A quantitative trait locus for cold tolerance at the booting stage on rice chromosome 8

A quantitative trait locus (QTL) for cold tolerance at the booting stage of a cold-tolerant rice breeding line, Hokkai-PL9, was analyzed. A total of 487 simple sequence repeat (SSR) markers distributed throughout the genome were used to survey for polymorphism between Hokkai-PL9 and a cold-sensitive breeding line, Hokkai287, and 54 markers were polymorphic. Single marker analysis revealed that markers on chromosome 8 are associated with cold tolerance. By interval mapping using an F₂ population between Hokkai-PL9 and Hokkai287, a QTL for cold tolerance was detected on the short arm of chromosome 8. The QTL explains 26.6% of the phenotypic variance, and its additive effect is 11.4%. Substitution mapping suggested that the QTL is located in a 193-kb interval between SSR markers RM5647 and PLA61. We tentatively designated the QTL as qCTB8 (quantitative trait locus for cold tolerance at the booting stage on chromosome 8).     

A point mutation in LTT1 enhances cold tolerance at the booting stage in rice

The cold tolerance of rice at the booting stage is a main factor determining sustainability and regional adaptability. However, relatively few cold tolerance genes have been identified that can be effectively used in breeding programmes. Here, we show that a point mutation in the low-temperature tolerance 1 (LTT1) gene improves cold tolerance by maintaining tapetum degradation and pollen development, by activation of systems that metabolize reactive oxygen species (ROS). Cold-induced ROS accumulation is therefore prevented in the anthers of the ltt1 mutants allowing correct development. In contrast, exposure to cold stress dramatically increases ROS accumulation in the wild type anthers, together with the expression of genes encoding proteins associated with programmed cell death and with the accelerated degradation of the tapetum that ultimately leads to pollen abortion. These results demonstrate that appropriate ROS management is critical for the cold tolerance of rice at the booting stage. Hence, the ltt1 mutation can significantly improve the seed setting ability of cold-sensitive rice varieties under low-temperature stress conditions, with little yield penalty under optimal temperature conditions. This study highlights the importance of a valuable genetic resource that may be applied in rice breeding programmes to enhance cold tolerance.     

Revision of the relationship between anther morphology and pollen sterility by cold stress at the booting stage in rice

Background and AimsCold stress in rice (Oryza sativa) plants at the reproductive stage prevents normal anther development and causes pollen sterility. Tapetum hypertrophy in anthers has been associated with pollen sterility in response to cold at the booting stage. Here, we re-examined whether the relationships between anther abnormality and pollen sterility caused by cold stress at the booting stage in rice can be explained by a monovalent factor such as tapetum hypertrophy.MethodsAfter exposing plants to a 4-d cold treatment at the booting stage, we collected and processed anthers for transverse sectioning immediately and at the flowering stage. We anatomically evaluated the effect of cold treatment on anther internal morphologies, pollen fertilities and pollen numbers in the 13 cultivars with various cold sensitivities.Key ResultsWe observed four types of morphological anther abnormalities at each stage. Pollen sterility was positively correlated with the frequency of undeveloped locules, but not with tapetum hypertrophy as commonly believed. In cold-sensitive cultivars grown at low temperatures, pollen sterility was more frequent than anther morphological abnormalities, and some lines showed remarkably high pollen sterility without any anther morphological alterations. Most morphological anomalies occurred only in specific areas within large and small locules. Anther length tended to shorten in response to cold treatment and was positively correlated with pollen number. One cultivar showed a considerably reduced pollen number, but fertile pollen grains under cold stress. We propose three possible relationships to explain anther structure and pollen sterility and reduction due to cold stress.ConclusionsThe pollen sterility caused by cold stress at the booting stage was correlated with the frequency of entire locule-related abnormalities, which might represent a phenotypic consequence, but not a direct cause of pollen abortion. Multivalent factors might underlie the complicated relationships between anther abnormality and pollen sterility in rice.     

粳稻孕穗期耐冷性NILs的形态评价和分子验证

2004年在海拔1916m昆明两种冷害（水温19.5±0.7℃,低田温冷泉水温17.8±1.1℃)、阿子营冷害(海拔2150m,水温18.2±0.22℃)条件下对5个亲本及其25个近等基因系进行耐冷性鉴定,用Statistica对17个农艺性状进行形态聚类和SSR分子标记聚类分析。结果表明：(1)已培育的穗期耐冷性NILs与轮回亲本十和田的形态极为相似,但与耐冷性相关的性状(穗颈长、实粒数、结实率、花药长和花药体积)有明显的差异;(2)从78个SSR标记筛选出了7个标记在十和田和NILs间存在多态性, 其中RM7452标记与耐冷基因连锁,各个近等基因系间遗传背景相似,但与十和田耐冷性差异大,证明了这些NILs是水稻穗期耐冷基因精细定位和克隆的理想材料。     

Mapping of QTLs associated with cold tolerance during the vegetative stage in rice

Low-temperature stress is an important factor affecting the growth and development of rice (Oryza sativa L.) in temperate and high-elevation areas. Cold stress may cause various seedling injuries, delayed heading and yield reduction due to spikelet sterility. In this study, 181 microsatellite marker loci were used to identify quantitative trait loci (QTLs) associated with cold tolerance at the vegetative stage in 191 recombinant inbred lines (RILs) derived from a cross of a cold-tolerant temperate japonica cultivar (M-202) with a cold-sensitive indica cultivar (IR50). Different temperature regimes were applied in growth chambers on 191 RILs. The temperature regimes imposed in the growth chamber simulated cold-stress injuries at the seedling and late vegetative stages. In this study a major QTL was identified on chromosome 12, designated as qCTS12a, that was closely associated with cold-induced necrosis and wilting tolerance, and accounted for 41% of the phenotypic variation. A number of QTLs with smaller effects were also detected on eight rice chromosomes.     

Fine mapping a QTL qCTB7 for cold tolerance at the booting stage on rice chromosome 7 using a near-isogenic line

Low temperature at the booting stage is a serious abiotic stress in rice, and cold tolerance is a complex trait controlled by many quantitative trait loci (QTL). A QTL for cold tolerance at the booting stage in cold-tolerant near-isogenic rice line ZL1929-4 was analyzed. A total of 647 simple sequence repeat (SSR) markers distributed across 12 chromosomes were used to survey for polymorphisms between ZL1929-4 and the cold-sensitive japonica cultivar Towada, and nine were polymorphic. Single marker analysis revealed that markers on chromosome 7 were associated with cold tolerance. By interval mapping using an F₂ population from ZL1929-4 × Towada, a QTL for cold tolerance was detected on the long arm of chromosome 7. The QTL explained 9 and 21% of the phenotypic variances in the F₂ and F₃ generations, respectively. Recombinant plants were screened for two flanking markers, RM182 and RM1132, in an F₂ population with 2,810 plants. Two-step substitution mapping suggested that the QTL was located in a 92-kb interval between markers RI02905 and RM21862. This interval was present in BAC clone AP003804. We designated the QTL as qCTB7 (quantitative trait locus for cold tolerance at the booting stage on chromosome 7), and identified 12 putative candidate genes.     

Mapping QTLs of root morphological traits at different growth stages in rice

Roots are a vital organ for absorbing soil moisture and nutrients and influence drought resistance. The identification of quantitative trait loci (QTLs) with molecular markers may allow the estimation of parameters of genetic architecture and improve root traits by molecular marker-assisted selection (MAS). A mapping population of 120 recombinant inbred lines (RILs) derived from a cross between japonica upland rice 'IRAT109' and paddy rice 'Yuefu' was used for mapping QTLs of developmental root traits. All plant material was grown in PVC-pipe. Basal root thickness (BRT), root number (RN), maximum root length (MRL), root fresh weight (RFW), root dry weight (RDW) and root volume (RV) were phenotyped at the seedling (I), tillering (II), heading (III), grain filling (IV) and mature (V) stages, respectively. Phenotypic correlations showed that BRT was positively correlated to MRL at the majority of stages, but not correlated with RN. MRL was not correlated to RN except at the seedling stage. BRT, MRL and RN were positively correlated to RFW, RDW and RV at all growth stages. QTL analysis was performed using QTLMapper 1.6 to partition the genetic components into additive-effect QTLs, epistatic QTLs and QTL-by-year interactions (Q x E) effect. The results indicated that the additive effects played a major role for BRT, RN and MRL, while for RFW, RDW and RV the epistatic effects showed an important action and Q x E effect also played important roles in controlling root traits. A total of 84 additive-effect QTLs and 86 pairs of epistatic QTLs were detected for the six root traits at five stages. Only 12 additive QTLs were expressed in at least two stages. This indicated that the majority of QTLs were developmental stage specific. Two main effect QTLs, brt9a and brt9b, were detected at the heading stage and explained 19% and 10% of the total phenotypic variation in BRT without any influence from the environment. These QTLs can be used in breeding programs for improving root traits.     

Enhanced chilling tolerance at the booting stage in rice by transgenic overexpression of the ascorbate peroxidase gene, OsAPXa

Low temperatures during the booting stage reduce rice yields by causing cold-induced male sterility. To determine whether antioxidant capacity affects the ability of rice plants to withstand chilling at the booting stage, we produced transgenic rice plants that overexpress OsAPXa and have increased APX activity. The effect of increased APX activity on the levels of H₂O₂ and lipid peroxidation were determined by measuring H₂O₂ levels and malondialdehyde (MDA) contents in spikelets during cold treatments at the booting stage. The levels of H₂O₂ and the MDA content increased by 1.5-fold and twofold, respectively in WT plants subjected to a 12°C treatment for 6 days. In contrast, transgenic lines showed small changes in H₂O₂ levels and MDA content under cold stress, and H₂O₂ levels and MDA content were significantly lower than in WT plants. APX activity showed negative correlations with levels of H₂O₂ and MDA content, which increased during cold treatment. Cold tolerance at the booting stage in transgenic lines and WT plants was evaluated. Spikelet fertility was significantly higher in transgenic lines than in WT plants after a 12°C treatment for 6 days. These results indicate that higher APX activity enhances H₂O₂-scavenging capacity and protects spikelets from lipid peroxidation, thereby increasing spikelet fertility under cold stress.     

Identification and analysis of QTLs controlling cold tolerance at the reproductive stage and validation of effective QTLs in cold-tolerant genotypes of rice (Oryza sativa L.)

Low temperature or cold stress is one of the major constraints of rice production and productivity in temperate rice-growing countries and high-altitude areas in the tropics. Even though low temperature affects the rice plant in all stages of growth, the percent seed set is damaged severely by cold and this reduces the yield potential of cultivars significantly. In this study, a new source of cold-tolerant line, IR66160-121-4-4-2, was used as a donor parent with a cold-sensitive cultivar, Geumobyeo, to produce 153 F₈ recombinant inbred lines (RILs) for quantitative trait locus (QTL) analysis. QTL analysis with 175 polymorphic simple sequence repeat (SSR) markers and composite interval mapping identified three main-effect QTLs (qPSST-3, qPSST-7, and qPSST-9) on chromosomes 3, 7, and 9. The SSR markers RM569, RM1377, and RM24545 were linked to the identified QTLs for cold tolerance with respect to percent seed set using cold-water (18–19°C) irrigation in the field and controlled air temperature (17°C) in the greenhouse. The total phenotypic variation for cold tolerance contributed by the three QTLs was 27.4%. RILs with high percent seed set under cold stress were validated with linked DNA markers and by haplotype analysis that revealed the contribution of progenitor genomes from the tropical japonica cultivar Jimbrug (Javanica) and temperate japonica cultivar Shen-Nung89-366. Three QTLs contributed by the cold-tolerant parent were identified which showed additive effect on percent seed set under cold treatment. This study demonstrated the utility of a new phenotyping method as well as the identification of SSR markers associated with QTLs for selection of cold-tolerant genotypes to improve temperate rice production.     

Mapping quantitative trait loci for heat tolerance at the booting stage using chromosomal segment substitution lines in rice

High temperature stress is a major obstacle in rice productivity. Considerable progress has been made on studying heat tolerance (HT) at different stages. However, the genetic basis of HT at the booting stage is poorly understood. In this study, we analyzed the morphological features of a heat-sensitive japonica cultivar Sasanishiki under natural high temperature stress at the booting stage. The anthers became smaller and the number, and fertility, of pollen grains were decreased significantly. As a result, there was a dramatic reduction in spikelet fertility. In contrast, the indica cultivar Habataki showed high HT and normal spikelet fertility under high temperature stress. Additonally, a set of chromosome segment substitution lines, derived from Sasanishiki and Habataki, were evaluated for HT related quantitative trait loci (QTLs) across two environments in the natural field. A total of 12 QTLs associated with HT were detected, of which, 5 were identified in two environments, and 7 in one environment. Furthermore, one of the major-effect QTLs (qHTB3-3) detected on the long arm of chromosome 3, was confirmed using overlapping substituted lines. qHTB3-3 was finally mapped between the two markers RM3525 and 3-M95, approximately 2.8 Mb apart. These findings and further gene cloning of qHTB3-3 will help us better understand the molecular control of HT in rice, and may contribute to the development of high HT rice varieties.     

黑龙江省水稻空壳率与孕穗期低温的关系

对黑龙江省6个主要水稻品种(龙稻3号、垦稻12号、空育131、龙稻7号、龙粳16号和松粳6号)进行孕穗期低温(处理温度分别为15℃、17℃、19℃,低温持续时间分别为2、4、6和8d)处理,采用线性内插和统计回归方法,分析了水稻单穗空壳率与孕穗期低温的关系.结果表明:研究区敏感性水稻品种和耐冷性较强水稻品种的障碍型冷害临界温度分别为17℃和16℃;孕穗期水稻对低温最敏感的时期为抽穗前14～18d.15℃低温处理8d时,松粳6号、垦稻12号的空壳率明显增加,空育131空壳率小幅升高,表明松粳6号和垦稻12号对低温反应较敏感、耐冷性较弱,而空育131对低温反应迟钝,耐冷性较强;垦稻12号、龙粳16号、空育131的冷积温与空壳率存在显著的相关关系(P0.01),随着冷积温的增加,水稻空壳率明显升高,但品种间的增幅不同.     

三种酰胺类新农药对水稻孕穗期稻纵卷叶螟的防效试验

单季杂交晚稻孕穗期稻纵卷叶螟Cnaphalocrocis medinalis Guenée发生不整齐,为害重,在主治药剂氟虫腈即将禁用之际,急需防治高龄幼虫的长效药剂,酰胺类农药是满足这一条件的新一代农药。3种酰胺类农药试验结果,在单季稻孕穗期防治稻纵卷叶螟,20%氟虫双酰胺(WDG)150g/hm2处理速效性与持效性表现最好,药后3d防效达90.3%,药后15d防效高达96.2%;20%氯虫苯甲酰胺(SC)150mL/hm2处理速效性略低于氟虫双酰胺,持效接近,药后3d、15d防效分别达75.2%、91.2%;40%氯虫·噻虫嗪(WDG)120mL/hm2处理速效介于氟虫双酰胺和氯虫苯甲酰胺之间,持效略低于前二者,药后3d、15d防效分别为83.2%、88.3%。对照药剂5%氟虫腈(SC)750mL/hm2和90%杀虫单3000g/hm2处理药后3d、15d的防效均低于上述3种酰胺类新药剂。保叶效果以氯虫苯甲酰胺、氟虫双酰胺处理最高,药后15d保叶率分别为83.6%和85.0%,二者无显著差异,其次为氯虫·噻虫嗪处理为59.6%,3种酰胺类新药剂保叶效果均显著高于对照药剂氟虫腈和杀虫单。结果表明,3种酰胺类农药孕穗期防治稻纵卷叶螟的药效、持效性和保叶效果均高于当前主治药剂氟虫腈、杀虫单,可在生产上推广应用。     

杂交粳稻花优14及其亲本孕穗期剑叶的基因表达谱芯片分析

本文以孕穗期的杂交粳稻花优14及其母本申9A和父本繁14的剑叶为材料,利用Affymetrix水稻基因组芯片检测了3个样品中的基因表达谱,并用生物信息学方法对差异表达基因进行了分析。结果表明：与其亲本相比,杂交粳稻花优14中共有2057个基因的表达水平出现了2倍(变化倍数≥2或≤0.5)以上的变化。通过对这些差异表达基因进行GO(Gene Ontology)功能分类,发现差异表达基因在光合系统Ⅰ、叶绿体膜和叶绿体被膜等与叶绿体相关的细胞组分中显著富集;同时差异表达基因还在叶绿素合成、叶绿素代谢和类胡萝卜素合成等生物学过程中富集。光合作用效率的改变可能和花优14杂种优势的形成相关。与已报道结果不同,本文在代谢途径分析结果中并没有发现花优14中差异表达基因在碳固定和光合作用等途径中显著的富集,但是发现差异表达基因在光合作用-天线蛋白以及淀粉和蔗糖的代谢途径中显著富集。该结果表明,在不同的杂交组合中,参与杂种优势形成的基因或者代谢途径可能是不同的。     

普通野生稻中增产相关QTL的发掘

普通野生稻(Oryza Rufipogon)是重要的遗传资源,发掘其优良等位基因将对水稻遗传改良产生重要影响。文章从以珍汕97为轮回亲本,普通野生稻为供体的BC2F1群体中选择一个与珍汕97表型明显不同的单株BC2F1-15,经过连续自交获得回交重组自交系BC2F5群体。均匀分布于12条染色体的126个多态性SSR(Simplesequence repeats)标记基因型分析,发现BC2F1-15单株在30%的标记位点为杂合基因型;利用该群体共检测到4个抽穗期、3个株高、4个每穗颖花数、2个千粒重和1个单株产量QTL。在第7染色体RM481-RM2区间,检测到抽穗期、每穗颖花数和产量QTL,野生稻等位基因表现增效作用;其他3个每穗颖花数QTL位点,野生稻等位基因也均具有增效作用。结果表明野生稻携带有增产相关的等位基因,这些有利等位基因无疑是水稻遗传改良可资利用的新资源。     

孕穗期低温对黑龙江省主栽水稻品种空壳率的影响

障碍型冷害对黑龙江省水稻生产影响巨大,明确主栽水稻品种耐冷性及障碍型冷害的量化指标,可为当地水稻安全生产提供科学依据。本文对黑龙江省4个主栽水稻品种(垦稻12、龙稻3号、龙稻7号和空育131)在孕穗期进行不同低温处理,分析了各处理对水稻空壳率的影响。结果表明:在平均温度为15℃时,孕穗期低温的持续天数是水稻形成障碍型冷害的关键因素;在低温持续时间相同的条件下,昼夜恒温15℃使各品种水稻受低温影响最严重;在不同低温处理条件下,不同品种水稻发生一般障碍型冷害的临界时长为1～4d或5～8d,发生严重障碍型冷害的临界时长为5～8d或8d以上,品种间临界时长有所差异;供试品种中,垦稻12和龙稻3号耐冷性较弱,空育131耐冷性较强,龙稻7号耐冷性居中。     

New QTLs identified for leaf correlative traits in rice seedlings under cadmium stress

Cadmium (Cd) is a non-essential toxic metal that is primarily released into the environment from artificial sources in recent decades. To investigate the genetics of Cd toxicity tolerance at the seedling stage in rice, a QTL analysis was carried out under cadmium stress conditions with two toxicity-linked traits—leaf rolling (LR) and the green leaf ratio (GLR). Using 127 rice lines of doubled haploid (DH) population derived from a cross between a japonica JX17 and indica ZYQ8, two QTLs for LR (qLR-1 and qLR-9) and one QTL for GLR (qGLR-3) were detected. Among them, the phenotypic variation of qLR-1 and qGLR-3 were 19.27 and 16.09, values which are useful for marker-assistant selection in breeding elite rice cultivars that have the capacity to tolerate Cd. The results further demonstrate that visual measurements of both LR and GLR in seedlings are effective methods for screening tolerant rice germplasm in cadmium stress scenarios.     

Comparative mapping of QTLs for agronomic traits of rice across environments by using a doubled-haploid population

We report here the RFLP mapping of quantitative trait loci (QTLs) which affect some important agronomic traits in cultivated rice. An anther culture-derived doubled-haploid (DH) population was established from a cross between indica and japonica rice varieties. A molecular linkage map comprising 137 markers was constructed based on this population which covered the rice genome at intervals of 14.8 cM on average. The linkage map was used to locate QTLs for such important agronomic traits as heading date, plant height, number of spikelets per panicle, number of grains per panicle, 1 000-grain weight and the percentage of seed set, by interval mapping. Evidence of genotype-by-environment interaction was found by comparing QTL maps of the same population grown in three diverse environments. A total of 22 QTLs for six agronomic traits was detected which were significant in at least one environment, but only seven were significant in all three environments; seven were significant in two environments and eight could only be detected in a single environment. However, QTLs-by-environment interaction was trait dependent. QTLs for spikelets and grains per panicle were common across environments while traits like heading date and plant height were more sensitive to environment. Received: 22 February 1996 / Accepted: 10 May 1996     

Mapping QTLs for root traits under different nitrate levels at the seedling stage in maize (Zea mays L.)

Nitrogen (N) loss is a worldwide problem in crop production. Apart from reasonable N fertilizer application, breeding N efficient cultivars provides an alternative way. Root architecture is an important factor determining N acquisition. However, little is known about the molecular genetic basis for root growth in relation to N supply. In the present study, an F₈ maize (Zea may L.) recombinant inbred (RI) population consisting of 94 lines was used to identify the QTLs for root traits under different nitrate levels. The lateral root length (LRL), axial root length (ARL), maximal axial root length (MARL), axial root number (ARN) and average axial root length (AARL) were evaluated under low N (LN) and high N (HN) conditions in a hydroponics system. A total of 17 QTLs were detected among which 14 loci are located on the same chromosome region as published QTLs for root traits. A major QTL on chromosome 1 (between bnlg1025 and umc2029) for the AARL under LN could explain 43.7% of the phenotypic variation. This QTL co-localizes with previously reported QTLs that associate with root traits, grain yield, and N uptake. Our results indicate that longer axial roots are important for efficient N acquisition and the major QTL for AARL may be used as a marker in breeding N efficient maize genotypes.     

A novel functional gene associated with cold tolerance at the seedling stage in rice

Identification and cloning of cold‐tolerant genes that can stably express under different cold environments are crucial for molecular rice breeding for cold tolerance. In the previous study, we identified a cold‐tolerant QTL at the seedling stage, qCTS‐9 which could be detected under different cold environments using a recombinant inbred line (RIL) population derived from a cold‐tolerant variety Lijiangxintuanheigu (LTH) and a cold‐sensitive variety Shanhuangzhan 2 (SHZ‐2). In this study, eight candidate genes within the qCTS‐9 interval were identified through integrated analysis of QTL mapping with genomewide differential expression profiling of LTH. The qRT‐PCR assay showed that only Os09g0410300 exhibited different expression patterns between LTH and SHZ‐2 during cold stress, and significantly positive correlation was found between cold induction of Os09g0410300 and seedling cold tolerance in the RI lines. Five SNPs and one InDel in the promoters of Os09g0410300 were detected between LTH and SHZ‐2, and the InDel marker ID410300 designed based on the insertion–deletion polymorphism in the promoter was significantly associated with seedling cold tolerance in RIL population. Further, Os09g0410300 over‐expression plants exhibited enhanced cold tolerance at the seedling stage compared with the wild‐type plants. Thus, our results suggest that Os09g0410300 is the functional gene underlying qCTS‐9. To our knowledge, it is a novel gene contributed to enhance cold tolerance at the seedling stage in rice. Identification of the functional gene underlying qCTS‐9 and development of the gene‐specific marker will facilitate molecular breeding for cold tolerance at the seedling stage in rice through transgenic approach and marker‐assisted selection (MAS).