Optimisation of breeding for agronomic traits in fibre hemp (Cannabis sativa L.) by study of parent-offspring relationships

Summary In the framework of a comprehensive Dutch research project the feasibility of hemp cultivation for paper pulp production is being investigated. This project also includes a hemp breeding programme which was initiated at CPRO-DLO in 1990. Hemp breeding is primarily aimed at improving bast fibre production since bast fibre is qualitatively superior to woody core fibre for paper pulp production. The progress of the ongoing breeding programme is hampered by the lack of knowledge on the inheritance of agronomic traits.The following traits were examined on 252 parental plants in 1991 and subsequently on their progenies in 1992: fibre content, plant height, date of flowering, stem diameter and cannabinoid contents. For each trait heritabilities were estimated and direct and indirect effects of artificial selection were studied.The heritability of bast fibre content was high and mass selection proved to be an efficient method causing no undesirable influence on other characters. Characters not directly related to bast fibre yield such as date of flowering, plant height and stem diameter were shown to have disadvantages as selection criteria for the improvement of bast fibre yield.The cannabinoids THC and CBD were studied, as the acceptance of hemp cultivation requires a low level. The content of THC, the psychoactive component can be successfully reduced by mass selection, but it is not certain that mass selection is the most efficient method.     

Predicting the chemical composition of fibre and core fraction of hemp (Cannabis sativa L.)

Summary Fibre formation in hemp (Cannabis sativa L.) is a dynamic process. In order to follow this process, the chemical composition of the hemp stem was studied during plant development using the acid and neutral detergent fibre and lignin methods. Additionally, near infra-red spectroscopy was carried out. To predict the chemical composition of the stem samples partial least square (PLS) analysis was carried out. The developed PLS models can predict the level of %ADF and %NDF in the hemp bast and core samples. For %ADL, the models are only suited for either core or bast tissues and can not be used for both tissues simultaneously.     

Life Cycle Analysis of field production of fibre hemp, the effect of production practices on environmental impacts

Summary Life Cycle Assessment (LCA) was used to assess the environmental impacts of field production of fibre hemp and seven other crops in France. The production of 1 ha of hemp yielded a eutrophication potential of 20.5 kg PO₄-equivalents, a global warming potential of 2330 kg CO₂-equivalents, an acidification potential of 9.8 kg SO₂-equivalents, a terrestrial ecotoxicity potential of 2.3 kg 1,4-dichlorobenzene-equivalents, an energy use of 11.4 GJ, and a land use of 1.02 ha.year. A comparison of hemp (low impacts), wheat (intermediate impacts) and sugar beet (high impacts) revealed that the crops were similar for the relative contributions of emitted substances and resources used to impacts, and for the relative contribution of processes to impacts. A reduction of the impacts of hemp production should focus on eutrophication, and consider the reduction of climate change, acidification and energy use as secondary objectives. Given this objective, the overall environmental effect of the substitution of mineral fertiliser by pig slurry is negative. The introduction of reduced tillage is of interest, as it decreases energy use, acidification and climate change. Measures leading to a reduction in NO₃ leaching are highly interesting, as they strongly decrease eutrophication. Implications for hemp breeding are discussed.     

Interaction between the eui gene and thermo-sensitive genic male sterility in rice

Panicle enclosure is a typical phenotype of almost all male-sterile rice lines. An elongated uppermost internode (eui) mutant exhibited notably rapid elongation of the uppermost internode at the heading stage; this is considered as a potential mechanism to eliminate panicle enclosure. We developed thermo-sensitive genic male-sterile (TGMS) eui mutants that were characterized by notably elongated uppermost internodes. The elongation of the uppermost internode in the TGMS eui mutant Changxuan 3S (CX) is mainly attributed to an increase in cell number and cell elongation, the latter being the more significant process. Temperature treatments revealed that the effects of temperature on panicle exsertion were similar to those on fertility and that the most temperature-sensitive stage coincides with the period from the formation of the pollen mother cell to meiosis during panicle initiation. These results indicate that elongation increases as temperature decreases and that the expression of the eui gene is more efficient at low temperatures than at high temperatures. In hybrid rice seed production using the TGMS eui mutant, the temperature range should be optimized at 24–28°C in order to preserve the development of completely male-sterile pollen and to eliminate panicle enclosure. Consequently, by using TGMS eui rice lines, gibberellin application can be avoided, thereby reducing the cost of hybrid seed production. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

水稻花器官数目突变体mf2的鉴定和基因定位

水稻的花器官发育影响着水稻的产量与品质。本研究通过¹²C重离子诱变航恢7号获得一个水稻花器官突变体multi-floret 2 (mf2), 其稃片、浆片、雄蕊、雌蕊增多, 多数小穗内具2~3朵类似小花。mf2内外稃不能很好勾合, 而且形状和维管束的数目都产生了一定程度的变化。电镜扫描幼穗发现花器官的变异在幼穗分化期的各花器官原基分化时就已形成。另外, 该突变体的抽穗期推迟, 株高降低, 穗数增多, 表明其营养生长也受到一定的影响。遗传分析表明mf2突变体表型受单隐性核基因控制。利用SSR、InDel分子标记将MF2定位于第1染色体的标记SSR39108和InD39210之间, 区间大小约为102 kb。     

A simple,rapid and reliable bioassay for evaluating seedling vigor under submergence in <Emphasis Type="Italic">indica</Emphasis> and <Emphasis Type="Italic">japonica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Submergence is a major stress causing yield losses particularly in the direct-seeded rice cultivation system and necessitates the development of a simple, rapid and reliable bioassay for a large scale screening of rice germplasms with tolerance against submergence stress. We developed two new bioassay methods that were based primarily on the seedling vigor evaluated by the ability of fast shoot elongation under submerged conditions, and compared their effectiveness with two other available methods. All four bioassay methods using cultivars of 7 indica and 6 japonica types revealed significant and consistent cultivar differences in seedling vigor under submergence and/or submergence tolerance. Japonica cultivars were more vigorous than indica cultivars, with Nipponbare being the most vigorous. The simplest test tube method showed the highest correlations to all other methods. Our results suggest that seedling vigor serves as a submergence avoidance mechanism and confers tolerance on rice seedlings to flooding during early crop establishment. A possible relationship is discussed between seedling vigor based on fast shoot elongation and submergence tolerance defined by recovery from submergence stress.     

Modification of plant height via RNAi suppression of OsGA20ox2 gene in rice

GA 20-oxidase (GA20ox) is a regulatory enzyme for the syntheses of biologically active GAs in plants. The loss-of-function mutations in OsGA20ox2 of rice (Oryza sativa L.) generate the well-known Green Revolution gene sd-1, which cause the semi-dwarfism phenotype. In our present investigation, semi-dwarf plants were generated from a taller rice variety QX1 by RNAi suppression on the expression of OsGA20ox2. The 531bp-fragment of OsGA20ox2 was amplified by PCR from genomic DNA of QX1 and used to construct the hairpin RNAi vector pCQK2. The wild type QX1 was transformed with pCQK2 by Agrobacterium-mediated transformation and some independent transgenic RNAi lines exhibited semi-dwarfism. RT-PCR and Northern blot analyses showed that the expression of OsGA20ox2 was specifically suppressed in the RNAi semi-dwarf lines. Endogenous GA assays revealed that the contents of the GA20ox2-catalyzed products GA₁₉, GA₂₀ and the down-stream biologically active GA₁ were drastically reduced in the RNAi semi-dwarf lines. We further showed that the RNAi semi-dwarf lines could be restored to normal plant height by applying exogenous GA₃. The results indicated that the semi-dwarfism of the RNAi semi-dwarf lines was associated with the decreased expression of OsGA20ox2 gene and the reduced content of endogenous biologically active GA₁. Analyses of panicle length, seeds per panicle and 1000-grain weight suggested that the RNAi semi-dwarf lines showed stable grain yield compared with the wild type plants. It is demonstrated that the RNAi approach could be useful for plant breeding purposes in the future. Qing Yang, Chun-Lian Wang have contributed equally to this work.     

Identification of quantitative trait loci (QTLs) for the characters of vascular bundles in peduncle related to indica-japonica differentiation in rice (Oryza sativa L.)

The number of vascular bundles in peduncle and the ratio of vascular bundles to primary rachis branches (V/R ratio)distinguishable between indica andjaponica, are the traits associated with the processes of differentiation between indica and japonica inrice (Oryza sativa L.). In this paper a doubled-haploid population derived from the F₁ hybrid of a cross between anindica cultivar and a japonicacultivar was used to map quantitative trait loci(QTLs) controlling numbers of vascular bundles in peduncle, primary rachis branches and the V/R ratio. For vascular bundles, three QTLs were detected and they collectively explained 58.8% of the total variation. Among them, the QTLqVB-8 with the largest effect,located on chromosome 8, individually accounted for 31.1% of the total variation. Two QTLs controlling primary rachis branches, located on chromosome 8and 10 respectively, were identified and they individually explained 10.5% and18.0% of the total variation respectively. Three QTLs for the V/R ratio, mapped on chromosome 1, 2 and 8, respectively,jointly explained 61.3% of the total variation. Of the three QTLs, the QTL qV/R-1 with the largest additive effect,explained 25.3% of the total variation,was located on chromosome 1 and found to be closely linked to the gene sh-2, a major gene underlying grain-shattering ability. In addition, four and two pairs of significant epistatic QTLs were detected for vascular bundles and the V/R ratio,respectively, but none for rachis branches. Our results suggested that the numbers of vascular bundles and primary rachis branches were independently controlled by different polygenic systems, but the two polygenic systems shared a fraction of quantitative trait loci. The present study also demonstrated that the chromosome region carrying the QTL qV/R-1 for the V/R ratio and the gene sh-2 might play an important role in the processes ofindica-japonica differentiation in rice (Oryza sativa L.). This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular mapping of a new gene for resistance to rice blast (Pyricularia grisea Sacc.)

A single dominant blast resistance gene conferring resistance to a Korean rice blast isolate was identified in rice variety `Suweon 365'. We report the chromosomal localization and molecular mapping of this blast resistance gene designated as Pi-18, which confers resistance to Korean isolate `KI-313' of the blast pathogen. To know whether there is a relationship among genes conditioning resistance to location-specific isolates of the blast pathogen and thereby to identify linked markers to resistance gene for isolate KI-313 collected in Korea, RFLP markers previously reported to be linked to major blast resistance genes in different rice germplasm and other markers mapped to nearby regions were surveyed for polymorphism between a resistant (`Suweon 365') and a susceptible (`Chucheongbyeo') parent. Linkage associations of the RFLP markers with the resistance gene were verified using an F2 and F3 segregating population of known blast reaction. RFLP analysis showed that Pi-18 was located near the end of chromosome 11, linked to a single copy clone RZ536 at a distance of 5.4 centiMorgans (cM) and that this gene was different from Pi-1(t). An allelism test revealed that this gene was also different from Pi-k. Currently, a combination of RAPD and microsatellite primers is being employed to find additional markers in this region. Tightly linked DNA markers will facilitate selection for resistant genotypes in breeding programs and provide the basis for map based cloning of this new blast resistance gene. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular mapping and characterization of traits controlling fiber quality in cotton

Cotton (Gossypium spp) is the world's leading natural fiber crop. Genetic manipulation continues to play a key role in the improvement of fiber quality properties. By use of DNA-based molecular markers and a polymorphic mapping population derived from an inter specific cross between TM-1 (G. hirsutum) and 3-79 (G. barbadense), thirteen quantitative trait loci (QTLs) controlling fiber quality properties were identified in 3-79, an extra long staple (ELS) cotton. Four QTLs influenced bundle fiber strength, three influenced fiber length, and six influenced fiber fineness. These QTLs were located on different chromosomes or linkage groups and collectively explained 30% to 60%of the total phenotypic variance for each fiber quality property in the F₂ population. The effects and modes of action for the individual QTLs were characterized with 3-79 alleles in TM-1 genetic background. The results indicated more recessive than dominant, with much less additive effect in the gene mode. Transgressive segregation was observed for fiber fineness that could be beneficial to improvement of this trait. Molecular markers linked to fiber quality QTLs would be most effective in marker-assisted selection (MAS) of these recessive alleles in cotton breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

QTL analysis of seed dormancy in rice (Oryza sativa L.)

Effective cumulative temperature (ECT) after heading would be a more reasonable parameter for seed sampling of pre-harvest sprouting/seed dormancy (SD) tests in segregating populations than the days after flowering. SD is an important agronomic trait associated with grain yielding, eating quality and seed quality. To identify genomic regions affecting SD at different grain-filling temperatures, and to further examine the association between SD and ECT during grain-filling, 127 double haploid (DH) lines derived from a cross between ZYQ8 (indica)/JX17 (japonica) by anther culture were analyzed. The quantitative trait loci (QTLs) and their digenic epistasis for SD were identified using a molecular linkage map of this population. A total of four putative QTLs for SD (qSD-3, qSD-5, 6 and 11) were detected on chromosomes 3, 5, 6 and 11, together explaining 41.4% of the phenotypic variation. Nine pairs of digenic epistatic loci were associated with SD on all but chromosome 9, and their contributions to phenotypic variation varied from 2.87%–8.73%. The SD QTL on chromosome 3 was identical to the QTLs found in other mapping populations with different genetic backgrounds, which could be a desirable candidate for gene cloning and marker-assisted selection in rice breeding.     

低植酸突变体水稻灌浆过程中籽粒淀粉合成与茎鞘物质转运特性

以协青早(XQZ)及其低植酸突变系(os-lpa1)为材料,通过对灌浆过程籽粒中植酸含量、蔗糖和淀粉积累变化动态的比较分析,并结合叶、茎、鞘等器官中碳水化合物的转运和籽粒淀粉合成代谢有关关键酶生理活性的测定,对os-lpa1灌浆充实不良、千粒重明显降低的生理原因进行了分析。结果表明,灌浆期os-lpa1剑叶、茎、鞘等器官中的蔗糖和可溶性总糖含量均高于其对照亲本XQZ,但os-lpa1籽粒中的淀粉积累量却明显降低。胚乳中ADPG焦磷酸化酶(ADPG-Ppase) 和淀粉磷酸化酶(SPase)的活性降低,可能是os-lpa1灌浆充实不良、籽粒淀粉积累量明显下降的主要原因之一。此外,os-lpa1灌浆期间叶鞘中的蔗糖磷酸合酶(SPS)和果糖-1,6-二磷酸酶(FBPase)活性也显著低于其相应灌浆期的对照亲本XQZ,导致茎鞘等器官中淀粉积累量增多和“流”不畅,这可能也是其产量水平降低的一个重要因素。     

水稻早衰突变体esl6的鉴定与基因定位

自然衰老提高了植物对环境的适应性,是其生长发育的重要生命历程,但在农业生产中,叶片一旦早衰,将极大影响作物的产量和品质。为探索水稻叶片衰老的分子机理,我们对EMS诱变获得的一个早衰突变体esl6进行了研究。田间种植情况下,四叶期之前,esl6与野生型无明显差异,之后心叶发育成完整叶后叶尖黄化,叶基部保持正常绿色,一直持续到开花期;在灌浆期,esl6的所有叶片均不同程度地黄化早衰,且叶片上部的衰老程度明显严重于叶片基部。衰老部位细胞结构异常,主要表现为细胞膜破裂、液泡变大和细胞器不完整等,叶绿体中基质类囊体破裂,含有较多的淀粉粒。与野生型相比,esl6叶尖衰老部位的SOD、CAT和POD活性以及超氧阴离子O₂^?、H₂O₂和羟自由基·OH含量均极显著升高。早衰不仅导致esl6叶片光合色素含量和净光合速率极显著降低,还引起esl6的植株变矮和叶片变短,倒一和倒二节间极显著变短是导致esl6植株矮化的主要原因。遗传分析表明该性状受一对隐性核基因调控,利用西大1A/esl6的F₂分离群体,最终将调控基因定位在第9染色体203 kb的物理范围内,为下一步基因的克隆和功能研究奠定了基础,有利于水稻叶片衰老分子机理的阐释。     

Heterosis in CMS hybrids of cotton for photosynthetic and chlorophyll fluorescence parameters

Summary The characterization of photosynthetic, chlorophyll fluorescence parameters and yield and yield components was compared between two newly developed CMS cotton hybrids (H1, H2) and their parents, as well as currently planted hybrid and conventional cultivars (C1, artificially produced hybrid CCRI 29 and C2, conventional cultivar CCRI 12) under the field condition. The results showed that P_n, F_v/F_m, Φ_PSII and qP of hybrids was significantly higher than that of C1 and C2. Furthermore, heterosis was found on photosynthetic parameters in hybrids over their parents at the first four growth stages, while only heterosis over mean of parents (HOMP) was detected in chlorophyll fluorescence parameters. It was clearly shown that one male-sterile line M2 was higher than the hybrids in F_v/F_m, Φ_PSII and qP over all growth stages. Significantly positive correlations (P < 0.01) were found between LAI or Yield and P_n, C_s, T_r, F_v/F_m, Φ_PSII, or qP, while significantly negative correlations between C_i and LAI or Yield, and no significance between qN and Chl a, C_s and qP were found. It was concluded that CMS hybrid cottons showed better potential to maintain relatively higher photosynthetic ability during the growth, which contributes to the increased lint yield.     

Through the genetic bottleneck: O. rufipogon as a source of trait-enhancing alleles for O. sativa

This paper summarizes results from a decade of collaborative research using advanced backcross (AB) populations to a) identify quantitative trait loci (QTL) associated with improved performance in rice and to b) clone genes underlying key QTLs of interest. We demonstrate that AB-QTL analysis is capable of (1) successfully uncovering positive alleles in wild germplasm that were not obvious based on the phenotype of the parent (2) offering an estimation of the breeding value of exotic germplasm, (3) generating near isogenic lines that can be used as the basis for gene isolation and also as parents for further crossing in a variety development program and (4) providing gene-based markers for targeted introgression of alleles using marker-assisted-selection (MAS). Knowledge gained from studies examining the population structure and evolutionary history of rice is helping to illuminate a long-term strategy for exploiting and simultaneously preserving the well-partitioned gene pools in rice.     

Validating a segment on the short arm of chromosome 6 responsible for genetic variation in the hull silicon content and yield traits of rice

Rice is a typical silicon-accumulating plant and the beneficial effect of silicon on rice has long been recognized. In a previous study using 244 recombinant inbred lines (RILs) of an indica rice cross, Zhenshan 97B/Milyang 46 grown in 2003, four QTLs were detected for hull silicon content. QTL qHUS-6 had the largest effect among these, and the same interval also had significant effects on yield traits in the same population. The primary objective of this study was to validate the QTL effect in this region on HUS and yield traits. The same RIL population and another RIL population of lower heterogeneity were grown in 2004. QTL qHUS-6 was found to have significant additive effects on hull silicon content with a consistent direction in the two populations. From a residual heterozygous line selected from RILs of the same cross, 15 F_2:3 lines that differed only in a 2.15-Mb segment extending from RM587 to RM6119 on the short arm of chromosome 6 were derived. In these lines, qHUS-6 displayed a major effect, so did QTLs for yield traits previously detected in the same region. Two more QTLs for HUS detected in 2003, qHUS-1-1 and qHUS-1-2, also had consistent effects in the Zhenshan 97B/Milyang 46 RIL population in 2004. Thus this study verified three candidate regions for fine mapping HUS QTLs and determining the genetic relationship between silicon content and yield traits in rice.     

以盒维数法分形分析水稻根系形态特征及初探其与锌吸收积累的关系

利用特定根盒装土, 培养4个水稻品种(MADHUKAR、IR8192-200、IR26、IR8192-31)植株, 用钉板法结合透明塑料膜固定获得近似原位根系样品, 扫描得到根系的二维平面图像, 以分形理论为基础, 利用盒维数法结合根系图像分形分析程序计算根系构型的分形维数和分形丰度, 比较各品种根系的形态特征, 并对分形参数、根系长度和 植株锌含量间的相关关系做了初步探讨。结果表明, 根系分形维数和分形丰度以MADHUKAR最大, IR8192-200最小, 说明MADHUKAR根系分支多, 在土壤中拓展体积大。分形维数、分形丰度与根系总长度之间均呈明显正相关, 而且根系总长度与分形丰度相关系数高于与分形维数的相关系数。分形维数和分形丰度与植株地上部干重、单位Zn浓度所产出的地上部生物量、地上部Zn吸收总量之间均呈显著正相关, 与地上部Zn浓度呈负相关。水稻根系形态和构型的变化影响植株生长, 影响植株Zn吸收积累及体内Zn的利用效率。盒维数法分形分析模型可用于研究水稻根系形态和构型, 为其提供新方法。     

不同粒型稻米碾磨特性及蛋白质分布的比较

以3个具不同品质和粒形特点的典型水稻品种,比较研究了稻米碾磨特性及蛋白质含量与组分在籽粒内部的分布情况。碾磨程度和达到相应碾磨程度所需碾磨时间之间的非线性关系显示糠层由外到内硬度不断增加;淀粉胚乳层硬度保持不变,且高于糠层硬度。不同品种达到相同碾磨程度所需时间差异较大,说明淀粉等物质的沉积密度在品种中存在较大差异。蛋白质含量在籽粒不同部位不是均匀分布的,3个品种糙米中均约85%的蛋白质分布在胚乳层中,清芦占11的蛋白质含量以外层胚乳最高,苏御糯和扬辐粳4901蛋白质含量均以糠层最高。随碾磨程度的提高,籽粒蛋白质含量由表及里呈降低趋势,核心层胚乳只有糠层的1/2左右,但不同类型水稻品种的降低趋势不同。SDS-PAGE分析表明,不同种子贮藏蛋白组分在籽粒内部的分布是相对均匀的,说明不同蛋白质组分在积累过程中,其遗传表达相对同步。     

QTL-based analysis of genotype-by-environment interaction for grain yield of rice in stress and non-stress environments

Use of DNA-based markers can accelerate cultivar development in variable cultivation environments since, in contrast to phenotype, DNA markers are environment-independent. In an effort to elucidate the genetic basis of genotype-by-environment interaction (G × E) for yield of rice (Oryza sativa L.), the associations between 139 AFLP markers and grain yield were determined for rice grown in fresh water (EC of 0.65 dS m⁻¹) and saline conditions (EC of 4–8 dS m⁻¹) with 0 kg ha⁻¹ or 100 kg ha⁻¹ nitrogen fertilizer in the years 2000 and 2001. A population of recombinant inbred lines of rice, developed from an IR29 × Pokkali cross, was used in the study. Both genotype × salinity and genotype × nitrogen level interactions were significant, with the genotype × salinity interaction being stronger. Through multiple regression analysis using a stepwise procedure for selecting markers, 36 markers were detected for grain yield in the four test conditions and of these 28 were detected in only one test condition implying strong environmental specificity for yield QTL expression. However, the fact that eight QTLs were detected in more than one test condition points to the existence of wide-adaptability genes in this cross. Markers with significant associations with yield explained between 37% and 48% of the yield variation in each test condition. Superior genotypes of rice were identified in all four test conditions based on their marker signatures. Furthermore, across N fertilizer regimes, yield predicted from summed additive effects of QTLs were significantly correlated with observed yield in the same year and across years. Thus marker-assisted selection can help breeders overcome the problem of low selection efficiency encountered during phenotypic selection for yield in stress environments.     

栽培稻种内rDNA基因IGS序列分析及系统学意义

以普通野生稻为对照, 将rDNA基因间区(IGS)序列用于栽培稻种内不同亚种以及亚种内不同品种的亲缘关系分析。结果表明, 栽培稻种内IGS序列长度为2 130~2 145 bp, G+C含量为74.59%~75.29%, 变异位点229个, 占10.70%, 信息位点76个, 占3.51%。IGS序列中籼亚种和粳亚种之间有38个亚种标志性碱基差异, 主要分布在IGS 5′ 端近400 bp的序列中。用IGS序列构建的系统树能将栽培稻的籼亚种和粳亚种分为两大类, 亚种内不同亲缘关系的品种也能区分开。本研究结果支持爪哇稻为栽培稻中一个独立亚种的观点。