Evolutionary and comparative analyses of the soybean genome

The soybean genome assembly has been available since the end of 2008. Significant features of the genome include large, gene-poor, repeat-dense pericentromeric regions, spanning roughly 57% of the genome sequence; a relatively large genome size of ~1.15 billion bases; remnants of a genome duplication that occurred ~13 million years ago (Mya); and fainter remnants of older polyploidies that occurred ~58 Mya and >130 Mya. The genome sequence has been used to identify the genetic basis for numerous traits, including disease resistance, nutritional characteristics, and developmental features. The genome sequence has provided a scaffold for placement of many genomic feature elements, both from within soybean and from related species. These may be accessed at several websites, including http://www.phytozome.net, http://soybase.org, http://comparative-legumes.org, and http://www.legumebase.brc.miyazaki-u.ac.jp. The taxonomic position of soybean in the Phaseoleae tribe of the legumes means that there are approximately two dozen other beans and relatives that have undergone independent domestication, and which may have traits that will be useful for transfer to soybean. Methods of translating information between species in the Phaseoleae range from design of markers for marker assisted selection, to transformation with Agrobacterium or with other experimental transformation methods.     

Cover crops and interrow tillage for weed control in short season maize (Zea mays)

Weed competition can cause substantial maize (Zea mays L.) yield reductions. Interseeding maize with cover crops or a combination of interrow cultivation and interseeded cover crops are possible alternative methods of weed control. This study was conducted to examine the potential of interrow cultivation plus cover crops to reduce weed density in maize without reducing the grain yield. Field experiments were conducted in 1993 and 1994 at two sites in Québec to determine the effects of planting 12 cover crops with maize on weed control. Fall rye (Secale cereal L.), hairy vetch (Vicia villosa Roth), a mixture of red clover (Trifolium pratense L.) and ryegrass (Lolium multiflorum Lam), a mixture of white clover (Trifolium repens L.) and ryegrass, subterranean clover (Trifolium subterraneum L.), yellow sweet clover (Meliotus officinalis Lam), black medic (Medicago lupulina L.), Persian clover (Trifolium resupinatum L.), strawberry clover (Trifolium fragiferum L.), crimson clover (Trifolium incarnatum L.), alfalfa (Medicago sativa L.), and berseem clover (Trifolium alexandrinum L.) were seeded at two planting dates, 10 and 20 days after maize emergence. Interrow cultivation was carried out weekly until forage seeding, with a final cultivation being conducted just prior to cover crop seeding. Cover crop planting date did not affect maize yields or the ability of interrow tillage plus cover crops to suppress the development of weed populations. Maize yield was less affected by the interseeded cover crops under conditions of adequate rainfall. Corn planted in fields heavily infested with weeds resulted in substantial yield reductions even when rainfall was adequate. Except for 1993 at l'Assomption interrow tillage plus cover crop treatments had consistently lower weed biomass when compared to the weedy control. Most of the weed control was due to the interrow cultivation performed prior to seeding of the cover crops. The lowest weed density occurred in the herbicide treated plots. The ability of interrow tillage plus cover crops to suppress the development of weeds was affected by the level of weed infestation, the growing conditions and location. The cover crops provide additional weed control but the interrrow tillage or some herbicide application may still be necessary.     

Development and application of PCR markers specific to the 1Ns chromosome of Psathyrostachys huashanica Keng with leaf rust resistance

Wheat–Psathyrostachys huashanica Keng disomic addition line 12-3 was developed and characterized using genomic in situ hybridization (GISH), expressed sequence tag–sequence tagged site (EST–STS), and sequence characterized amplified region (SCAR) markers. Mitotic and meiotic GISH analyses indicated that it contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. Eight EST–STS multiple-loci markers located on the homoeologous group 1 chromosomes of wheat amplified polymorphic bands in the 1Ns disomic addition line 12-3, which were unique to P. huashanica. These markers suggested that the introduced Ns chromosomes belonged to homoeologous group 1. Furthermore, diagnostic fragments of random amplified polymorphic DNA marker OPAG10₉₈₆ and simple sequence repeat marker Xgwm601 ₁₃₅ were cloned, sequenced, and converted into SCAR markers, i.e., RHS153 and SHS10, respectively, which were validated using a range of distinct plant species and a complete set of wheat–P. huashanica disomic addition lines (1Ns–7Ns, 2n = 44 = 22 II). The results demonstrated that the SCAR markers targeted the Ns genome of P. huashanica and they were linked to the 1Ns chromosome. In addition, 12-3 was evaluated to test its leaf rust resistance in the adult stages and its agronomic traits. These newly developed EST–STS and SCAR markers will be powerful tools for wheat breeders who want to screen for genotypes containing the 1Ns chromosome, with low costs and high throughput.     

Effects of species diversity on seasonal variation in herbage yield and nutritive value of seven binary grass-legume mixtures and pure grass under cutting

Intensively managed sown temperate grasslands are generally of low species diversity, although swards based on grass-legume mixtures may have superior productivity and herbage quality than grass-only swards. We conducted a cutting experiment over two years to test the effect of species composition and diversity on herbage yield, contents of N, neutral detergent fibre (NDF) and in vitro organic matter digestibility (IVOMD). Perennial ryegrass (PR, Lolium perenne) was sown alone and with each of four forage legumes: red clover (RC, Trifolium pratense), lucerne (LU, Medicago sativa), birdsfoot trefoil (BT, Lotus corniculatus) and white clover (WC, Trifolium repens); WC was also sown with hybrid ryegrass (HR, Lolium × boucheanum), meadow fescue (MF, Festuca pratensis) and timothy (TI, Phleum pratense). Herbage productivity was lowest in pure PR followed by PR/BT, and highest in PR/RC; this mixture had the highest legume proportion, N content and N yield. There was less WC in swards with HR and MF than with PR and TI. These differences were reflected in N contents of herbage of the mixtures. Legumes had higher N and lignin and lower NDF contents and IVOMD than grasses. Among legumes, NDF content was highest and IVOMD lowest in LU, followed by BT and the clovers. The highest N content was in WC. Among grasses, PR and HR had lower NDF contents and a higher IVOMD than MF; the highest N content was in PR. The grass component of mixtures had less effect than the legume component on herbage yield and quality. Results are discussed in terms of their potential to contribute to forage resources in farming practice and enhance resource use efficiency and ecosystem services.     

Development of a core set of SSR markers for the characterization of <Emphasis Type="Italic">Gossypium</Emphasis> germplasm

Molecular markers such as simple sequence repeats (SSR) are a useful tool for characterizing genetic diversity of Gossypium germplasm. Genetic profiles by DNA fingerprinting of cotton accessions can only be compared among different collections if a common set of molecular markers are used by different laboratories and/or research projects. Herein, we propose and report a core set of 105 SSR markers with wide genome coverage of at least four evenly distributed markers per chromosome for the 26 tetraploid cotton chromosomes. The core marker set represents the efforts of ten research groups involved in marker development, and have been systematically evaluated for DNA polymorphism on the 12 genotypes belonging to six Gossypium species [known collectively as the cotton marker database (CMD) panel]. A total of 35 marker bins in triplex sets were arranged from the 105 markers that were each labeled with one of the three fluorescent dyes (FAM, HEX, and NED). Results from this study indicated that the core marker set was robust in revealing DNA polymorphism either between and within species. Average value of polymorphism information content (PIC) among the CMD panel was 0.65, and that within the cultivated cotton species Gossypium hirsutum was 0.29. Based on the similarity matrix and phylogenetic analysis of the CMD panel, the core marker set appeared to be sufficient in characterizing the diversity within G. hirsutum and other Gossypium species. The portability of this core marker set would facilitate the systematic characterization and the simultaneous comparison among various research efforts involved in genetic diversity analysis and germplasm resource preservation.     

Generation of interspecific hybrids between <Emphasis Type="Italic">Trifolium vesiculosum</Emphasis> and <Emphasis Type="Italic">T. alexandrinum</Emphasis> using embryo rescue

Interspecific hybrids were developed between Trifolium alexandrinum cultivar Wardan × Trifolium vesiculosum and T. alexandrinum cultivar BL1 × T. vesiculosum through embryo rescue, as the crosses failed to set seed under natural conditions. Trifolium vesiculosum was used as a donor/male parent in this study as it is reported to possess tolerance to stem rot and high forage yield. Fertilization in crossed florets of the crosses was manifested from the recovery of swollen ovaries (< 7.80%) and confirmed from the presence of one degenerated ovule in most (> 93.00%) of the swollen ovaries. The hybrid embryos at various developmental stages (heart, torpedo and cotyledonary) were rescued at a frequency of 2.56% from Wardan × T. vesiculosum and 6.12% from BL1 × T. vesiculosum. Differentiation occurred only in the cotyledonary stage embryos, resulting in 17 putative interspecific hybrid plantlets. The assessment of plantlet hybridity through SSR markers (for the alleles inherited from the donor parent), micromorphological leaf traits (leaf texture and stomata) and morphological characters (plant height, leaflet length and width) confirmed production of two interspecific hybrids designated as AV1 and BV3 representing both the crosses. AV1 displayed moderate resistance and BV3 was resistant to stem rot.     

Effect of spring fertilization on ecosystem services of organic wheat and clover relay intercrops

Nitrogen (N) deficiency and weed infestation are main factors limiting yield and yield stability in organic wheat. Organic fertilizers may be used to improve crop performance but off-farm input costs tend to limit profitability. Instead, forage legumes may be inserted into the crop rotation to improve the N balance and to control weed infestation. In opposition to simultaneous cropping, relay intercropping of legumes in organic winter wheat limits resource competition for the legume cover crop, without decreasing the performance of the associated wheat.The aim of this study is to evaluate the effect of spring organic fertilization on the performance of intercropped legumes and wheat, and on services provided by the legume cover.Two species of forage legumes (Trifolium pratense L. and Trifolium repens L.) were undersown in winter wheat (Triticum aestivum L. cv Lona) in five organic fields during two consecutive crop seasons. Organic fertilizer was composed of feather meal and applied on wheat at legume sowing. The cover crop was maintained after the wheat harvest and destroyed just before sowing maize.Spring organic nitrogen fertilization increased wheat biomass (+35%), nitrogen (+49%), grain yield (+40%) and protein content (+7%) whatever the intercropping treatment. At wheat harvest, red clover biomass was significantly higher than white clover one (1.4 vs. 0.7 t ha⁻¹). Nitrogen fertilization decreased forage legume above-ground biomass at wheat harvest, at approximately 0.5 t ha⁻¹ whatever the specie. No significant difference in forage legume biomass production was observed at cover killing. Nitrogen accumulation in legume above-ground tissues was significantly higher for white clover than for red clover. Both red and white clover species significantly decreased weed infestation at this date. Nitrogen fertilization significantly increased weed biomass whatever the intercropping treatment and decreased nitrogen accumulation in both clover species (−12%).We demonstrated that nitrogen fertilization increased yield of wheat intercropped with forage legume while the performance of legumes was decreased. Legume growth was modified by spring fertilization whatever the species.     

Development of EST-SSR markers and construction of a linkage map in faba bean (Vicia faba)

To develop a high density linkage map in faba bean, a total of 1,363 FBES (Faba bean expressed sequence tag [EST]-derived simple sequence repeat [SSR]) markers were designed based on 5,090 non-redundant ESTs developed in this study. A total of 109 plants of a ‘Nubaria 2’ × ‘Misr 3’ F₂ mapping population were used for map construction. Because the parents were not pure homozygous lines, the 109 F₂ plants were divided into three subpopulations according to the original F₁ plants. Linkage groups (LGs) generated in each subpopulation were integrated by commonly mapped markers. The integrated ‘Nubaria 2’ × ‘Misr 3’ map consisted of six LGs, representing a total length of 684.7 cM, with 552 loci. Of the mapped loci, 47% were generated from multi-loci diagnostic (MLD) markers. Alignment of homologous sequence pairs along each linkage group revealed obvious syntenic relationships between LGs in faba bean and the genomes of two model legumes, Lotus japonicus and Medicago truncatula. In a polymorphic analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES markers showed polymorphisms. Along with the EST-SSR markers, the dense map developed in this study is expected to accelerate marker assisted breeding in faba bean.     

Identification of QTLs for agronomic traits using association mapping in lentil

A diverse panel of 96 genotypes of lentil was used in this study to identify QTL for nine agronomic traits through marker-trait association analysis. This study showed significant genetic variability among the lentil genotypes for nine agronomic traits and had medium to large broad sense heritability estimates (h² =?0.58–0.95). Screening of 534 SSR markers resulted in 266 polymorphic loci that generated 697 alleles ranging from 2 to 16 alleles per locus across the genotypes. The model-based population structure analysis identified two distinct subpopulations among lentil genotypes and each subpopulation did not show any admixture. Marker trait association (MTA) analysis following ML model resulted in the identification of 24 MTAs for nine traits at P?<?0.01. The per cent of phenotypic variation explained by each associated marker with particular agronomic trait ranged from 7.3 to 25.8%. The highest proportion of total phenotypic variation (23.1–25.8%) was explained by the QTLs controlling the primary branches/per plant. In the present study, few EST-SSR markers showed significant association with days to maturity, pods/plant, secondary branches/plant, 100 seed weight, yield/plant and reproductive duration and explained large phenotypic variation (7.3–23.8%). Hence, these markers can be used as functional markers in lentil breeding program for developing improved cultivars.     

A new DNA marker for sex identification in purple asparagus

Garden asparagus (Asparagus officinalis L.) is an economically-important perennial crop. This plant is dioecious, as there are both male and female individuals; male individuals are preferred over females for agricultural production. To reduce the time required for garden asparagus breeding, various male-specific DNA markers are utilized. Male-specific DNA markers, such as Asp1-T7sp and MSSTS710, are currently available for sex identification in many asparagus cultivars. In the current study, we found that these markers are not suitable for sex identification in the purple asparagus cultivar ‘Pacific Purple’, as male-specific amplification of this marker was detected in some male individuals of this cultivar but not in other males. The Asp1-T7sp marker is suitable for use in sex identification in various Asparagus species related to A. officinalis, indicating that the region around this marker is conserved among these species. Thus, we isolated a DNA fragment around this marker by inverse PCR and produced a new DNA marker, MspHd, based on this sequence. However, like Asp1-T7sp and MSSTS710, MspHd was not suitable for sex identification in the cultivar ‘Pacific Purple’. Since all ‘Pacific Purple’ males have morphologically similar male flowers with functional stamens, we produced a new male-specific marker based on the sex determination gene, MSE1/AspMYB35/AspTDF1, which is responsible for stamen development. This marker, named AspMSD, is suitable for sex identification in ‘Pacific Purple’. In addition, this marker can be utilized for sex identification in various asparagus cultivars and some related Asparagus species.     

Genetic dissection of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) tiller,plant height,and grain yield based on QTL mapping and metaanalysis

Tiller number per plant (TN) and plant height (PH) are important agronomic traits related to grain yield (GY) in rice (Oryza sativa L.). A total of 30 additive quantitative trait loci (A-QTL) and 9 significant additive × environment interaction QTLs (AE-QTL) were detected, while the phenotypic and QTL correlations confirmed the intrinsic relationship of the three traits. These QTLs were integrated with 986 QTLs from previous studies by metaanalysis. Consensus maps contained 7156 markers for a total map length of 1112.71 cM, onto which 863 QTLs were projected; 78 meta-QTLs (MQTLs) covering 11 of the 30 QTLs were detected from the cross between Dongnong422 and Kongyu131 in this study. A total of 705 predicted genes were distributed over the 21 MQTL intervals with physical length <0.3 Mb; 13 of the 21 MQTLs, and 34 candidate genes related to grain yield and plant development, were screened. Five major QTLs, viz. qGY6-2, qPH7-2, qPH6-3, qTN6-1, and qTN7-1, were not detected in the MQTL intervals and could be used as newly discovered QTLs. Candidate genes within these QTL intervals will play a meaningful role in molecular marker-assisted selection and map-based cloning of rice TN, PH, and GY.     

Marker assisted backcross breeding approach to improve blast resistance in Indian rice (Oryza sativa) variety ADT43

Marker assisted backcrossing breeding has become one of the essential tools in transferring novel genes to adapted varieties and was employed to pyramid three blast resistance genes Pi1, Pi2 and Pi33 to a popular susceptible rice variety ADT43. Gene pyramiding process was facilitated by marker aided selection for both foreground as well as background genotype. Previously reported linked molecular markers were deployed to survey resistant and susceptible genotypes. In the BC₃F₁ generation four lines viz, AC-B3-11-7, AC-B3-11-36, AC-B3-11-57, AC-B3-11-83 were identified to be pyramided with three genes and subjected to background analysis and a genome recovery up to 95 % was observed and advanced to further generations. Morphological, yield and grain quality traits were significantly similar to ADT43. The introgressed lines with three gene combinations were highly resistant to the blast pathogen compared to genotypes with single genes and the susceptible checks under blast nursery screening at two epiphytotic locations; Coimbatore and Gudalur. The selected three gene pyramided backcross lines in the desirable background were advanced to obtain an improved ADT 43 with resistance to blast disease.     

A skeletal linkage map of Hordeum bulbosum L. and comparative mapping with barley (H. vulgare L.)

A restriction fragment length polymorphism (RFLP) based linkage map of a cross between two diploid Hordeum bulbosum (2n = 2x = 14) clones, PB1 and PB11, was constructed from 46 recombinant progeny clones. Since both parents are heterozygous, separate and combined parental maps were constructed. All of the RFLP markers screened had previously been mapped in barley (H. vulgare L.) so that comparative maps could be produced. The PB1 linkage map consists of 20 RFLP marker loci assigned to four linkage groups covering 94.3 cM. The PB11 linkage map consists of 27 RFLP marker loci assigned to six linkage groups covering 149.1 cM. Thirteen markers polymorphic in both parents were used as ‘anchors’ to create a combined linkage map consisting of 38 loci assigned to six linkage groups and covering a genetic distance of 198 cM. Marker order was highly conserved in a comparison with the linkage map of H. vulgare (Laurie etal., 1995). However, in contrast, the genetic distances for the same markers were very different being 649 cM and 198 cM respectively, a genetic distance ratio of 1: 3.3. Thus although the map was short, it can be presumed to cover half the genome of H. bulbosum. This study provides further confirmation of the close relationship between the two species and gives a basis for the development of marker mediated introgression through interspecific hybridisation between the two species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Performance of Timothy-based Grass/Legume Mixtures in Cold Winter Region

This study sought to identify grass/legume mixtures that increase the yield and persistence of forage stands with improved nutritive quality in cold‐winter regions, compared with the standard mixture of timothy (Phleum pratense L.)/red clover (Trifolium pratense L.)/alsike clover (Trifolium hybridum L.). Timothy was mixed with either perennial ryegrass (Lolium perenne L.), meadow fescue (Festuca pratensis L.) or Kentucky bluegrass (Poa pratensis L.). The legumes in mixtures were red clover, alfalfa (Medicago sativa L.) or white clover (Trifolium repens L.). Averaged over three production years, the majority of mixtures had greater dry matter (DM) yields than the standard (8.35 t ha^?1). Timothy, grown alone and in three mixtures, outyielded the standard by 19–30 %. Yield reductions in mixtures over the 3‐year period were greatest with red clover, and least with bluegrass. Mixtures with alfalfa were highest in nitrogen (28.4 g kg^?1), while grasses grown alone (24.6 g kg^?1) and the standard mixture (25.1 g kg^?1) were the lowest in N. Mixtures with red clover or alfalfa had the least neutral detergent fibre (NDF), averaging 418 and 429 g kg^?1 respectively. Mixtures including white clover were initially low in NDF at 347 g kg^?1 in year 1 but increased to 550 g kg^?1 in year 3 as white clover composition declined in the sward.     

An SSR-based genetic map of pepper (Capsicum annuum L.) serves as an anchor for the alignment of major pepper maps

Of the Capsicum peppers (Capsicum spp.), cultivated C. annuum is the most commercially important, but has lacked an intraspecific linkage map based on sequence-specific PCR markers in accord with haploid chromosome numbers. We constructed a linkage map of pepper using a doubled haploid (DH) population derived from a cross between two C. annuum genotypes, a bell-type cultivar ‘California Wonder’ and a Malaysian small-fruited cultivar ‘LS2341 ({"type":"entrez-nucleotide","attrs":{"text":"JP187992","term_id":"347495878"}}JP187992)’, which is used as a source of resistance to bacterial wilt (Ralstonia solanacearum). A set of 253 markers (151 SSRs, 90 AFLPs, 10 CAPSs and 2 sequence-tagged sites) was on the map which we constructed, spanning 1,336 cM. This is the first SSR-based map to consist of 12 linkage groups, corresponding to the haploid chromosome number in an intraspecific cross of C. annuum. As this map has a lot of PCR-based anchor markers, it is easy to compare it to other pepper genetic maps. Therefore, this map and the newly developed markers will be useful for cultivated C. annuum breeding.     

Development of NBS-related microsatellite (NRM) markers in hexaploid wheat

NBS (nucleotide binding site) genes, one type of the most important disease-resistance genes in the plant kingdom, are usually found clustered in genome. In this study, a total of 2288 full-length NBS protein-coding sequences were isolated from the wheat (Triticum aestivum L.) genome, and 903 TaNBSs of which were found expressed in wheat. Meanwhile, 2203 microsatellite loci were detected within 1061 scaffolds containing TaNBS. The distribution of these microsatellite loci across wheat homologous groups (HG) is 20% HG2, 16% HG7, 15% HG1, 15% HG6, 12% HG4, 12% HG5 and 10% HG3. We developed 1830 NBS-related microsatellite (NRM) markers for the microsatellite loci on TaNBS-scaffold sequences.Among them, 342 NRM markers were developed for HG2 with the largest number of microsatellite loci, and 69 out of these markers were anchored to the wheat genetic map using mapping population. Then, a total of 26 2AS-NRM markers, nine 2BL-NRM markers and nine 2DL-NRM markers were integrated into the genetic maps carrying Yr69, Pm51 and Pm43, respectively. Finally, candidate sequences, within the gene clusters where Yr5 and Sr21 located, were analyzed according to the genomic position information of TaNBS and NRM markers. These NRM markers have clear chromosome locations and are correlated with potential disease resistance sequences, which can be manipulated to mapping or adding linkage markers of disease-resistance genes or QTLs, especially for those in the NBS gene clusters.