Development of Isogenic Lines of Morphological Markers in Indica Rice

A total of 28 accessions of rice (Oryza sativa L.) germplasms were collected from International Rice Research Institute (IRRI), Philippine and Gene Pool of China National Rice Research Institute (CNRRI). These germplasms were attributed as a Donor Gene Pool, consisting of 27 diverse morphological marker lines with each having a unique valuable trait (gene), and the recurrent parent-Zhefu 802, an early-season indica cultivar in south of China in the 1980s and 1990s. A set of 27 isogenic lines with 2-3 markers on every chromosome were obtained after more than 10 successive backcrosses. Moreover, 15 accessions of double-marker isogenic lines were developed by further pyramiding markers on the same chromosome. Key agronomic traits such as heading date, plant height, tillering number and panicle length in this set of lines were similar to that of Zhefu 802 except the marker traits.

Establishment of Near Isogenic Lines of Huangzaosi Maize with Resistance against Maize Head Smut

[ Objective ] The paper was to establish the near isogenic lines of Huangzaosi maize with resistance against maize head smut, and to provide basis for its wide application in breeding and production. [Method] Combing with hybrid, backcress and self-cross method, as well as molecular markers, Huangzaosi maize was successfully introduced with head smut resistance, and its near isogenic lines were studied. The characteristics of resistant and susceptible variation and the changes of combining ability of near isogenic lines after backcrossing breeding were observed. [ Result ] The incidence rate of Huangzansi maize in control was 46% ; the incidence rates of the selected 24 near isogenic lines of resistant Huangzaosi were all lower than 10%, and the selected rate was 92.6% ; the combining ability of most near isogenic lines of resistant Huangzaosi maize had no significant difference with that of H^i maize in control; the yields of hybrids prepared by several inbred lines such as M135 and M140 were outstanding, which exceeded the control Zhengdan 958, showing a higher combining ability. [ Conclusion] The phenotype and combining ability of near isngenic lines of Huangzaosi maize with resistance against maize head smut obtained in the test were very close to Huan- gzaosi, and its resistance against maize head smut was greatly increased, thus solving the problems of Huangzaesi without resistance against maize head smut.

Integrating transcriptomic and proteomic analyses of photoperiod-sensitive in near isogenic maize line under long-day conditions

As a short-day plant species, maize requires an optimal photoperiod for inducing reproductive growth. However, there is a lack of information regarding photoperiod-induced changes in maize mRNA and protein levels. In this study, a photoperiod-insensitive maize inbred line and its near isogenic photoperiod-sensitive line were used. By integrating RNAbased transcriptomic and iTRAQ LC-MS/MS-based proteomic approaches, we generated a comprehensive inventory of the transcripts and proteins with altered abundances in response to a long photoperiod(LP) during growth stage transitions. We detected 22 000 transcripts in RNA-sequence runs and 5 259 proteins from an iTRAQ-based analysis. A weak correlation between mRNA-and protein-level changes was observed, suggesting the LP-induced transition between maize growth stages is largely regulated post-transcriptionally. Differentially expressed genes influenced by LP conditions were associated with several regulatory processes in both maize inbred lines, especially phosphate ion transport and the circadian rhythm. Additionally, 31 transcripts and six proteins related to photoperiodic flowering in maize were identified by comparing transcriptomic and proteomic data. This transcriptomic and proteomic analysis represents the first comprehensive and comparative study of gene/protein-level changes occurring in photoperiod-sensitive and-insensitive maize inbred lines during growth stage transitions under LP conditions.

Construction of Near Isogenic Lines for Pericarp Color and Evaluation on Their Near isogenicity in Rice

The inheritance of purple pericarp was studied through genetic analysis using F2, BC1F1 and F3 from a cross between a purple pericarp rice variety Zixiangnuo and a white pericarp rice variety Chunjiangnuo 2. Seven pairs of near isogenic lines （NILs） for pericarp color of rice were further constructed. Genetic analysis indicated that the purple pericarp was controlled by two complementary genes （Pb and Pp）. Agronomic trait analysis and polymorphism analysis using SSR markers on these seven pairs of NILs were used in the evaluation of near-isogenicity. No significant differences in agronomic traits were found except 1000-grain weight between the NILs. The polymorphic SSR markers for the parents were only detected in target segments of the five pairs of NILs different in Pb, which revealed the ideal near isogenicity of them. However, for the two pairs of NILs different in Pp, the polymorphic markers for the two parents were detected as well as in non-targeted segments of chromosomes 11 and 12, respectively.

Development of branchless watermelon near isogenic lines by marker assisted selection

Pruning is time-consuming and laborious in watermelon cultivation,which can not meet the needs for simplified cultivation in the future.The development of branchless lines will provide important germplasms for breeding watermelon varieties and is an important method for genetic improvement.In this study,the watermelon accession,Wu Cha Zao(WCZ)is a branchless inbred line that carries the branchless gene Clbl,which was used as the donor parent to develop branchless near isogenic lines(NILs).To construct the NILs of Clbl,WCZ crossed with the normal branching watermelon inbred line WT20 which was used as the recurrent parent.The co-segregating markers dCAPS10 and Indel1 with Clbl were used for foreground selection,and a total of 108 SSR markers was selected with good polymorphism between two parental lines for background selection which had relatively uniform distribution across 11 chromosomes.Using these markers to select individuals from the BC_(1)F_(1),BC_(2)F_(1),and BC_(2)F_(2) generations,three NILs with a proportion of recurrent parent genome(PRPG)>99%were finally obtained.The lateral branch and plant height phenotypes did not significantly differ between the NILs and WCZ,indicating that the NILs of Clbl under the genetic background of WT20 has been successfully developed.These results provide ideal materials for further in-depth analysis of the genetic mechanisms of lateral branch development and ideal plant architecture breeding in watermelon.

Dissection of QTLs for Yield Traits Using Near Isogenic Lines Derived from Residual Heterozygous Lines in Rice

Three residual heterozygous lines (RHLs) carrying heterozygous segments in the intervals RM587–RM225, RM204–RM6119 and RM6119–RM402 on the short arm of rice chromosome 6, respectively, were selected from a rice population derived from an RHL for the interval RM587–RM402. Ten maternal homozygotes, 10 paternal homozygotes and 20 heterozygotes were selected from each of the F2 populations derived from the three RHLs. The three sets of near isogenic lines (NILs) were grown to detect the grain yield per plant, number of panicles per plant, number of filled grains per panicle and 1000-grain weight. With analysis on the phenotypic differences among the three genotype groups in each NIL set and those among overlapping chromosome segment substitution lines, three QTLs for number of filled grains per panicle and two QTLs for grain yield per plant were resolved. They were located in the intervals ranging from 0.66 Mb to 2.49 Mb. The additive effect was higher than the dominance effect at each locus. The allele for increasing the trait value was derived from the paternal parent at qNFGP6-1, and from the maternal alleles at other QTLs. Based on the present study, an approach for constructing new genetic resource to facilitate fine mapping of QTLs in rice was proposed.

Identification of AFLP Markers Linked to Leaf Rust Resistance Genes Using Near Isogenic Lines of Wheat

The present investigation was undertaken to find molecular markers linked to leaf rust resistance genes, Lr9 and Kharchia local mutant KLM4-3B. Preliminary AFLP analysis was carried out with different stocks, a survey of primer combinations with different selective nucleotide indicated that for each primer combination, the number of scorable loci ranged from 34 to 123. Only a limited primer combination used in the set of parental and near isogenic lines showed a high level of polymorphism for AFLP marker. Putative AFLP marker were found to be linked to Lr9, Lr19 and KLM4-3B. The alien genes were readily identified.

Effect of high-molecular-weight glutenin subunit deletion on soft wheat quality properties and sugar-snap cookie quality estimated through near-isogenic lines

High-molecular-weight glutenin subunits（HMW-GSs） play a critical role in determining the viscoelastic properties of wheat dough. The HMW-GSs are encoded by Glu-A1, Glu-B1, and Glu-D1 loci on the long arms of chromosomes 1A, 1B, and 1D, respectively. In the present study, four near-isogenic lines with different HMW-GS deletions and compositions at the Glu-A1 and Glu-D1 loci in Yangmai 18 background were used for quality analysis. Deletion in Glu-D1 showed much weaker gluten quality and dough strength than null Glu-A1 genotype and wild genotype（WT）, based on the measurements of sodium dodecyl sulfate（SDS）-sedimentation, lactic acid solvent retention capacity（SRC）, gluten index, development time, stability time, and alveograph P and L values. The deletion of Glu-D1 did not significantly affect grain hardness, grain protein content, water SRC, sodium carbonate SRC, and sucrose SRC. Double null genotype in Glu-A1 and Glu-D1 and single null genotype in Glu-D1 showed significantly higher cookie diameter, crispness, and lower cookie height compared with single null genotype in Glu-A1 and WT. These indicate that the null Glu-D1 genotype is useful for improvement of biscuit quality, and use of this germplasm would be a viable strategy to develop new wheat varieties for biscuit processing.

Mapping a Novel Gene of Cold Tolerance at Booting Stage by Using Near-Isogenic Lines in japonica Rice

Genetic analysis showed that cold tolerance at booting stage of near-isogenic lines （NILs） of Kunmingxiaobaigu was controlled by a gene with large phenotypic variance. One hundred and sixty-four simple sequence repeats （SSR） distributed over 12 chromosomes were used to screen polymorphism between Towata （recurrent parent, RP） and near-isogenic line pool （NILP）, and two SSR markers at the long arm of chromosome 5 showed polymorphism in comparison with RP genome. Of the two markers, RM31 was found possibly linked with the cold tolerance gene at booting stage through one-way ANOVA. Twelve SSR markers around RM31 were then used to detect polymorphism between RP and NIL, and only RM7452 had polymorphism. The gene of cold tolerance at booting stage was further mapped on chromosome 5 between RM7452 and RM31 with genetic distances of 4.8 cM and 8.0 cM, respectively. This gene explained 10.50% of phenotypic variance and 5.10% of phenotypic variance of fully filled grains, and was tentatively designated as Ctb（t）.

Physiological Macro-lesions Enhanced Resistance to Blast (Magnaporthe grisea) in Rice Near-isogenic Lines

Roll-leaf-1 （rl-1） and spot-leaf-1 （spl-1） were two near-isogenic lines, which were obtained after 3 to 4 backcrosses with early season indica rice Zhefu 802 as recurrent parent. Henna macro-lesions, referred as physiological or morphological markers, began to appear on leaves at 4.5- to 6.0-leaf stage. The rice seedlings were inoculated at 3.5-, 5.0- and 7.0-leaf stages with high pathogenic races Zhong A1 and Zhong B1 of Magnaporthe grisea, respectively. The resistance of rl-1, spl-1 and Zhefu 802 against blast was significantly different. The seedlings of Zhefu 802 at 3.5- to 7.0-leaf stage were susceptible to races Zhong A1 and Zhong B1 of M. grisea, whereas those of rl-1 and spl-1 at 3.5-, 5.0- and 7.0-leaf stages were susceptible, moderately resistant and resistant, respectively. These results suggested that the enhanced resistance of rl-1 and spl-1 related to the appearance of their morphological marker lesions. The experiment provided a basis for studying lesion mimic and hypersensitive response in association with disease resistance.

Identification of Molecular Markers Linked to Leaf Rust Resistance Genes in Wheat and Their Detection in the Local Near-Isogenic Line

Sixty-five random amplified polymorphic DNA (RAPD) primers were used for the detection of polymorphism among recipient and donor parents and their isogenic lines linked to leaf rust resistance genes, Lr9 and the resistant gene in kharchia local mutant KLM4-3B. Three primers showed polymorphism among recurrent parent, donor parent and isogenic lines.

孕穗期NILs的耐冷相关性状的相关分析及聚类分析

用耐冷性极强的地方品种与十和田配制的NILs及亲本共84份材料,分别种在昆明和阿子营,通过相关和聚类分析,结果表明:(1)在昆明,结实率与花药长、倒二节长、实粒数、秕粒数之间相关系数较大,结实率、花药长、倒二节长、实粒数、秕粒数这几个性状与耐冷性的关系较密切。(2)在阿子营,结实率与花药长、株高、穗长、实粒数、秕粒数、总粒数之间相关达极显著水平,表明移栽至成熟期极端冷害下造成结实率降低,对整个植株生长发育均有抑制作用。(3)在昆明,84个材料18个性状聚类没有明显的类间区别,13个性状聚类可划分为2大类;在阿子营,78个材料8个性状聚类可划分为4类。(4)结实率作为耐冷性指标是较可靠的,在极端冷害下,可参考花药长等其他耐冷性指标。

粳稻孕穗期耐冷性NILs的形态评价和分子验证

2004年在海拔1 916 m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2 150 m,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,从分子水平上验证了近等基因系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。

Study on the Quality of NILs of Wheat cv. Longfumai 3 Possessing HMW-GS Null and 1 Subunits

To determine the genetic differences between high molecular weigh glutenin subunits （HMW-GS） null and 1. HMW-GS 1 was introduced into Longfumai 3 （N, 7＋8, 5＋10） by 5 consecutive backcrosses with biochemical marker assisted selection. The near isogenic lines （NILs） of HMW-GS null and 1 were obtained and planted in the experimental field of Crop Breeding Institute of Heilongjiang Academy of Agricultural Science in 2004 and 2005. The field experiments were designed using the method of two-line contrast arrangement with four replicates. The result of two years experiment showed that the statistic differences of quality parameters between Longfumai 3 with subunit 1 and with null were not significant in flour protein content, dry gluten content, and water absorption. However the gluten index,＇ Zeleny sedimentation, the ratio of sedimentation/dry gluten, the development time, stability, and the breakdown time in the NIL with 1 subunit were increased by 5.8% （P〈0.01）, 9.3% （P〈0.01）, 8.6% （P=0.01）, 127.3% （P〈0.01）, 79.2% （P〈0.01）, and 53.6% （P〈0.01）, and the ratio of wet gluten/dry gluten and the degree of softening were decreased by 1.7% （P = 0.05） and 16.5% （P = 0.13）, respectively. The impact of the HMW-GS 1 on the gluten strength was highly positive in NILs containing HMW-GS 5 ＋ 10, suggesting that HMW-GS 1 can be an indispensable subunit for breeding strong gluten wheat.

Qualitative Difference Between HMW-GS 5+10 and 2+12 NILs of Four Spring Wheat Cultivars with High-Quality Genetic Background

To investigate the effect of high molecular weight glutenin subunits (HMW-GS) 5+10 inhigh quality spring wheat, four near-isogenic lines (NILs) developed by 6 consecutivebackcrosses sharing the same genetic background and differing only at the Glu-D1 locusin each pair were employed in this study. The results showed that gluten indexes were allincreased by 3.1-27.7% (P<0.01) after the 5+10 subunits introduced into the fourcultivars. Their stability time was increased by 128, 104, 233 and 36%, where the maximumresistance raised by 130, 56.04, 95.10 and 16.33% in Kefeng6, Longmai20, Longfumai10and Xiaobingmai33, respectively.

QTL-Seq Identified a Major QTL for Grain Length and Weight in Rice Using Near Isogenic F_2 Population

Mapping and isolation of quantitative trait loci(QTLs)or genes controlling grain size or weight is very important to uncover the molecular mechanisms of seed development and crop breeding.To identify the QTLs controlling grain size and weight,we developed a near isogenic line F_2(NIL-F_2)population,which was derived from a residual heterozygous plant in an F_7 generation of recombinant inbred line(RIL).With the completion of more than 30×whole genome re-sequencing of the parents,two DNA bulks for large and small grains,a total of 58.94 Gb clean nucleotide data were generated.A total of455 262 single nucleotide polymorphisms(SNPs)between the parents were identified to perform bulked QTL-seq.A candidate genomic region containing SNPs strongly associated with grain length and weight was identified from 15 to 20 Mb on chromosome 5.We designated the major QTL in the candidate region as q TGW5.3.Then,q TGW5.3 was further validated with PCR-based conventional QTL mapping method through developing simple sequence repeat and Insertion/Deletion markers in the F_2 population.Furthermore,recombinants and the progeny tests delimited the candidate region of q TGW5.3 to 1.13 Mb,flanked by HX5009(15.15 Mb)and HX5003(16.28 Mb).A set of NILs,selected from the F_2 population,was developed to evaluate the genetic effect of q TGW5.3.Significant QTL effects were detected on grain length,grain width and 1000-grain weight of H12-29 allele with 1.14 mm,-0.11 mm and 3.11 g,which explained 99.64%,95.51%and 97.32%of the phenotypic variations,respectively.

大麦抗赤霉病近等基因系的转录组分析

赤霉病是大麦主要病害之一,严重影响大麦产量与品质。为研究大麦赤霉病抗性分子机制,以2个大麦赤霉病抗、感近等基因系N53和T66为材料,利用转录组测序技术(RNA-Seq),分析N53、T66在接种禾谷镰刀菌后不同时间点的基因表达特征和差异。结果表明:以接种0 h作为对照,N53和T66分别鉴定出15230和15106个差异表达基因。不同接种时间点上、下调的基因数目和富集到GO及KEGG通路的基因数目在两材料间存在显著差异。不同处理组间共筛选出421个共同差异表达基因,GO功能注释分类分成3大类42个亚类,KEGG通路共涉及到21个,并在信号转导(signal transduction)、植物-病原体相互作用(plant-pathogen interaction)、植物激素信号转导(plant hormone signal transduction)、MAPK-信号通路-植物(MAPK signaling pathway-plant)、苯丙烷类生物合成(phenylpropanoid biosynthesis)通路显著富集。综上,这一研究从转录组水平初步探索大麦对赤霉病的响应机制,可为进一步挖掘大麦赤霉病抗性候选基因、明晰大麦赤霉病抗性分子机制提供参考。

Puroindolineb位点近等基因系对小麦面粉及面包和馒头品质的影响

明确不同硬度等位基因与加工品质的关系对小麦品质改良具有重要意义。本文以7个Puroindoline b位点近等基因系为材料,研究了不同硬度等位基因对小麦面粉及面包和馒头品质的影响。结果表明,Pina-D1b/Pinb-D1a基因型的籽粒硬度值、蛋白质含量以及破损淀粉含量较高;而Pina-D1a/Pinb-D1d基因型的出粉率、面粉亮度较高,具有较好的磨粉品质。和面仪参数中的峰高、峰宽和8min尾高均以Pina-D1b/Pinb-D1a基因型数值最高,Pina-D1a/Pinb-D1d基因型最低,且两者之间的差异均达到显著水平;Pina-D1b/Pinb-D1a基因型的衰落角最小。Pina-D1a/Pinb-D1c和Pina-D1a/Pinb-D1d基因型具有较高馒头色泽和张弛性评分,较好的馒头制作品质;Pina-D1a/Pinb-D1e和Pina-D1a/Pinb-D1g基因型次之。Pina-D1a/Pinb-D1f基因型的面包总评分略优于其他基因型。

Puroindoline b位点近等基因系对小麦籽粒淀粉含量和组分及其特性的影响

为了研究不同硬度等位基因与小麦籽粒淀粉组分含量及特性的关系,为小麦品质改良提供依据,以7个硬度Puroindoline b位点近等基因系为试材,研究了不同硬度基因型对小麦籽粒淀粉组分含量及特性的影响。结果表明,Pina-D1b/Pinb-D1a基因型的籽粒硬度值、支链淀粉含量最高;Pina-D1b/Pinb-D1a基因型的淀粉溶解度最高,而Pina-D1a/Pinb-D1d溶解度最低,两者差异达显著水平;不同基因型之间籽粒淀粉酶解力没有显著差异;冻融失水率以基因型Pina-D1a/Pinb-D1b最低,表明Pina-D1a/Pinb-D1b基因型冻融稳定性最好,可能较适宜冷冻食品的制作;对于抗性淀粉,Pina-D1a/Pinb-D1b基因型的抗性淀粉含量最高、Pina-D1a/Pinb-D1d最低,表明Pina-D1a/Pinb-D1b基因型有助于籽粒中抗性淀粉含量的提高。

利用F2-NIL定位一个控制水稻穗颖花数、株高和抽穗期的多效QTL

利用重组自交系合适世代（F7或F8）的单株具有一定杂合率的特点,在重组自交系的一个家系内,选择到水稻每穗颖花数（SPP）、株高（PH）和抽穗期（HD）3个性状同时分离的2个单株。以这2个单株为亲本快速构建F2群体（F2-NIL）。由于这2个单株背景高度一致,故这个群体就是快速构建的近等基因系群体。后代测验结果为3个数量性状中的每一个性状的3种基因型分离数目均相同（29,63,33）,且卡方测验符合1∶2∶1的孟德尔分离比例,表明3个数量性状——每穗颖花数、抽穗期和株高均表现为质量性状分离。利用这个群体定位了一个位于第8染色体上、同时控制水稻每穗颖花数、抽穗期和株高3个性状的多效区段RM310-RM126,这个QTL可以解释这3个性状的很大的表现型方差（66.0%-81.1%）。利用后代测验结果,定位了控制这3个性状的基因位置。控制3个性状的基因和QTL位于相同的位置,QTL遗传效应的方向也相同,表明控制3个性状的遗传机制更可能是一因多效的结果,而非3个紧密连锁基因单独控制的结果 同时也表明利用重组自交系群体构建近等基因系的方法是快速构建近等基因系的有效方法。