Physical mapping and putative candidate gene identification of a quantitative trait locus <Emphasis Type="Italic">Ctb1</Emphasis> for cold tolerance at the booting stage of rice

Norin-PL8 is a cold-tolerant variety of rice (Oryza sativa L.) that was developed by introgressing chromosomal segments from a cold-tolerant tropical japonica variety, Silewah, into a template japonica variety, Hokkai241. We previously identified two closely linked quantitative trait loci, Ctb1 and Ctb2, for cold tolerance at the booting stage of Norin-PL8 in the long arm of chromosome 4. We report here the physical mapping of Ctb1 and the identification of the candidate genes. A total of 2,008 segregating individuals were screened for recombination in the Ctb1 region by a PCR-based screening, and a series of near-isogenic lines (NILs) were developed from progenies of recombinants. A comparison of the degrees of cold tolerance of the NILs indicated that Ctb1 is located in the 56-kb region covered by a bacterial artificial chromosome clone, OSJNBa0058 K23, that had been sequenced by the International Rice Genome Sequence Project. We found seven open reading frames (ORFs) in the 56-kb region. Two ORFs encoded receptor-like protein kinases that are possibly involved in signal transduction pathways. Proteins that may be associated with a ubiquitin-proteasome pathway were encoded by three ORFs, two of which encoded F-box proteins and one of which encoded a protein with a BAG domain. The other two ORFs encoded a protein with an OTU domain and an unknown protein. We were also able to show that Ctb1 is likely to be associated with anther length, which is one of major factors in cold tolerance at the booting stage.     

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。     

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.     

Identification and pyramiding of QTLs for cold tolerance at the bud bursting and the seedling stages by use of single segment substitution lines in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Cold stress is one of the main constraints in rice production, and damage from cold can occur at different developmental stages in rice. Understanding the genetic basis of cold tolerance is the key for breeding cold-tolerant variety. In this study, we used single segment substitution lines (SSSLs) derived from a cross between cold-tolerant japonica variety “Nan-yang-zhan” and a popular indica variety “Hua-jing-xian 74” to detect and pyramid QTLs for cold tolerance at the bud bursting and the seedling stages. Evaluation of cold tolerance of these SSSLs and their recurrent parent helped identify two cold-tolerant QTLs (qCTBB-5 and qCTBB-6) at the bud bursting stage and two cold-tolerant QTLs (qCTS-6 and qCTS-12) at the seedling stage. The SSSLs carrying these QTLs showed stronger cold tolerance than their recurrent parent HJX74 did in three independent experiments. The qCTBB-6 and qCTS-6 were mapped to the same chromosomal region. QTL pyramiding was performed by intercrossing of SSSLs carrying the respective QTLs for cold tolerance at the bud bursting stage and the seedling stage and marker-assisted selection (MAS). The selected pyramiding line SC1-1 with different cold-tolerant QTLs showed cumulative effects on cold tolerance. Our results suggest that different genes (QTLs) control cold tolerance at bud bursting and seedling stages, and pyramiding of stable expression QTLs for cold tolerance at different developmental stages through MAS is a good strategy to prevent cold damage in rice.     

Fine mapping of three quantitative trait loci for late blight resistance in tomato using near isogenic lines (NILs) and sub-NILs

An earlier study identified quantitative trait loci (QTLs) lb4, lb5b, and lb11b for quantitative resistance to Phytophthora infestans (late blight) in a backcross population derived from crossing susceptible cultivated tomato (Lycopersicon esculentum) with resistant L. hirsutum. The QTLs were located in intervals spanning 28–47 cM. Subsequently, near-isogenic lines (NILs) were developed for lb4, lb5b, and lb11b by marker-assisted backcrossing to L. esculentum. Sub-NILs containing overlapping L. hirsutum segments across each QTL region were selected and used to validate the QTL effects, fine-map QTLs, and evaluate potential linkage drag between resistance QTLs and QTLs for horticultural traits. The NILs and sub-NILs were evaluated for disease resistance and eight horticultural traits at three field locations. Resistance QTLs were detected in all three sets of NIL lines, confirming the BC₁ mapping results. Lb4 mapped near TG609, and between TG182 and CT194, on chromosome 4, a 6.9-cM interval; lb5b mapped to an 8.8-cM interval between TG69a and TG413 on chromosome 5, with the most likely position near TG23; and lb11b mapped to a 15.1-cM interval on chromosome 11 between TG194 and TG400, with the peak centered between CT182 and TG147. Most QTLs for horticultural traits were identified in intervals adjacent to those containing the late blight resistance QTLs. Fine mapping of these QTLs permits the use of marker-assisted selection for the precise introgression of L. hirsutum segments containing late blight resistance alleles separately from those containing deleterious alleles at horticulturally important QTLs.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by D.B. Neale     

Mapping of loci affecting copper tolerance in wheat—The possible impact of the vernalization gene Vrn-A1

Pot experiments with copper-treated soil and a control were performed in a greenhouse to determine QTLs for copper tolerance in wheat, using deletion, introgression and single chromosome recombinant lines. Genetic and physical mapping identified loci for copper tolerance on the long arm of chromosomes 5A and 5D, while loci with minor effects were found on the long and short arms of chromosome 5B. Tests on ‘Chinese Spring’–Aegilops tauschii introgression lines revealed a locus influencing copper tolerance on chromosome 3DS. QTLs for copper tolerance on chromosome 5A were mapped genetically and physically to exactly the same position as the gene for vernalization requirement (Vrn-A1). It is therefore suggested that Vrn-A1 may have a pleiotropic effect on copper tolerance may be due to the control of Cbf genes.     

Genome-wide association mapping of tan spot resistance (Pyrenophora tritici-repentis) in European winter wheat

The effect of low temperature on the physiology of maize has been well studied, but the genetics behind cold tolerance is poorly understood. To better understand the genetics of cold tolerance we conducted a quantitative trait locus (QTL) analysis on a segregating population from the cross of a cold-tolerant (EP42) and a cold-susceptible (A661) inbred line. The experiments were carried under cold (15 °C) and control (25 °C) conditions in a phytotron. Cold temperature reduced the shoot dry weight, number of survival plants and quantum yield of electron transport at photosystem II (ΦPSII) and increased the anthocyanin content in maize seedlings. Low correlations were found between characteristics under low and optimum temperature. Ten QTLs were identified, six of them at control temperatures and four under cold temperatures. Through a meta-QTL analysis we identified three genomic regions in chromosomes 2, 4 and 8 that regulate the development of maize seedlings under cold conditions and are the most promising regions to be the target of future marker-assisted selection breeding programs or to perform fine mapping to identify genes involved in cold tolerance in maize.     

Fine mapping of a yield-enhancing QTL cluster associated with transgressive variation in an <Emphasis Type="Italic">Oryza sativa </Emphasis>× <Emphasis Type="Italic">O. rufipogon</Emphasis> cross

A high-resolution physical map targeting a cluster of yield-related QTLs on the long arm of rice chromosome 9 has been constructed across a 37.4 kb region containing seven predicted genes. Using a series of BC₃F₄ nearly isogenic lines (NILs) derived from a cross between the Korean japonica cultivar Hwaseongbyeo and Oryza rufipogon (IRGC 105491), a total of seven QTLs for 1,000-grain weight, spikelets per panicle, grains per panicle, panicle length, spikelet density, heading date and plant height were identified in the cluster (P ≤ 0.0001). All seven QTLs were additive, and alleles from the low-yielding O. rufipogon parent were beneficial in the Hwaseongbyeo background. Yield trials with BC₃F₄ NILs showed that lines containing a homozygous O. rufipogon introgression in the target region out-yielded sibling NILs containing Hwaseongbyeo DNA by 14.2–17.7%, and out-yielded the Hwaseongbyeo parent by 16.2–23.7%. While higher yielding plants containing the O. rufipogon introgression were also taller and later than controls, the fact that all seven of the QTLs were co-localized in the same 37.4 kb interval suggests the possibility that a single, pleiotropic gene acting as a major regulator of plant development may control this suite of agronomically important plant phenotypes. Xiaobo Xie and Fengxue Jin have contributed equally to this study.     

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).