Detection and validation of QTLs associated with seed longevity in rice (Oryza sativa L.)

Seed longevity in rice is a major determinant in seed production and germplasm preservation. In this paper, a recombinant inbred line (RIL) population consisting of 172 lines derived from the cross between Xiang743 and ‘Katy’ was used to map quantitative trait loci (QTLs) for seed longevity (SL) after seed storage for 18 and 30 months under ambient conditions. Two putative QTLs, qSL‐2 and qSL‐8, were detected and located on chromosomes 2 and 8, respectively. qSL‐2 is an allele from Xiang743 allele and increases seed longevity. qSL‐8 was a novel QTL from ‘Katy’ allele and increases seed longevity. qSL‐8 explained 15.29% and 17.35% of the phenotypic variance after seed storage for 18 and 30 months, respectively. Furthermore, qSL‐8 was validated in a secondary population developed by self‐pollination of a residual heterozygous line (RHL) selected from the RIL population, which explained 25.93% of the phenotypic contribution. These results provide an opportunity for map‐based cloning of qSL‐8. Furthermore, qSL‐8 may be a target for improving seed longevity by marker‐assisted selection (MAS) in rice.     

Genome-wide association study reveals loci associated with seed longevity in common wheat (Triticum aestivum L.)

Seed longevity could significantly determine seed regeneration cycle and greatly affect wheat production. With the 90 K chip assays, a genome-wide association study was performed to identify seed longevity-related markers and loci in common wheat. Seed germination ratios (GR) under artificially ageing of 166 wheat accessions across three environments were evaluated to assess seed longevity. Totally, 23 longevity-related loci were identified in the study, explaining 6.7%–11.4% of the phenotypic variations. Of these, QlgGR.cas-1A and QlgGR.cas-2B.2 were deemed as stable loci associated with wheat seed longevity. Fifteen loci were found overlapped with known quantitative trait loci or genes. Besides, QlgGR.cas-1A, QlgGR.cas-2B.2, QlgGR.cas-3D.1, QlgGR.cas-3D.2, QlgGR.cas-4A.2, QlgGR.cas-5A.1, QlgGR.cas-5A.2 and QlgGR.cas-6A.1 were colocated with seed dormancy-related loci. Significant additive effects were obtained for seed longevity by pyramiding favourable alleles. Several candidate genes were found involved in signal transduction and stress resistance pathways by sequencing analysis of significantly longevity-related molecular markers. These results might provide new sights into the genetic architecture of seed longevity.     

Breeding of tetra null soybean (Glycine max) for lipoxygenase,kunitz trypsin inhibitor,lectin, and 7S α' subunit proteins

Soybean seed includes various bioactive substances. Also, they contain a variety of antinutritional factors including lipoxygenase, Kunitz trypsin inhibitor (KTI), lectin, and 7S α' subunit proteins. The genetic removal of these proteins will improve the nutritional value of soybean seed. The objective of this research was to breed new soybean with tetra recessive alleles (lox1lox2lox3/lox1lox2lox3‐ti/ti‐le/le‐cgy1/cgy1) for lipoxygenase, KTI, lectin, and 7S α' subunit proteins. Seven parents were used to breed tetra null strain. SDS‐PAGE and Western blot analysis were used to determine the presence or absence of lipoxygenase, 7S α' subunit, KTI, and lectin proteins in mature seed. Tetra null soybean line has a purple flower, determinate growth habit, tan pod, and yellow seed coat colour. Stem height of the breeding line was 62.3 cm. The 100‐seed weight of the breeding line was 27.1 g and yield (t/ha) was 2.84. This is the first soybean strain with lox1lox2lox3/lox1lox2lox3‐ti/ti‐le/le‐cgy1/cgy1 genotype (absence of lipoxygenase, KTI, lectin, and 7S α' subunit proteins).     

Mapping of quantitative trait loci controlling seed longevity of rice (Oryza sativa L.) after various periods of seed storage

Seed longevity varies considerably in cultivated rice (Oryza sativa L.), but the underlying genetic mechanism of longevity has not been well elucidated. Quantitative trait loci (QTL) that control seed longevity after various periods of seed storage were sought using recombinant inbred lines derived from a combination involving ‘Milyang23’(Indica‐type) and ‘Akihikari’ (Japonica‐type). In all, 12 QTLs for germination and normal seedling growth were detected as indices of seed longevity on chromosome 7 (one region) and chromosome 9 (two regions) in treated seeds that had been stored under laboratory conditions for 1, 2 or 3 years.‘Milyang23’ alleles of all QTLs promoted germination and normal seedling growth after all durations of storage. These QTL regions were detected repeatedly in more than one seed condition. Therefore, we infer that these regions control seed longevity.     

Variation of seed storage proteins in landraces of common bean (Phaseolus vulgaris L.) from Basilicata,Southern Italy

Summary Twenty landraces of common bean (Phaseolus vulgaris L.) from Sarconi and Rotonda two locations of Basilicata, a Southern Italy region, were screened for variation in seed storage proteins (phaseolin and phytohemagglutinin) by polyacrylamide gel electrophoresis (SDS/PAGE and IEF-SDS/PAGE). No variation of the main seed protein fractions was observed within each landrace. Phaseolin patterns type C and T were exhibited from the landraces; the type C resulted predominant with a frequency of 70%. Only the C type was observed for the landraces of Rotonda, T and C for those from Sarconi. Two variants were observed for the phytohemagglutinin by SDS-PAGE, one was common to eighteen landraces. However, these variants submitted to IEF-SDS/PAGE resulted similar to the type T_G2described by Brown. The importance to safeguard these landraces is stressed by the observation that the more spread commercial cultivars of common bean growing in Southern-Italy had a T phaseolin pattern. A reduction of C type diffusion could produce the loss a typical trait of common bean in Mediterranean regions.     

Genetic mapping within the wheat D genome reveals QTL for germination, seed vigour and longevity, and early seedling growth

Quantitative trait loci (QTL) controlling germination, seed vigour and longevity, and early seedling growth were identified using a set of common wheat lines carrying known D genome introgression segments. Seed germination (capacity, timing, rate and synchronicity) was characterized by a standard germination test, based either on the 1 mm root protrusion (germination sensu stricto) or the development of normal seedlings. To quantify seed vigour, the same traits were measured from batches of seed exposed for 72 h at 43°C and high (ca. 100%) humidity. Seed longevity was evaluated from the relative trait values. Seedling growth was assessed both under non-stressed and under osmotic stress conditions. Twenty QTL were mapped to chromosomes 1D, 2D, 4D, 5D, and 7D. Most of the QTL for germination sensu stricto clustered on chromosome 1DS in the region Xgwm1291–Xgwm337. A region on chromosome 7DS associated with Xgwm1002 harboured loci controlling the development of normal seedlings. Seed vigour-related QTL were present in a region of chromosome 5DL linked to Xgwm960. QTL for seed longevity were coincident with those for germination or seed vigour on chromosomes 1D or 5D. QTL for seedling growth were identified on chromosomes 4D and 5D. A candidate homologues search suggested the putative functions of the genes within the respective regions. These results offer perspectives for the selection of favourable alleles to improve certain vigour traits in wheat, although the negative effects of the same chromosome regions on other traits may limit their practical use.     

水稻种子萌发过程中胚蛋白质差异表达分析

采用IEF-SDS-PAGE双向电泳技术,对金稻1号水稻种子未萌发和萌发1-4d胚进行蛋白质分离。发现金稻1号水稻种子未萌发和萌发1-4d胚在PDQuest图像分析软件可识别的蛋白质点约215、201、187、162、132个,其中表达量变化2.5倍以上的蛋白质点有22个。选取表达量变化2.5倍以上的12个差异蛋白质点进行胶内酶解,并进行肽质量指纹图谱及其生物信息分析,初步鉴定出6个蛋白质,分别为硫氧还蛋白; 热休克蛋白;ATP合酶;LEA蛋白12;核苷二磷酸激酶;乙二醛酶。对这些蛋白质在水稻种子萌发过程中的作用进行了讨论。     

Fine mapping of qSS‐9, a major and stable quantitative trait locus,for seed storability in rice (Oryza sativa L.)

Seed storability in rice (Oryza sativa L.) is an important agronomic trait. We previously showed a quantitative trait locus of seed storability, qSS‐9, on chromosome 9 in a backcross population of ‘Koshihikari’ (japonica) / ‘Kasalath’ (indica) // ‘Koshihikari’. In this study, fine mapping of the chromosomal location of qSS‐9 was performed. Effect of ‘Kasalath’ allele of qSS‐9 was validated using a chromosome segment substitution line, SL36, which harboured the target quantitative trait loci (QTL) from ‘Kasalath’ in the genetic background of ‘Nipponbare’ under different ageing treatments in different environments. Subsequently, an F₂ population from a cross between ‘Nipponbare’ and SL36 was used for fine mapping of qSS‐9. Simultaneously, four subnear isogenic lines (sub‐NILs) that represented different recombination breakpoints across the qSS‐9 region were developed from F₃ progeny. Finally, the qSS‐9 locus was located between the Indel markers Y10 and Y13, which delimit a region of 147 kb in the ‘Nipponbare’ genome. These results provide a springboard for map‐based cloning of qSS‐9 and possibilities for breeding rice varieties with strong seed storability.     

Heat stress during seed development affects forage brassica (Brassica napus L.) seed quality

In two consecutive seasons, forage rape (Brassica napus L.) plants were exposed to short periods (240°C hr) of heat stress (30°C day/25°C night) during seed filling (80% seed moisture content = S1), at physiological maturity (50% seed moisture content = S2) and at both S1 plus S2 (=S3) in a Biotron before being returned to the field until seed harvest. Seeds were hand harvested at 14% seed moisture content and their quality assessed by measuring germination, seed vigour (using the accelerated ageing and conductivity tests) and seed mass (as determined by thousand seed weight). Heat stress at both S1 and S2 caused a small (<10%) but significant reduction in germination in both seasons. There was a significant heat stress timing interaction in the first but not the second season. Reductions in germination were a result of increased abnormal seedling production not seed death. All three heat stress treatments significantly reduced seed vigour, with S3>S2>S1. Seed mass was reduced by heat stress at S1 but not at S2. Variable seed vigour in high‐germinating New Zealand‐produced forage rapeseed lots is most likely explained by short periods of heat stress during seed development.     

QTL analysis of seed storability in rice

A double haploid population, which consists of 127 lines derived from anther culture of a typical indica and japonica hybrid ‘ZYQ8’/‘JX17’, was used in this study. Seed storability was investigated by using the storage property measured by the difference of seed germination rates before and after treatment of the rice seeds under 40°C and 95% relative humidity for 10 days in a phytotron. Three QTLs related to rice seed storability were detected on chromosomes 9, 11 and 12, with the LOD scores 2.76, 4.83 and 2.54, respectively, together explaining 35.4% of the genetic variation. The ‘JX17’ allele at qLS‐9 and the ‘ZYQ8’ alleles at qLS‐11 and qLS‐12 could enhance the rice seed storability. The effects of the ‘ZYQ8’ alleles of qLS‐11 and qLS‐12 were also verified using chromosome segment substitution lines.     

Quantitative trait loci controlling rice seed germination under salt stress

Salt tolerance of rice (Oryza sativa L.) at the seed germination stage is one of the major determinants for the stable stand establishment in salinity soil. One population of recombinant inbred lines (RILs, F_2:9), derived from a cross between a japonica rice landrace tolerant to salt stress and a sensitive indica rice variety, was used to determine the germination traits including imbibition rate and germination percentage under control (water) and salt stress (100 mM NaCl) for 10 days at 30 °C. The multiple interval mapping (MIM) were applied to conduct QTL for the traits. The results showed that seed germination was a quantitative trait controlled by several genes, and strongly affected by salt stress. A total of 16 QTLs were detected in this study, and each QTL could explain 4.6–43.7% of the total phenotypic variance. The expression of these QTLs might be developmentally regulated and growth stage-specific. In addition, only one digenic interaction was detected under salt stress, showing small effect on germination percentage with R² 2.7%. Among sixteen QTLs detected in this study, four were major QTLs with R² > 30%, and some novel alleles of salt tolerance genes in rice. The results demonstrated that the japonica rice Jiucaiqing is a good source of gene(s) for salt tolerance and the major or minor QTLs identified could be used to improve the salt tolerance by marker-assisted selection (MAS) in rice.     

赤霉素对盐胁迫抑制水稻种子萌发的缓解作用的蛋白质组分析

用粳稻日本晴(Oryza sativa L. cv.Nipponbare),研究了盐胁迫对水稻种子萌发的抑制作用和赤霉酸(GA₃)对盐胁迫的缓解作用;分别以H₂O (对照）,5 g L^-1 NaCl (处理I),5 g L^-1 NaCl + 100 μmol L^-1 GA₃(处理II)培养水稻种苗48 h,提取芽中的蛋白质,利用双向电泳(2-DE)和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术分析了水稻蛋白质组的变化。结果表明,在盐胁迫条件下,日本晴种子的萌发显著受到抑制,而GA₃能显著缓解这种抑制作用;用ImageMaster软件分析2-DE凝胶,发现有4个蛋白质斑点表现出显著的变化,在盐胁迫下斑点S1、S2和S3表达下调而斑点S4消失,在GA₃与盐共处理时,这4个蛋白质点的表达均有不同程度的恢复;经MALDI-TOF MS分析,其中2个蛋白质斑点(S1,S3)分别被鉴定为isoflavone reductase-like蛋白与葡萄糖磷酸变位酶,这些蛋白可能与GA₃提高水稻耐盐性途径相关。     

Incongruity in the interspecific crosses of Vitis L. Reproductive expression in the F1 progeny

Summary F₁ progeny from three classes of crosses were studied for indications of incongruity in the form of reproductive dysfunction. Percent fruit set was found to be approximately equivalent to the additive inverse of percent aborted flowers, as the effect of shot berries was small and not significant. In interspecific crosses, barriers were manifested as reduced fruit set and seed number per berry. These two characters were used to create an index of reproductive efficiency. Nine F₁s from crosses of most divergent class, V. riparia × V. vinifera (R × V), displayed significantly tower reproductive efficiency, percent fruit set, and seed number per berry. Four female F₁s from the least divergent class, V. riparia × V. riparia (R × R), had the highest reproductive efficiency, percent fruit set and seed number per berry, when pollinated by two V. riparia pollen sources. Seventeen F₁s from the highly-intercrossed class, V. riparia × French Hybrid (R × FH), displayed intermediate levels of reproductive efficiency, percent fruit set, and seed number per berry. The R × FH class involved V. vinifera, V. rupestris, V. aestivalis, V. labrusca, V. berlandieri, V. cinerea, and V. riparia. It is proposed that the complex intercrossing in the pedigrees of the R × FH class ameliorated incongruity. In the most divergent class, R × V, progenies of one of the V. riparia parents displayed fewer incongruity effects than progenies of the other V. riparia parents. Progeny testing of many proposed parental combinations may be used to uncover useful congruent combinations.Minnesota Agricultural Experiment Station, Scientific Journal Series No. 20835     

Out-crossing frequency and genetic analysis of hybrids between transgenic glufosinate herbicide-resistant rice and the weed, red rice

The potential of transferring herbicide resistance from transgenic rice (Oryza sativa L.) varieties to sexually compatible weeds is of paramount importance for development of effective weed control strategies. The objective of this research was to determine the genetic control and frequency of natural outcrossing between a transgenic, glufosinate-resistant rice line and a Louisiana biotype of red rice (Oryza sativa L.). Molecular and phenotypic data showed that outcrossing in field plots between a non-transgenic purple marker line and red rice did occur within one field season, but at a low rate of <1%. Similarly, molecular and phenotypic data demonstrated that glufosinate resistance was transferred from the transgenic line to the red rice biotype in the field within one year at a low frequency of 0.3%. Compared to parental lines, the transgenic-red rice hybrids were extremely late, tall, and never set seed during the normal field season. Genetic analyses in all F₂ populations showed glufosinate resistance behaved in a Mendelian fashion as a single, dominant gene. Presence of the bar gene for glufosinate resistance did not increase fitness or seed fecundity in hybrids or subsequent progeny. The genetic analyses and outcrossing results from this study suggest that an effective management program can be developed to prolong the usefulness of transgenic, glufosinate herbicide technology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Low Temperature Effects during Seed Filling on Chickpea Genotypes (Cicer arietinum L.): Probing Mechanisms affecting Seed Reserves and Yield

Chickpea is sensitive to cold conditions (<15 °C), particularly at its reproductive phase and consequently it experiences significant decrease in the seed yield. The information about the effects of cold stress on chickpea during the seed filling phase is lacking. Moreover, the underlying metabolic reasons associated with the low temperature injury are largely unknown in the crop. Hence, the present study was undertaken with the objectives: (i) to find out the possible mechanisms leading to low temperature damage during the seed filling and (ii) to investigate the relative response of the microcarpa (Desi) and the macrocarpa (Kabuli) chickpea types along with elucidation of the possible mechanisms governing the differential cold sensitivity at this stage. At the time of initiation of the seed filling (pod size ∼1 cm), a set of plants growing under warm conditions of the glasshouse (temperature: 17/28 ± 2 °C as average night and day temperature) was subjected to cold conditions of the field (2.3/11.7 ± 2 °C as average night and day temperature), while another set was maintained under warm conditions (control). The chilling conditions resulted in the increase in electrolyte leakage, the loss of chlorophyll, the decrease in sucrose content and the reduction in water status in leaves, which occurred to a greater extent in the macrocarpa type than in the microcarpa type. The total plant weight decreased to the same level in both the chickpea types, whereas the rate and duration of the seed filling, seed size, seed weight, pods per plant and harvest index decreased greatly in the macrocarpa type. The stressed seeds of both the chickpea types experienced marked reduction in the accumulation of starch, proteins, fats, crude fibre, protein fractions (albumins, globulins, prolamins and glutelins) with a larger decrease in the macrocarpa type. The accumulation of sucrose and the activity levels of the enzymes like starch synthase, sucrose synthase and invertase decreased significantly in the seeds because of the chilling, indicating impairment in sucrose import. Minerals such as calcium, phosphorous and iron as well as several amino acids (phenylalanine, tyrosine, threonine, tryptophan, valine and histidine) were lowered significantly in the stressed seeds. These components were limited to a higher extent in the macrocarpa type indicating higher cold sensitivity of this type.     

Comparative Morphological Studies of Microspore Derived and F2 Plants Obtained from an Interspecific Rice Hybrid Oryza sativa Linn. ×O. rufipogon Griff.)*

Anther culture of an interspecific rice hybrid from a cross of Oryza sativa× O. rufipogon was attempted. Of the 117 regenerated pollen clones, 56 could survive to maturity. A majority of these were either haploids or doubled haploids and very few turned out to be chromosomal variants. Comparative study of doubled haploids and the seed derived F₂ plants indicate the distinct advantages of anther culture techniques. (1) Androgenic plants, though few in number, showed greater ariation for all the traits with the exception of ear bearing tillers. (2) Predominance of recombinants with wild traits was observed in F₂ segregation. (3) It was possible to recover indica type recombinants among the anther-derived plants with one or two traits introgressed from O. rufipogon. These results suggest the feasibility and utility of anther culture in distant hybridization for incorporation of alien variation into cultivated rice.     

Exploiting the natural variation of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">thaliana</Emphasis> for the seed-specific production of proteins

The seed-specific expression of recombinant proteins in transgenic plants offers several interesting advantages over other production platforms. The aim of this study was to select accessions of Arabidopsis thaliana with the highest potential as a platform for seed-specific production of recombinant proteins. A. thaliana was chosen because of its flexibility, high seed yield per m2, high natural protein content and its non-food status. Seven characteristics were measured for 96 accessions; days to first flower bud, days to complete senescence, rosette size, number of main bolts, dry biomass of plant, seed yield and protein content of seeds. Three characteristics (length of life cycle, seed yield and protein content) were used to select accessions with a maximal yield. A variation of length of life cycle between 87 ± 11 days (Ler-1) and more than 200 days (several accessions) was registered. Seed yields per accession varied between 18 ± 16 mg (Wa-1) and 274 ± 76 mg (Mr-0). Protein content ranged between 30% (Ws-2) and 38% (Cvi-0). Based on the results of this study, accession Nok-3 is selected as the accession best suited for exploitation as a seed-based platform for the production of recombinant proteins. Nok-3 has a high seed yield (194 ± 66 mg) combined with a moderate protein content of 34.8% and short life cycle of 126 ± 17 days, resulting in a calculated protein yield per year three times higher than reference accession Col-0. In conclusion, this study illustrates the unexploited variability present in the Arabidopsis gene pool that can be used directly for further optimization of Arabidopsis seeds as production platform. In combination with A. thaliana’s rapid life cycle, flexibility, and high fertility, this makes it an attractive platform for the production of specific groups of recombinant proteins, such as high-purity products produced on a relatively small scale.     

Phaseolus coccineus Storage Proteins II. Electrophoretic Analysis and Erythroagglutinating Activity in Various Cultivars

Biochemical analyses of seed storage proteins of Phaseolus coccineus have been carried out to identify seventeen different cultivars. The electrophoretic patterns in native polyacrylamide gel electrophoresis (PAGE) and denaturing conditions (SDS-PAGE); evidenced qualitative and quantitative differences for the three major protein components: legumin, vicilin and phytohaemagglutinin (PHA). The results were confirmed by isoelectrofocusing (IEF) analyses. Erythroagglutination tests showed the presence of high agglutinating activity particularly in cultivars with low vicilin content. The experimental results allow one to distinguish all the cultivars by their electrophoretic spectra and agglutinating activities.     

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.     

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。