USSR5回交群体为供试材料，构建了相应的分子连锁图谱，分别以高温处理下直接小穗育性及小穗育性热敏感指数为指标，对水稻孕穗期高温耐热性及其相对耐热性进行数量性状位点（QTLs）分析。结果表明，在第2、4和5染色体上检测到孕穗期耐热性相关的QTL各一个，对表型变异的解释率为6.4%~15.8%；在第4、8染色体上分别检测到与孕穗期相对耐热性相关的QTL，qhts-4和qhts-8，LOD值分别为3.81和2.86，对表型变异的解释率分别为16.8%和9.9%。对其进一步的上位性分析表明，有8条染色体的4对位点存在基因间互作，小穗育性耐热性除受主效QTL控制外，还受基因间互作及修饰基因的影响。     

Genetic variation in root morphology and microsatellite DNA loci in upland rice (Oryza sativa L.) from Vietnam

Drought is a major constraint to the productivity of rice in upland ecosystems. The rice root system plays an important role in the regulation of water uptake and extraction from deep soil layers. The aim of this research was to study the variation in root morphology and the genetic diversity in upland rice accessions. Thirty-three upland rice accessions originated from Vietnam along with 13 selected upland rice lines from several other countries were used in this study. Variation in root morphology was observed in most of the investigated root traits such as maximum root length, total root dry weight, deep root to shoot ratio, and total root to shoot ratio. Most of the traits showed significant correlation and appeared interrelated. Genetic diversity among upland rice accessions was studied with microsatellite markers. Forty-one alleles were detected with 14 rice microsatellite primer pairs among all the rice accessions. Two dendrograms have been created based on 35 microsatellite alleles and 10 morphological traits data for 38 accessions and compared. These results provided useful information for the selection of suitable cross combinations for mapping quantitative trait loci (QTLs) related to drought resistance in upland rice.     

Genetic Variation for Salinity Tolerance in Pakistani Rice (Oryza sativa L.) Germplasm

Soil salinity is one of the major production constraints. Development and planting of salt‐tolerant varieties can reduce yield losses due to salinity. We screened 185 rice genotypes at germination stage in petri dishes under control, 50, 100 and 150 mm salt stress, and at seedling stage in Yoshida's hydroponic nutrient solution under control, 50 and 100 mm salt stress. At germination stage, 15 genotypes including Nona Bokra, Sonahri Kangni, 7421, 7423 and 7467, whereas at seedling stage, 28 genotypes including Nona Bokra, Jajai‐77, KSK‐133, KSK‐282, Fakhr‐e‐Malakand, Pakhal, IR‐6, Khushboo‐95, Shahkar and Shua‐92 were found salt tolerant. Basmati‐370, Mushkan, Homo‐46 and accessions 7436, 7437 and 7720 were sensitive to salinity at both germination and seedling stage. We further screened a subset of 33 salt‐tolerant and salt‐sensitive genotypes with SSR markers. Four SSR markers (RM19, RM171, RM172 and RM189) showed significant association with two or more of the studied traits under 50, 100 and 150 mm salt stress. These markers may be further tested for their potential in marker‐assisted selection. The salt‐tolerant genotypes identified in this study may prove useful in the development of salt‐tolerant rice varieties in adapted genetic background.     

Genetic characteristics of Korean weedy rice (Oryza sativa L.) by RFLP analysis

Summary The genetic characteristics and classification of 24 strains of Korean weedy rice, two strains of foreign red rice, three Japonica cultivars, one Tongil cultivar and one Indica cultivar (Oryza sativa L.) were investigated at the DNA level using the restriction fragment length polymorphism (RFLP) method. Eighty-three random combinations between six restriction enzymes and forty genomic DNA probes (RG# and KR#) were assayed. Thirty-seven (92.5%) out of the forty probes used showed polymorphisms among the 31 accessions assayed. A high level of polymorphism was found between short and long grain type Korean weedy rices, whereas fewer polymorphisms were presented among strains within each grain type. A dendrogram summarizing genetic similarity coefficients among thirty-one accessions was constructed based on their DNA polymorphisms. The Korean weedy rice strains were classified into two groups identical to the short and long grain types classified by morphological and physiological characters. From the RFLP analysis, it was deduced that the short grain strains of Korean weedy rice belonged to Japonica, while the long grain strains were closer to Indica than to Japonica, and were differentiated into a local ecotype surviving in the growth conditions in the southern part of the Korean peninsula.     

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.     

Random amplified polymorphic DNA analysis of Australian rice (Oryza sativa L.) varieties

Summary The genetic relationships between rice varieties were analysed by using the polymerase chain reaction (PCR), with arbitrary oligonucleotide primers in the random amplified polymorphic DNA (RAPD) method. PCR with 22 arbitrary primers applied to 37 varieties produced 144 useful markers, of which 67% were polymorphic. Thus, with selected primers sufficient polymorphism could be detected to allow identification of individual varieties. Visual examination of electrophoresis gels and analysis of banding patterns confirmed that commercial Australian and USA lines and their relatives were very closely related, with similarity indices of 88–97%. Three varieties originating from more distant geographical centres were easily distinguished, producing variety-specific amplification profiles and expressing a lower similarity index of 80% to all other varieties tested. PCR offers a potentially simple, rapid and reliable method for rice genotype identification and recognition of lines that could contribute genetic diversity to new commercial varieties.Abbreviations PCR Polymerase Chain Reaction - RAPD Random Amplified Polymorphic DNA     

Genetic variation of germination cold tolerance in Japanese rice germplasm

Low temperatures at the initial stages of rice development prevent fast germination and seedling establishment and may cause significant productivity losses. In order to develop rice cultivars exhibiting cold tolerance, it is necessary to investigate genetic resources, providing basic knowledge to allow the introduction of genes involved in low temperature germination ability from accessions into elite cultivars. Japanese rice accessions were evaluated at the germination under two conditions: 13°C for 28 days (cold stress) and 28°C for seven days (optimal temperature). The traits studied were coleoptile and radicle length under optimal temperature, coleoptile and radicle length under cold and percentage of the reduction in coleptile and radicle length due to low temperature. Among the accessions studied, genetic variation for traits related to germination under low temperatures was observed and accessions exhibiting adequate performance for all investigated traits were identified. The use of multivariate analysis allowed the identification of the genotypes displaying cold tolerance by smaller reductions in coleoptile and radicle lenght in the presence of cold and high vigour, by higher coleoptile and radicle growth under cold.     

Genetic progress of upland rice (Oryza sativa L.) lines for disease resistance

The rice crop is affected by diseases throughout its cycle, impacting negatively on grain yield and quality. The control of the disease impact can be accomplished via crop breeding, using highly multiple resistant genotypes. This study aimed to evaluate the efficiency of multiple-character and specific selection of multiple resistance to major culture-associated diseases (neck blast, leaf scald and grain discoloration) in rice lines of the Upland Rice Genetic Breeding Program. The experiments were conducted in 35 sites during 12 agricultural years, where 124 lines were evaluated for the severity of fungal diseases, under natural field conditions. Multiple parameters were calculated based on the diseases´ scores: genetic, phenotypic and environmental variances, heritability, selection gain, renewal rate, and genetic and renewal progress. Genetic variance for the disease resistance was identified in the population, and the selection gain for multiple-character selection was of 3.16 year⁻¹ throughout the breeding process with a renewal rate of over 35%. The programme has showed efficiency in selecting multiple resistant genotypes to the mentioned diseases, highlighting genotypes with high potential for market release.     

Anther culture of tropical japonica × indica hybrids of rice (Oryza sativa L.)

Summary Nine japonica × indica F1 hybrids of rice involving 6 indica and 3 japonica tropical varieties, were large scale anther cultured. The frequency of callusing anthers averaged 18.7%. The microspore-derived calli produced green plants with a mean frequency of 8.7%. Albino plants represented 61% of the shoot forming calli. Monitoring of the green and albino plant regenerating capabilities of calli arising between week 4 and week 8 of incubation of the anthers showed no increase of the albino/green ratio and a slow decrease of the shoot forming ability of the transferred calli after the sixth week of culture. Spontaneous doubled haploids (SDH) represented 46% of the regenerated green plants in 4 hybrids. However, a high frequency of partially sterile regenerants was noticed among 132 SDH plants generated from a hybrid.     

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.