Saturation mapping of QTL regions and identification of putative candidate genes for drought tolerance in rice

We have developed 85 new markers (50 RFLPs, 5 SSRs, 12 DD cDNAs, 9 ESTs, 8 HSP-encoding cDNAs and one BSA-derived AFLP marker) for saturation mapping of QTL regions for drought tolerance in rice, in our efforts to identify putative candidate genes. Thirteen of the markers were localized in the close vicinity of the targeted QTL regions. Fifteen of the additional markers mapped, respectively, inside one QTL region controlling osmotic adjustment on chromosome 3 ( oa3.1) and 14 regions that affect root traits on chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 10 and 12. Differential display was used to identify more putative candidate genes and to saturate the QTL regions of the genetic map. Eleven of the isolated cDNA clones were found to be derived from drought-inducible genes. Two of them were unique and did not match any genes in the GenBank, while nine were highly similar to cDNAs encoding known proteins, including a DnaJ-related protein, a zinc-finger protein, a protease inhibitor, a glutathione-S-transferase, a DNA recombinase, and a protease. Twelve new cDNA fragments were mapped onto the genetic linkage map; seven of these mapped inside, or in close proximity to, the targeted QTL regions determining root thickness and osmotic adjustment capacity. The gene I12A1, which codes for a UDP-glucose 4-epimerase homolog, was identified as a putative target gene within the prt7.1/brt7.1 QTL region, as it is involved in the cell wall biogenesis pathway and hence may be implicated in modulating the ability of rice roots to penetrate further into the substratum when exposed to drought conditions. RNAs encoding elongation factor 1, a DnaJ-related protein, and a homolog of wheat zinc-finger protein were more prominently induced in the leaves of IR62266 (the lowland rice parent of the mapping materials used) than in those of CT9993 (the upland rice parent) under drought conditions. Homologs of 18S ribosomal RNA, and mRNAs for a multiple-stress induced zinc-finger protein, a protease inhibitor, and a glutathione-S-transferase were expressed at significantly higher levels in CT9993 than in IR62266. Thus several genes involved in the regulation of DNA structure and mRNA translation were found to be drought-regulated, and may be implicated in drought resistance.Communicated by R. Hagemann     

Quantitative trait loci associated with leaf and neck blast resistance in recombinant inbred line population of rice (Oryza sativa).

Blast is an economically important disease of rice. To map genes controlling blast resistance, recombinant inbred lines (RIL) were developed from Khao Dawk Mali 105, an aromatic, blast-susceptible cultivar and the blast resistance donor, CT 9993-5-10-M (CT). A linkage map encompassing 2112 cM was constructed from 141 RILs using 90 restriction fragment length polymorphisms (RFLPs) and 31 simple sequence repeats (SSR). Virulent isolates of blast fungus were identified by screening differential host sets against 87 single-spore isolates collected from the north and northeast of Thailand. Fifteen virulent blast isolates were selected for leaf blast screening. Neck blast was evaluated both under natural conditions and controlled inoculations. Quantitative trait loci (QTLs) for broad resistance spectrum (BRS) to leaf blast were located on chromosomes 7 and 9. In particular, the QTL(ch9) was mapped near the Pi5(t) locus. The QTL(ch7) was located close to a previously mapped partial resistance QTL. Both loci showed significant allelic interaction. Genotypes having CT alleles at both QTL(ch7) and QTL(ch9) were the most resistant. Two neck-blast QTLs were mapped on chromosomes 5 and 6. The inconsistent map locations between the leaf and neck blast QTLs indicate the complexity of fixing both leaf and neck blast resistance. The coincidence of BRS and field resistance QTLs on chromosome 7 supports the idea that BRS may reflect the broad resistance spectrum to leaf blast in rice. These findings laid the foundation for the development of a marker-assisted scheme for improving Khoa Dawk Mali 105 and the majority of aromatic Thai rice varieties that are susceptible to blast.     

Improving QTL Mapping Resolution Based on Genotypic Sampling—a Case Using a RIL Population

The QTL mapping results were compared with the genotypically selected and random samples of the same size on the base of a RIL population. The results demonstrated that there were no obvious differences in the trait distribution and marker segregation distortion between the genotypically selected and random samples with the same population size. However, a significant increase in QTL detection power, sensitivity, specificity, and QTL resolution in the genotypically selected samples were observed. Moreover, the highly significant effect was detected in small size of genotypically selected samples. In QTL mapping, phenotyping is a more sensitive limiting factor than genotyping so that the selection of samples could be an attractive strategy for increasing genome-wide QTL mapping resolution. The efficient selection of samples should be more helpful for QTL maker assistant selection, fine mapping, and QTL cloning.     

Gas exchange,chlorophyll fluorescence,and osmotic adjustment in two mango cultivars under drought stress

The responses of photosynthetic gas exchange, chlorophyll fluorescence, content of pigments, main osmolytes, and malondialdehyde (MDA) to water-withholding for 15 days and re-hydration in seedlings of two mango cultivars (Mangifera indica L. var. “Choke Anand’ and var. “Khieo Sawoei”) under 50% sunlight and full sunlight were investigated. For both cultivars, the water-witholding resulted in progressively decreases in leaf relative water content, net photosynthesis (P _n), stomatal conductance (g _s), and increases in the conversion of xanthophyll cycle pigments estimated by an index of leaf spectral reflectance (ΔPRI), carotenoid to chlorophyll ratio, non-photochemical quenching (NPQ), the contents of malondialdehyde (MDA) and compatible solutes (total soluble sugar and proline). The effect of the water stress was more pronounced in full sunlight than 50% sunlight. The maximum photochemistry efficiency measured at dawn was fairly constant during the period of the treatment for both cultivars under both light regimes. The water stress caused less pronounced inhibition of photosynthesis in “Choke Anand” than in “Khieo Sawoei” cultivar under both light regimes. After re-hydration, the recovery was relatively quicker in “Choke Anand” than in “Khieo Sawoei” cultivar. Both cultivars in both 50% and full sunlight showed complete recovery in photochemistry after 5 days of re-watering but photosynthesis did not show a complete recovery as indicated by gas exchange rates. As the results of lower NPQ, ΔPRI and osmotic adjustment in the cultivar “Khieo Sawoei” compared to the cultivar “Choke Anand”, the former cultivar was less tolerant to drought than the latter. Our study further showed that partial shading (e.g., 50% of sunlight) significantly alleviated the harmful effect of drought stress on mango cultivars but in fact stomata of seedlings grown in partial shade was more responsive to water deficit than in full light.     

Development of pre-isogenic lines for rice blast-resistance by marker-aided selection from a recombinant inbred population

To increase the available set of near-isogenic lines (NILs) for blast-resistance in rice, we have developed a general method for establishing NILs from populations of fixed recombinants that have been used for gene mapping. We demonstrated the application of this method by the selection of lines carrying genes from the rice cultivar Moroberekan. Moroberekan is a West African japonica cultivar that is considered to have durable resistance to rice blast. Multiple genes from Moroberekan conferring complete and partial resistance to blast have previously been mapped using a recombinant inbred (RI) population derived from a cross between Moroberekan and the highly and broadly susceptible indica cultivar CO39. To analyze individual blast-resistance genes, it is desirable to transfer them individually into a susceptible genetic background. This RI population, and the associated data sets on blast reaction and restriction fragment length polymorphism (RFLP) genotypes, were used for selection of lines likely to carry individual blast-resistance genes and a minimum number of chromosomal segments from Moroberekan. Because skewed segregation in the RI population favored CO39 (indica) alleles, resistant lines carrying 8.7–17.5% of Moroberekan alleles (the proportion expected after two or three backcrosses) could be selected. We chose three RI lines carrying different complete resistance genes to blast and two RI lines carrying partial resistance genes to blast as potential parents for the development of NILs. These lines were subjected to genetic analysis, which allowed clarification of some issues that could not be resolved during the initial gene-mapping study.     

水稻低温发芽性QTL的分子标记定位

利用1个粳／籼交来源的重组自交系群体,采用纸卷法在15℃低温条件下进行发芽试验,在发芽培养的6～14d中每天观测统计1次发芽率(％)。结合一张含有198个DNA标记的连锁图谱,用复合区间作图法定位水稻低温发芽性QTL。共检测到7个主效应QTL,分别位于水稻1、3、5、6和8号染色体上,单个QTL对性状的贡献率为5％～16％。其中,位于3号染色体标记区间RM148-RM85的qLTG-3-2和位于8号染色体标记区间RM223-RM210的qLTG-8-1对性状的贡献率最大,分别达16％和14％。QTL qLTG-3-2在发芽培养6～10d中表达,其效应由强渐弱,对性状的贡献率由发芽培养6d时的16．4％逐渐降低为发芽11d时的5．1％;而QTL qLTG-8-1则在发芽培养9～14d中起作用,其效应值由小逐渐增大,对性状的贡献率由发芽9d时的8．6％逐渐上升为发芽13～14d的14％。尽管这2个QTL加性效应的大小在低温发芽过程中按一定趋势变化,但加性效应的方向始终是一致的。QTL qLTG-3-2的增效基因来源于亲本特青,而QTL qLTG-8-1的增效基因来自于亲本Lemont。这2个QTL的增效等位基因有望作为分子标记辅助育种的操作对象,用于水稻品种低温发芽性的遗传改良。     

水稻叶绿素含量的QTL定位

利用由两个籼稻品种Acc8558和H359杂交构建的一个包含131个株系(F19)的重组自交系群体,及其相应的包含147个RFLP和78个AFLP标记的遗传图谱,采用多性状复合区间定位方法,对控制水稻叶绿素含量的QTL进行了定位分析。对叶绿素a和叶绿素b含量各检测到6个QTL,其中5个QTL在两性状问是相同的。这些QTL主要分布在第1和第4染色体上,因此这两条染色体对叶绿素含量是重要的。QTL qChlAlc／qChlBlb(二者位置相同)在4个观测时期均表现较大效应,且在最后的剑叶期贡献最大,因此对叶绿素含量最为重要。另两个QTL(qCh-LA4a／qChlB4a和qChlA4b／qChlB4b)只在第2次观测时期效应显著,表明它们只在特定发育阶段发挥作用。     

水稻加工品质数量性状基因座 (QTLs)分子定位研究

检测了Lemont/特青RI群体212个株系的糙米率（BR）,精米率（MR）和整精米率（HR）等3项加工品质性状,利用RFLP连锁图和线性模型的复合区间作图方法（QTLMapperV1.0)进行QTL定位研究。群体呈边境分布,双向超亲现象明显,HR较BR,MR变异范围更大并偏向低值方向;分别检测到1个MR,4个HR主效QTL,其中QHr6和QHr7等2个基因座具有较大遗传效应;分别检测到12对影响BR、5对影响MR,16对影响HR的上位性基因座,上位性效应的影响大于主效QTLs,不同性状或同一性状上位性效应通过共同的区间形成复杂的互相联系。     

小麦萌发期抗旱和耐盐性状的QTL分析

该研究以‘山农0431×鲁麦21’RIL群体及其父母本为材料,用20%PEG-6000溶液和100 mmol·L-1 NaCl溶液分别模拟干旱和盐环境,对12个小麦萌发期抗旱耐盐相关性状进行测定,结合已构建的分子标记遗传图谱对小麦萌发期抗旱、耐盐的相关性状进行QTL分析,为小麦抗旱、耐盐基因的克隆和分子标记辅助选择提供参考。结果表明:(1)正常、干旱和盐胁迫3种处理下共检测到143个QTL。检测到相对高频QTL(RHF-QTL)29个,平均贡献率范围为4.39%~13.28%,贡献率在10%以上的主效RHF-QTL有10个。(2)检测到胁迫下特异表达的RHF-QTL共17个,正常处理下特异表达的RHF-QTL为8个,稳定表达的RHF-QTL为4个。(3)QTL分析结果表明,7个RHF-QTL形成了3个QTL簇,且分布在2D、4D和5B等3条染色体上,其中:QC1位于2D染色体的wPt-6847~D-1172783区间,包括3个QTL(QRl-2D.2、QSdw-2D.3、QTdw-2D);QC2位于4D染色体短臂的D-2245724~D-1108531区间,包括2个QTL(QSl-4D、QShl-4D);QC3位于5B染色体的D-982263~S-1083095区间,包括2个QTL(QSl-5B.2、QTdw-5B.1)。     

Mapping quantitative trait loci for milling quality, protein content and color characteristics of rice using a recombinant inbred line population derived from an elite rice hybrid

Milling properties, protein content, and flour color are important factors in rice. A marker-based genetic analysis of these traits was carried out in this study using recombinant inbred lines (RILs) derived from an elite hybrid cross ’Shanyou 63’, the most-widely grown rice hybrid in production in China. Correlation analysis shows that the traits were inter-correlated, though the coefficients were generally small. Quantitative trait locus (QTL) analysis with both interval mapping (IM) and composite interval mapping (CIM) revealed that the milling properties were controlled by the same few loci that are responsible for grain shape. The QTL located in the interval of RM42-C734b was the major locus for brown rice yield, and the QTL located in the interval of C1087-RZ403 was the major locus for head rice yield. These two QTLs are the loci for grain width and length, respectively. The Wx gene plays a major role in determining protein content and flour color, and is modified by several QTLs with minor effect. The implications of the results in rice breeding were discussed. Received: 15 September 2000 / Accepted: 31 March 2001     

Mapping QTLs for root traits in a recombinant inbred population from two indica ecotypes in rice

Evaluation of root traits in rainfed lowland rice is very difficult. Molecular genetic markers could be used as an alternative strategy to phenotypic selection for the improvement of rice root traits. This research was undertaken to map QTLs associated with five root traits using RFLP and AFLP markers. Recombinant inbred lines (RILs) were developed from two indica parents, IR58821–23-B-1–2-1 and IR52561-UBN-1–1-2, that were adapted to rainfed lowland production systems. Using wax-petrolatum layers to simulate a hardpan in the soil, 166 RILs were evaluated for total root number (TRN), penetrated root number (PRN), root penetration index (RPI, the ratio of PRN to TRN), penetrated root thickness (PRT) and penetrated root length (PRL) under greenhouse conditions during the summer and the fall of 1997. A genetic linkage map of 2022 cM length was constructed comprising 303 AFLP and 96 RFLP markers with an average marker space of 5.0 cM. QTL analysis via interval mapping detected 28 QTLs for these five root traits, which were located on chromosomes 1, 2, 3, 4, 6, 7, 10 and 11. Individual QTLs accounted for between 6 and 27% of the phenotypic variation. Most of the favorable alleles were derived from the parent IR58821–23-B-1–2-1, which was phenotypically superior in root traits related to drought resistance. Three out of six QTLs for RPI were detected in both summer and fall experiments and they also were associated with PRN in both experiments. Out of eight QTLs for RPT, five were common in both seasons. Two genomic regions on chromosome 2 were associated with three root traits (PRN, PRT and RPI), whereas three genomic regions on chromosomes 2 and 3 were associated with two root traits (PRT and RPI). Two QTLs affecting RPI and two QTLs affecting PRT were also found in similar genomic regions in other rice populations. The consistent QTLs across genetic backgrounds and the common QTLs detected in both experiments should be good candidates for marker-assisted selection toward the incorporation of root traits in a drought resistance breeding program, especially for rainfed lowland rice. Received: 17 November 1999 / Accepted: 19 March 2000     

Genetic Dissection of Epistatic Interactions Contributing Grain Yield Variability in Rice under Drought

AimsThe aim of the present study was to evaluate the performance of ‘high’-‘low’ yielding pyramided lines (PLs), having the same combinations of qDTYs in Samba Mahsuri, MR219 and IR64-Sub1 genetic backgrounds, and to understand the genetic interactions among QTL and/with genetic background affecting grain yield.BackgroundEpistasis regulates the expression of traits governed by several major/minor genes/QTL. Multiple pyramided lines (PLs) with the same grain yield QTL (qDTYs) combinations but possessing grain yield variability under different levels of reproductive stage drought stress were identified in different rice genetic backgrounds at International Rice Research Institute (IRRI).ObjectivesThe objectives of the present study were to evaluate the performance pyramided lines (PLs) with drought QTL in the backgrounds of Samba Mahsuri, MR219 and IR64-Sub1 under reproductive stage drought stress (RS) and NS (non-stress) conditions, to understand the effect of epistatic interactions among qDTYs and with genetic background on GY under the differential level of stress and to identify the promising drought-tolerant lines with high yield under drought and higher background recovery in different genetic backgrounds.MethodsThe experiments were conducted in 2015 DS (dry season), 2015 WS (wet season) and 2017 DS at IRRI, Los Baños, Philippines, in a transplanted lowland ecosystem under lowland severe stress (LSS), lowland moderate stress (LMS) and lowland non-stress (LNS). The experiments were laid out in alpha lattice design with two replications.ResultsSeveral digenic interactions were found in different genetic backgrounds, 13 interactions in Samba Mahsuri, 11 in MR219 and 20 in IR64-Sub1 backgrounds. Among all digenic interactions, one QTL × QTL interaction, 17 QTL × background and 26 background × background interactions resulted in GY reduction in low yielding PLs in different genetic backgrounds under LSS or LMS. Negative interaction of qDTY_3.1, qDTY_4.1 and qDTY_9.1 with background markers and background × background interactions caused up to 15% GY reduction compared to the high yielding PLs under LMS in the Samba Mahsuri PLs. In MR219 PLs, the negative interaction of qDTY_2.2, qDTY_3.2, qDTY_4.1 and qDTY_12.1 with the background marker interval RM314-RM539, RM273-RM349 and RM445-RM346, RM473D-RM16, respectively resulted 52% GY reduction compared to the high yielding PLs under LSS. In IR64-Sub1 PLs, qDTY_6.1 interacted with background loci at RM16-RM135, RM228-RM333, RM202-RM287 and RM415-RM558A marker interval under LSS and at RM475-RM525 marker interval under LMS, causing GY reduction to 58% compared to the high yielding PLs.ConclusionHigh yielding PLs in Samba Mahsuri (IR 99734:1-33-69-1-22-6), MR219 (IR 99784-156-87-2-4-1) and IR64-Sub1 (IR 102784:2-89-632-2-1-2) backgrounds without any negative interactions were identified. The identified selected promising PLs may be used as potential drought-tolerant donors or may be released as varieties for drought-prone ecosystems in different countries.     

水稻低温发芽力QTL定位和遗传分析

以Kinmaze(粳稻)／DV85(籼稻)的重组自交系F10世代群体检测了影响水稻低温发芽力性状的数量性状基因座(QTL)。通过测定不同时期的低温发芽率,确定了15℃低温、第10d为检测低温发芽率的最适处理温度和时间,该条件下能够充分检测到品种的差异和分离群体的变异。通过设置对照,证明所检测的低温发芽率不受休眠及二次休眠的影响。15℃低温、第10d时,Kinmaze的发芽率达35％,DV85的发芽率只有7％,两亲本之间存在明显差异,该群体81个家系的低温发芽率变幅在0％～99％之间。QTL分析结果检测到5个与低温发芽力相关的基因座,分别位于第2、6、7、11和12染色体上。位于第2、6和11染色体上的qLTG-2、qLTG-6和qLTG-11贡献率分别为27．1％、17．1％和15．0％,对低温发芽力性状的增效基因来自DV85;位于第7、12染色体上qLTG-7和qLTG-12的贡献率分别为22．9％和8．8％,增效基因来自Kinmaze。其中,qLTG-6和qLTG-11在染色体上的位置与已报道的有关低温发芽力QTL位置相似,而qLTG-2、qLTG-7和qLTG-12为新检测的低温发芽力基因座。上位性分析结果显示,第3与第5染色体上存在影响低温发芽力的互作位点,其互作可以提高低温发芽力,而第7染色体上的两位点之间的互作降低了低温发芽力。     

Mapping genes controlling root morphology and root distribution in a doubled-haploid population of rice

 A deep thick root system has been demonstrated to have a positive effect on yield of upland rice under water stress conditions. Molecular-marker-aided selection could be helpful for the improvement of root morphological traits, which are otherwise difficult to score. We studied a doubled-haploid population of 105 lines derived from an indica×japonica cross and mapped the genes controlling root morphology and distribution (root thickness, maximum root length, total root weight, deep root weight, deep root weight per tiller, and deep root to shoot ratio). Most putative QTL activity was concentrated in fairly compact regions on chromosomes 1, 2, 3, 6, 7, 8 and 9, but was widely spread on chromosome 5 and largely absent on chromosomes 4, 10, 11 and 12. Between three and six QTLs were identified on different chromosomes for each trait. Individual QTLs accounted for between 4 and 22% of the variation in the traits. Multiple QTL models accounted for between 14 and 49%. The main QTLs were common between traits, showing that it should be possible to modify several aspects of root morphology simultaneously. There was evidence of interaction between marker locations in determining QTL expression. Interacting locations were mostly on different chromosomes and showed antagonistic effects with magnitudes large enough to mask QTL detection. The comparison of QTL locations with another population showed that one to three common QTLs per trait were recovered, among which the most significant was in one or other population. These results will allow the derivation of isogenic lines introgressed with these common segments, separately in the indica and japonica backgrounds. Received: 12 August 1996 / Accepted: 15 November 1996     

杂交水稻亲本明恢63对纹枯病水平抗性的QTL定位

利用240份源于组合珍汕97／明恢63的重组自交群体（F11、F12）,连续两年进行2重复的随机区组田间试验,运用改进的纹枯病人工接种鉴定的方法,调查其纹枯病病级,结合该组合F9群体构建的分子标记遗传连锁图谱,运用区间作图法对抗纹枯病QTLs进行了定位。两年在第5染色体的相邻区间C624－C246（1999年）和C246－RM26（2000年）上各检测到一个抗纹枯病QTL,两者一个LOD值置信区间存在较大的重叠,而且LOD峰值位点很接近,推测它们可能是同一个QTL。两年在第9染本上均检测到一个QTL,分别位于C472－R26389（1999年）和RM247－RM242（2000年）区间上,两区间相距9．8cM。两年检测到的所有抗纹枯病QTLs均业自明恢63。     

Mapping quantitative trait loci controlling seed dormancy and heading date in rice, Oryza sativa L., using backcross inbred lines

 To detect quantitative trait loci (QTLs) controlling seed dormancy, 98 BC₁F₅ lines (backcross inbred lines) derived from a backcross of Nipponbare (japonica)/Kasalath (indica)//Nipponbare were analyzed genetically. We used 245 RFLP markers to construct a framework linkage map. Five putative QTLs affecting seed dormancy were detected on chromosomes 3, 5, 7 (two regions) and 8, respectively. Phenotypic variations explained by each QTL ranged from 6.7% to 22.5% and the five putative QTLs explained about 48% of the total phenotypic variation in the BC₁F₅ lines. Except for those of the QTLs on chromosome 8, the Nipponbare alleles increased the germination rate. Five putative QTLs controlling heading date were detected on chromosomes 2, 3, 4, 6 and 7, respectively. The phenotypic variation explained by each QTL for heading date ranged from 5.7% to 23.4% and the five putative QTLs explained about 52% of the total phenotypic variation. The Nipponbare alleles increased the number of days to heading, except for those of two QTLs on chromosomes 2 and 3. The map location of a putative QTL for heading date coincided with that of a major QTL for seed dormancy on chromosome 3, although two major heading-date QTLs did not coincide with any seed dormancy QTLs detected in this study. Received: 10 October 1997 / Accepted: 12 January 1998     

水稻RIL群体苗期耐冷性QTL分析

水稻苗期冷害是影响早春季节和高纬度地区水稻成苗和秧苗生长的重要限制因素之一。为了鉴定控制水稻苗期耐冷性的QTL,研究采用了1个水稻“粳籼交”重组自交系(RIL)群体,结合1张高密度分子遗传图谱,对3叶期幼苗经过10℃冷处理3d、恢复培养2d和4d时的秧苗存活率进行复合区间作图。亲本Lemont和特青的苗期耐冷性具有极显著差异,Lemont的苗期耐冷性很强,而特青对低温敏感。在重组自交系群体中,苗期耐冷性表现为连续变异,在两个方向上均出现大量超亲分离。共检测到5个水稻苗期耐冷性QTL,分别位于水稻1、3、8和11号染色体上,单个QTL对性状的贡献率为7％～21％。其中,4个QTL的增效基因来源于亲本Lemont,另1个QTL的增效基因来源于亲本特青。2个主效QTL(qSCT-3和qSCT-8)分别位于3号染色体标记区间RM282-RM156和8号染色体标记区间RM230—RM264,对性状的贡献率达到或接近20％,被检测到的LOD值显著较高,其增效基因均来自于耐冷性亲本Lemont。研究结果进一步揭示了水稻苗期耐冷性QTL具有丰富的位点多样性,表明耐冷性普遍较强的粳稻是发掘苗期耐冷性优异基因的主要稻种资源。