Genomic regions underlying agronomic traits in linseed （Linum usitatissimum L.） as revealed by association mapping

The extreme climate of the Canadian Prairies poses a major chal enge to improve yield. Although it is possible to breed for yield per se, focusing on yield-related traits could be advantageous because of their simpler genetic architecture. The Canadian flax core col ection of 390 accessions was genotyped with 464 simple sequence repeat markers, and phenotypic data for nine agronomic traits including yield, bol s per area, 1,000 seed weight, seeds per bol , start of flowering, end of flowering, plant height, plant branching, and lodging col ected from up to eight environments was used for association mapping. Based on a mixed model （principal component analysis （PCA） t kinship matrix （K））, 12 significant marker-trait associations for six agronomic traits were identi-fied. Most of the associations were stable across environments as revealed by multivariate analyses. Statistical simulation for five markers associated with 1000 seed weight indicated that the favorable al eles have additive effects. None of the modern cultivars carried the five favorable al eles and the maximum number of four observed in any accessions was mostly in＆amp;nbsp;breeding lines. Our results confirmed the complex genetic architecture of yield-related traits and the inherent difficulties associated with their identification while il ustrating the potential for improvement through marker-assisted selection.     

Population structure and marker�Ctrait association analysis of the US peanut (Arachis hypogaea L.) mini-core collection

Peanut (Arachis hypogaea L.) is one of the most important oilseed and nutritional crops in the world. To efficiently utilize the germplasm collection, a peanut mini-core containing 112 accessions was established in the United States. To determine the population structure and its impact on marker-trait association, this mini-core collection was assessed by genotyping 94 accessions with 81 SSR markers and two functional SNP markers from fatty acid desaturase 2 (FAD2). Seed quality traits (including oil content, fatty acid composition, flavonoids, and resveratrol) were obtained through nuclear magnetic resonance (NMR), gas chromatography (GC), and high-performance liquid chromatography (HPLC) analysis. Genetic diversity and population structure analysis identified four major subpopulations that are related to four botanical varieties. Model comparison with different levels of population structure and kinship control was conducted for each trait and association analyses with the selected models verified that the functional SNP from the FAD2A gene is significantly associated with oleic acid (C18:1), linoleic acid (C18:2), and oleic-to-linoleic (O/L) ratio across this diverse collection. Even though the allele distribution of FAD2A was structured among the four subpopulations, the effect of FAD2A gene remained significant after controlling population structure and had a likelihood-ratio-based R ( 2 ) (R ( LR ) ( 2 ) ) value of 0.05 (oleic acid), 0.09 (linoleic acid), and 0.07 (O/L ratio) because the FAD2A alleles were not completely fixed within subpopulations. Our genetic analysis demonstrated that this peanut mini-core panel is suitable for association mapping. Phenotypic characterization for seed quality traits and association testing of the functional SNP from FAD2A gene provided information for further breeding and genetic research.     

Association analysis of fiber quality traits and exploration of elite alleles in Upland cotton cultivars/accessions （Gossypium hirsutum L.）

Exploring the elite al eles and germplasm acces-sions related to fiber quality traits wil accelerate the breeding of cotton for fiber quality improvement. In this study, 99 Gossypium hirsutum L. accessions with diverse origins were used to perform association analysis of fiber quality traits using 97 polymorphic microsatel ite marker primer pairs. A total of 107 significant marker-trait associations were detected for three fiber quality traits under three different environments, with 70 detected in two or three environments and 37 detected in only one environment. Among the 70 significant marker-trait associations, 52.86% were reported previously, implying that these are stable loci for target traits. Furthermore, we detected a large number of elite al eles associated simulta-neously with two or three traits. These elite al eles were mainly from accessions col ected in China, introduced to China from the United States, or rare al eles with a frequency of less than＆amp;nbsp;5%. No one cultivar contained more than half of the elite al eles, but 10 accessions were col ected from China and the two introduced from the United States did contain more than half of these al eles. Therefore, there is great potential for mining elite al eles from germplasm accessions for use in fiber quality improvement in modern cotton breeding.     

Analysis of Genetic Diversity and Relationships in a Tunisian Fig （Ficus carica） Germplasm Collection by Random Amplified Microsatellite Polymorphisms

The random amplified mirosatellite polymorphism method was performed in a set of Tunisian fig landraces using eighteen primer combinations. A total of sixty three random amplified microsatellite polymorphism （RAMPO） markers were scored and used either to assess the genetic diversity in these cultivars or to detect cases of mislabeling. Opportunely, data proved that the designed procedure constitutes an attractive and fast method with low costs and prevents radio exposure. As a result, we have identified the primer combinations that are the most efficient to detect genetic polymorphism in this crop. Therefore, the derived unweighted pair-group method with arithmetic averages （UPGMA） dendrogram illustrates the genetic divergence among the landraces studied and exhibits a typically continuous variation. Moreover, no evident correlation between the sexes of trees was observed. In addition, using these markers, discrimination between landraces has been achieved. Thus, random amplified mirosatellite polymor- phism is proved to be powerful for characterizing the local fig germplasm.     

Identification of genomic regions determining flower and pod numbers development in soybean （Glycine max L.）

Flower and pod numbers per plant are important agronomic traits underlying soybean yield.So far quantitative trait loci (QTL) detected for flower and pod-related traits have mainly focused on the final stage,and might therefore have ignored genetic effects expressed during a specific developmental stage.Here,dynamic expressions of QTL for flower and pod numbers were identified using 152 recombinant inbred lines (RILs) and a linkage map of 306 markers.Wide genetic variation was found among RILs;17 unconditional and 18 conditional QTL were detected for the two traits at different developmental stages over two years.Some QTL were detected only at one stage and others across two or more stages,indicating that soybean flower and pod numbers development may be governed by time-dependent gene expression.Three main QTL (qfn-Chr18-2,qfn-Chr20-1,and qfn-Chr19) were detected for flower number,and two main QTL (qpn-Chr11 and qpn-Chr20) were detected for pod number.The phenotypic variation explained by them ranged from 6.1% to 34.7%.The markers linked to these QTL could be used in marker-assisted selection for increasing soybean flower and pod numbers,with the ultimate aim of increasing soybean yield.Comparison of the QTL regions for flower and pod numbers traits with the related genes reported previously showed that seven and four related genes were located in the QTL regions of qfn-Chr11 and qfn-Chr19,respectively.Tbese results provide a basis for fine mapping and cloning of flower and pod development-related genes.     

Molecular characterization and phylogenetic analysis of three odorant binding protein gene transcripts in Dendrolimus species （Lepidoptera： Lasiocampidae）

Pine caterpillar moths, Dendrolimus spp. （Lepidoptera： Lasiocampidae）, are serious economic pest of pines. Previously, phylogenetic analyses of Dendrolimus using different methods yielded inconsistent results. The chemosensory systems of insects may play fundamental roles in promoting speciation. Odorant-binding proteins （OBPs） participate in the first step of odor detection. Studying the evolution of OBPs in closely related species may help us to identify their role in speciation. We identified three OBPs - one pheromone-binding protein and two general odorant-binding proteins - from male antennae of four Dendrolimus species, D. superans （Butler）, D. punctatus （Walker）, D. kikuchii Matsumura, and D. houi Lajonquiere, the olfactory recognition systems of which had not been previously investigated. We analyzed their molecular characteristics and compared their sequences to those of OBPs in D. tabulaeformis Tsai et Liu. Ka/Ks ratio analyses among the five Dendrolimus species indicate that PBP1 genes experienced more evolutionary pressure than the GOBPs. Phylogenetic relationships of PBP1 and GOBP1 both indicated that D. houi was the basal species, then branched D. kikuchii, while D. tabulaeformis, D. punctatus, and D. superans evolved more recently. These relationships are consistent with the changes in sex pheromone components of these five species. Dendrolimus tabulaeformis and D. punctatus are closely related sister species. However, the distances among GOBP2 sequences in the five Dendrolimus were very short, and the relationships of D. houi and D. la＇kuchii could not be resolved. Integrating our results with those of previous studies, we hypothesized that D. kikuchii, D. punctatus and D. superans evolved from the basal ancestor because of sex pheromone mutations and environmental pressure.     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A 18T 16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed ofa 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2gene on microspore development is discussed in the present paper.     

Genetic analysis of tolerance to photo-oxidative stress induced by high light in winter wheat （Triticum aestivum L.）

High light induced photooxidation (HLIP) usually leads to leaf premature senescence and causes great yield loss in winter wheat. In order to explore the genetic control of wheat tolerance to HLIP stress, a quantitative trait loci (QTL) analysis was conducted on a set of doubled haploid population, derived from two winter wheat cultivars. Actual values of chlorophyll content (Chl), minimum fluorescence level (Fo), maximum fluorescence level (Fm), and the maximum quantum efficiency of photosystem Ⅱ (Fv/Fm) under both HLIP and non-stress conditions as well as the ratios of HLIP to non-stress were evaluated. HLIP considerably reduced Chl, Fm, and Fv/Fm, but increased Fo, compared with that under non-stress condition. A total of 27, 16, and 28 QTLs were associated with the investigated traits under HLIP and non-stress and the ratios of HLIP to non-stress, respectively. Most of the QTLs for the ratios of HLIP to non-stress collocated or nearly linked with those detected under HLIP condition. HLIP-induced QTLs were mapped on 15 chromosomes, involving in 1A, 1B, 1D,2A, 2B, 2D, 3A, 3B, 4A, 4D, 5B, 6A, 6B, 7A, and 7D while those expressed under non-stress condition involved in nine chromosomes, including 1B, 1D, 2A, 2B, 3B, 4A, 5A, 5B, and 7A. The expression patterns of QTLs under HLIP condition were different from that under non-stress condition except for six loci on five chromosomes. The phenotypic variance explained by individual QTL ranged from 5.0% to 19.7% under HLIP, 8.3% to 20.8% under non-stress, and 4.9% to 20.2% for the ratios of HLIP to non-stress, respectively. Some markers, for example,Xgwm192 and WMC331 on 4D regulating Chl, Fo, Fm, and Fv/Fm under HLIP condition, might be used in marker assistant selection.     

Genetic analysis and molecular mapping of a novel gene for zebra mutation in rice （Oryza sativa L.）

A novel zebra mutant, zebra-15, derived from the restorer line JinhuilO （Oryza sativa L. ssp. indica） treated by EMS, displayed a distinctive zebra leaf from seedling stage to jointing stage. Its chlorophyll content decreased （55.4%） and the ratio of Chla/Chlb increased （90.2%） significantly in the yellow part of the zebra-15, compared with the wild type. Net photosynthetic rate and fluorescence kinetic parameters showed that the decrease of chlorophyll content significantly influenced the photosynthetic efficiency of the mutant. Genetic analysis of F2 segregation populations derived from the cross of XinonglA and zebra-15 indicated that the zebra leaf trait is controlled by a single recessive nuclear gene. Ninety-eight out of four hundred and eighty pairs of SSR markers showed the diversity between the XinonglA and the zebra-15, their F2 population was then used for gene mapping. Zebra-15 （Z-15） gene was primarily restricted on the short arm of chromosome 5 by 150 F2 recessive individuals, 19.6 cM from marker RM3322 and 6.0 cM from marker RM6082. Thirty-six SSR markers were newly designed in the restricted location, and the Z-15 was finally located between markers nSSR516 and nSSR502 with the physical region 258 kb by using 1,054 F2 recessive individuals.     

Mapping QTLs for seed yield and drought susceptibility index in soy-bean（Glycine max L.） across different environments

Drought stress has long been a major constraint in maintaining yield stability of soybean （Glycine max （L.） Merr.） in rainfed ecosystems. The identification of consistent quantitative trait loci （QTL） involving seed yield per plant （YP） and drought susceptibility index （DSI） in a population across different environments would therefore be important in molecular marker-assisted breeding of soybean cultivars suitable for rainfed regions. The YP of a recombinant line population of 184 F2：7：11 lines from a cross of Kefengl and Nannong1138-2 was studied under water-stressed （WS） and well-watered （WW） conditions in field （F） and greenhouse （G） trials, and DSI for yield was calculated in two trials. Nineteen QTLs associated with YP-WS and YP-WW, and 10 QTLs associated with DSI, were identi- fied. Comparison of these QTL locations with previous findings showed that the majority of these regions control one or more traits re- lated to yield and other agronomic traits. One QTL on molecular linkage group （MLG） K for YP-F, and two QTLs on MLG C2 for YP-G, remained constant across different water regimes. The regions on MLG C2 for YP-WW-F and MLG H for YP-WS-F had a pleiotropic effect on DSI-F, and MLG A1 for YP-WS-G had a pleiotropic effect on DSI-G. The identification of consistent QTLs for YP and DSI across different environments will significantly improve the efficiency of selecting for drought tolerance in soybean.     

Diversity characterization and association analysis of agronomic traits in a Chinese peanut (Arachis hypogaea L.) mini-core collection

Association mapping is a powerful approach for exploring the molecular basis of phenotypic variations in plants.A peanut(Arachis hypogaea L.)mini-core collection in China comprising 298 accessions was genotyped using 109 simple sequence repeat(SSR)markers,which identified 554 SSR alleles and phenotyped for 15 agronomic traits in three different environments,exhibiting abundant genetic and phenotypic diversity within the panel.A model-based structure analysis assigned all accessions to three groups.Most of the accessions had the relative kinship of less than 0.05,indicating that there were no or weak relationships between accessions of the mini-core collection.For 15 agronomic traits in the peanut panel,generally the Q t K model exhibited the best performance to eliminate the false associated positives compared to the Q model and the general linear model-simple model.In total,89SSR alleles were identified to be associated with 15 agronomic traits of three environments by the Q t K model-based association analysis.Of these,eight alleles were repeatedly detected in two or three environments,and 15 alleles were commonly detected to be associated with multiple agronomic traits.Simple sequence repeat allelic effects confirmed significant differences between different genotypes of these repeatedly detected markers.Our results demonstrate the great potential of integrating the association analysis and marker-assisted breeding by utilizing the peanut mini-core collection.     

花生金属硫蛋白基因AhMT3a的克隆及其表达

以鲁花14号花生为材料,从花生cDNA文库和基因组中筛选和克隆了花生的金属硫蛋白基因AhMT3a。该基因全长785 bp,有2个内含子,开放阅读框由201个碱基组成,编码66个氨基酸,其中包含13个半胱氨酸(Cys),预测其分子量为6.83kD,等电点为4.59。运用生物信息学手段对AhMT3a蛋白的信号肽、跨膜区、亚细胞定位和疏水性进行了预测。与拟南芥、棉花和草莓等植物type 3 MTs的序列比对结果表明,花生和其他不同植物的MT3在氨基酸序列上具有较高的同源性,从系统发育树中可以看出AhMT3a和蒿麦的金属硫蛋白亲缘关系较近。半定量RT-PCR和芯片杂交结果显示花生AhMT3a在花中表达量最高,在种子中表达量最低;在ABA、NaCl及PEG等不同处理下,表达量变化不大。     

花生叶片离体培养花器官分化及其开花结果研究

以花生幼叶为外植体进行离体培养,研究BA浓度对花器官分化的影响并进一步观察试管内花器官的发育.结果表明:经MSB 1mg/LBA 0.5mg/LKIN 2mg/LIAA培养基诱导的愈伤组织,转接到附加1～3mg/LBA的MSB培养基上培养,均能直接诱导分化花器官,但2mg/LBA的诱导效率最高达21.13%;诱导分化的花器官转接到MSB培养基继续培养,部分花器官可以在试管内开花、受精、成针、结实.试验实现了以花生幼叶为外植体,在试管内完成诱导花芽、开花、受精、形成果针、子房膨大,直至形成荚果等过程,为离体条件下研究花生花器官分化、荚果及种子发育提供了技术体系和材料.     

Assessment of transpiration efficiency in peanut (Arachis hypogaea L.) under drought using a lysimetric system

Transpiration efficiency (TE) is an important trait for drought tolerance in peanut ( Arachis hypogaea L.). The variation in TE was assessed gravimetrically using a long time interval in nine peanut genotypes (Chico, ICGS 44, ICGV 00350, ICGV 86015, ICGV 86031, ICGV 91114, JL 24, TAG 24 and TMV 2) grown in lysimeters under well-watered or drought conditions. Transpiration was measured by regularly weighing the lysimeters, in which the soil surface was mulched with a 2-cm layer of polythene beads. TE in the nine genotypes used varied from 1.4 to 2.9 g kg⁻¹ under well-watered and 1.7 to 2.9 g kg⁻¹ under drought conditions, showing consistent variation in TE among genotypes. A higher TE was found in ICGV 86031 in both well-watered and drought conditions and lower TE was found in TAG-24 under both water regimes. Although total water extraction differed little across genotypes, the pattern of water extraction from the soil profile varied among genotypes. High water extraction within 24 days following stress imposition was negatively related to pod yield ( r ²   =   0.36), and negatively related to water extraction during a subsequent period of 32 days ( r ²   =   0.73). By contrast, the latter, i.e. water extraction during a period corresponding to grain filling (24 to 56 days after flowering) was positively related to pod yield ( r ²   =   0.36). TE was positively correlated with pod weight ( r ²   =   0.30) under drought condition. Our data show that under an intermittent drought regime, TE and water extraction from the soil profile during a period corresponding to pod filling were the most important components.     

Diversity and compatibility of peanut (Arachis hypogaea L.) bradyrhizobia and their host plants

A high degree of genetic diversity among 125 peanut bradyrhizobial strains and among 32 peanut cultivars collected from different regions of China was revealed by using the amplified fragment length polymorphism (AFLP) technique. Eighteen different peanut bradyrhizobial genotypes and six peanut cultivars were selected for symbiotic cross-inoculation experiments. The genomic diversity was reflected in the symbiotic diversity. The peanut cultivars varied in their ability to nodulate with the strains used. Some cultivars had a more restricted host range than the others. Also the strains displayed a range of nodulation patterns. In yield formation there were clear differences between the plant cultivar/bradyrhizobium combinations. There was good compatibility between some peanut bradyrhizobial strains and selected cultivars, with inoculation resulting in well-nodulated, high-yielding symbiotic combinations, but no plant cultivar was compatible with all strains used. The strains displayed a varying degree of effectiveness, with some strains being fairly effective with all cultivars and others with selected ones. The AFLP genotypes of the strains did not explain the symbiotic behavior, whereas the yield formation of the plant cultivars was more related to the genotype. It is concluded that to obtain optimal nitrogen fixation efficiency of peanut in the field, compatible plant cultivar-bradyrhizobium combinations should be selected either by finding inoculant strains compatible with the plant cultivars used, or plant cultivars compatible with the indigenous bradyrhizobia.     

花生根部性状的遗传分析

利用花生RIL群体,分析了11个花生根部性状的遗传力,估算基因对数及性状间的相互关系,根据偏度系数(g1)和峰度系数(g2)估算控制性状的基因互作情况。结果表明:11个性状都是受多基因控制的数量性状,在RIL群体中基因型间的差异均表现为连续变异和明显的超亲分离。侧根干重的遗传力最高达0.60,其次是侧根鲜重,为0.58,而其他性状的遗传力均较低。控制主根长性状的多基因间存在互作,互作方式为重叠作用;控制主根粗(3cm)性状的基因间也存在一定的重叠作用,但是作用不明显;控制其他性状的基因都存在互作,表现为互补作用,但互补作用的强弱有差异。主根粗(1cm)、主根粗(3cm)、主根干重、主根鲜重、侧根干重和侧根鲜重之间都显著或极显著相关;根体积与主根粗(1cm)、主根粗(3cm)、侧根干重和侧根鲜重显著或极显著相关。     

Leaflet development, induction time, and medium influence somatic embryogenesis in peanut (Arachis hypogaea L.)

Factors affecting somatic embryogenesis in peanut (Arachis hypogaea L.) using leaflet explants of seedlings obtained from aseptically germinated embryo axes were evaluated. Somatic embryogenesis was influenced by developmental stage, leaflet size, induction medium, and time on induction medium. Leaflets that were 5–7 mm long had a greater embryogenic response than smaller or larger leaflets. Percent embryogenesis and mean number of embryos were related to the developmental stage of germinating seedlings. A greater response was obtained if leaflets were folded and closely appressed. Preselection of leaflets increased percent embryogenesis from 21% up to 67%. As leaflets unfolded, embryogenesis decreased; open leaflets lost the potential for embryogenesis. The optimal induction conditions were a 7-day incubation period on Murashige and Skoog medium with 136 μm 2,4-dichlorophenoxyacetic acid and 0.93 μm kinetin. Somatic embryos germinated to form plants that exhibited a normal morphology. Received: 29 December 1997 / Revision received: 9 April 1998 / Accepted: 24 April 1998     

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm2 embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).