Protocol: A high-throughput DNA extraction system suitable for conifers

<p>Abstract</p> <p>Background</p> <p>High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers.</p> <p>Results</p> <p>We have developed a high-throughput DNA extraction protocol for conifers using an automated liquid handler and modifying the Qiagen MagAttract Plant Kit protocol. The modifications involve change to the buffer system and improving the protocol so that it almost doubles the number of samples processed per kit, which significantly reduces the overall costs. We describe two versions of the protocol: one for medium-throughput (MTP) and another for high-throughput (HTP) DNA isolation. The HTP version works from start to end in the industry-standard 96-well format, while the MTP version provides higher DNA yields per sample processed. We have successfully used the protocol for DNA extraction and genotyping of thousands of individuals of several spruce and a pine species.</p> <p>Conclusion</p> <p>A high-throughput system for DNA extraction from conifer needles and seeds has been developed and validated. The quality of the isolated DNA was comparable with that obtained from two commonly used methods: the silica-spin column and the classic CTAB protocol. Our protocol provides a fully automatable and cost effective solution for processing large numbers of conifer samples.</p

Higher fine-scale genetic structure in peripheral than in core populations of a long-lived and mixed-mating conifer - eastern white cedar (Thuja occidentalis L.)

<p>Abstract</p> <p>Background</p> <p>Fine-scale or spatial genetic structure (SGS) is one of the key genetic characteristics of plant populations. Several evolutionary and ecological processes and population characteristics influence the level of SGS within plant populations. Higher fine-scale genetic structure may be expected in peripheral than core populations of long-lived forest trees, owing to the differences in the magnitude of operating evolutionary and ecological forces such as gene flow, genetic drift, effective population size and founder effects. We addressed this question using eastern white cedar (<it>Thuja occidentalis</it>) as a model species for declining to endangered long-lived tree species with mixed-mating system.</p> <p>Results</p> <p>We determined the SGS in two core and two peripheral populations of eastern white cedar from its Maritime Canadian eastern range using six nuclear microsatellite DNA markers. Significant SGS ranging from 15 m to 75 m distance classes was observed in the four studied populations. An analysis of combined four populations revealed significant positive SGS up to the 45 m distance class. The mean positive significant SGS observed in the peripheral populations was up to six times (up to 90 m) of that observed in the core populations (15 m). Spatial autocorrelation coefficients and correlograms of single and sub-sets of populations were statistically significant. The extent of within-population SGS was significantly negatively correlated with all genetic diversity parameters. Significant heterogeneity of within-population SGS was observed for 0-15 m and 61-90 m between core and peripheral populations. Average <it>Sp</it>, and gene flow distances were higher in peripheral (<it>Sp </it>= 0.023, σ_g= 135 m) than in core (<it>Sp </it>= 0.014, σ_g= 109 m) populations. However, the mean neighborhood size was higher in the core (<it>Nb </it>= 82) than in the peripheral (<it>Nb </it>= 48) populations.</p> <p>Conclusion</p> <p>Eastern white cedar populations have significant fine-scale genetic structure at short distances. Peripheral populations have several-folds higher within-population fine-scale genetic structure than core populations. Anthropogenic disturbances and population fragmentation presumably have significant effects on fine-scale genetic structure in eastern white cedar. Core populations have higher neighborhood size than peripheral populations, whereas gene flow distances are higher in peripheral than in core populations. The results of our study contribute to the knowledge of poorly-understood spatial genetic structure of core versus peripheral populations in plants. As well, the information is of significance for conservation of genetic resources of eastern white cedar and perhaps of other long-lived forest trees with mixed-mating system.</p

Restoration of Arid Grasslands: Issues and Strategies

Economy of the Thar Desert of India, lying between 24-29°N latitude and 70-76°E longitude, is closely linked with the raising of livestock which mainly depends upon the native rangelands for their sustenance. Pearl millet, moth bean, cluster bean, range grasses and legumes, trees and shrubs are the major components of arid ecosystem. Perennial grasses, viz., buffel grass, bird wood grass, sewan and gramna are the dominating pasture species of the region. Due to frequent droughts and overgrazing, the productivity of the natural grasslands of the region has been steadily decreasing leading to reduced carrying capacity between 0.2-0.5 ACU per ha, which needs to be enhanced through improved technological interventions

Phenotypic Variability of \u3cem\u3eCenchrus ciliaris\u3c/em\u3e L. Germplasm in Field Gene Bank

The world faces a continual need to increase forage crop productivity, and to develop new varieties more adapted to changing environmental and biological challenges, and evolving needs of Local communities. One of the main reasons for under-utilization of germplasm, according to curators, breeders and other users of plant genetic resources, is the lack of adequate passport, characterization and evaluation data (Biodiversity International, 2007). Buffel grass (Cenchrus ciliaris L.) is a C4 perennial grass of arid lands distributed over hotter and drier parts of India, Mediterranean region and tropical and southern Africa. This forage grass is under the focus of different ecological issues like, response to desertification, quality of forage and impact of invasion. The use of genetic resources by the researchers, gene bank managers and farmers will be limited by non-availability of essential information of their phenotypic and genotypic characters. Therefore, the accurate documentation of information about the origin, characterization and performance of germplasm is essential for effective conservation, use and also for the Intellectual Property Rights (IPR) issues

Response of fodder cropping sequences to irrigation scheduling in arid environment

A field experiment was conducted at Jodhpur during kharif, rabi and summer seasons for three consecutive years (2008-09, 2009-10 and 2010-11) to assess the fodder production potential, water use, water use efficiency and fodder quality of different cropping systems under variable moisture regimes. The main plot treatments consist of four cropping sequences, i.e. bajra–lucerne, cowpea– oat – bajra, Cenchrus ciliaris - C. ciliaris + lucerne, bajra + cowpea (1:1) – oat–sorghum and three irrigation levels, i.e. 50, 75 and 100 mm CPE in subplots with three replications. Among the crop sequences round the year, maximum mean green fodder yield was recorded from cowpea-oat-bajra sequence, which was at par with bajra + cowpea – oat – sorghum sequence but significantly higher than C. ciliaris +lucerne and bajra–lucerne sequences. Similarly, dry matter yield was also maximum from cowpea– oat –bajra sequence which was also at par with C.ciliaris–C. ciliaris+lucerne and bajra + cowpea – oat – sorghum sequences but was only significantly higher than bajra – lucerne sequence. During rabi season all crops produced significantly higher green fodder yield at 50 mm CPE level than 75 and 100 mm CPE levels but dry matter yield was at par at 50 and 75 mm CPE levels. Green and dry matter yields of summer crops were significantly higher at 50 mm CPE level than that of 75 and 100 mm CPE. This showed that irrigation at 75 CPE level optimized dry fodder yield during rabi season and 50 mm CPE during summer season. The fodder yield of C. ciliaris + lucerne system at 50 mm CPE and 75 mm CPE was at par but significantly higher than that of 100 mm CPE while fodder yield of bajra + cowpea (1:1)-oat–sorghum and cowpea –oat–bajra at 50 mm CPE was significantly higher than that of 75 mm CPE. Water use efficiency (WUE) and water productivity were higher with bajra, C. ciliaris and oat grown with sprinkler irrigation. Among cropping sequences • ciliaris -C. ciliaris+ lucerne system had maximum WUE and water productivity but statistically at par with cowpea- oat –bajra sequence. Water use was higher at 50 mm CPE irrigation level but water use efficiency and water productivity were higher with 75 and 100 mm CPE level. Protein yield was maximum with bajra – lucerne system followed by cowpea –oat– bajra while C. ciliaris-C. ciliaris + lucerne system had lowest protein yield being at par with bajra + cowpea–oat–sorghum sequence. Protein yield was higher with 50 mm CPE level as compared to 75 and 100 mm CPE in all cropping sequences. Irrigation at 75 mm CPE was best for C. ciliaris + lucerne intercropping system and 50 mm CPE for bajra + cowpea, oat – bajra, cowpea–oat–sorghum and bajra-lucerne crop sequence for getting higher productivity of quality fodder under arid conditions

Near-saturated and complete genetic linkage map of black spruce (Picea mariana)

<p>Abstract</p> <p>Background</p> <p>Genetic maps provide an important genomic resource for understanding genome organization and evolution, comparative genomics, mapping genes and quantitative trait loci, and associating genomic segments with phenotypic traits. Spruce (<it>Picea</it>) genomics work is quite challenging, mainly because of extremely large size and highly repetitive nature of its genome, unsequenced and poorly understood genome, and the general lack of advanced-generation pedigrees. Our goal was to construct a high-density genetic linkage map of black spruce (<it>Picea mariana</it>, 2n = 24), which is a predominant, transcontinental species of the North American boreal and temperate forests, with high ecological and economic importance.</p> <p>Results</p> <p>We have developed a near-saturated and complete genetic linkage map of black spruce using a three-generation outbred pedigree and amplified fragment length polymorphism (AFLP), selectively amplified microsatellite polymorphic loci (SAMPL), expressed sequence tag polymorphism (ESTP), and microsatellite (mostly cDNA based) markers. Maternal, paternal, and consensus genetic linkage maps were constructed. The maternal, paternal, and consensus maps in our study consistently coalesced into 12 linkage groups, corresponding to the haploid chromosome number (1n = 1x = 12) of 12 in the genus <it>Picea</it>. The maternal map had 816 and the paternal map 743 markers distributed over 12 linkage groups each. The consensus map consisted of 1,111 markers distributed over 12 linkage groups, and covered almost the entire (> 97%) black spruce genome. The mapped markers included 809 AFLPs, 255 SAMPL, 42 microsatellites, and 5 ESTPs. Total estimated length of the genetic map was 1,770 cM, with an average of one marker every 1.6 cM. The maternal, paternal and consensus genetic maps aligned almost perfectly.</p> <p>Conclusion</p> <p>We have constructed the first high density to near-saturated genetic linkage map of black spruce, with greater than 97% genome coverage. Also, this is the first genetic map based on a three-generation outbred pedigree in the genus <it>Picea</it>. The genome length in <it>P. mariana </it>is likely to be about 1,800 cM. The genetic maps developed in our study can serve as a reference map for various genomics studies and applications in <it>Picea a</it>nd Pinaceae.</p

Assembly and Annotation of Red Spruce (Picea rubens) Chloroplast Genome, Identification of Simple Sequence Repeats, and Phylogenetic Analysis in Picea

We have sequenced the chloroplast genome of red spruce (Picea rubens) for the first time using the single-end, short-reads (44 bp) Illumina sequences, assembled and functionally annotated it, and identified simple sequence repeats (SSRs). The contigs were assembled using SOAPdenovo2 following the retrieval of chloroplast genome sequences using the black spruce (Picea mariana) chloroplast genome as the reference. The assembled genome length was 122,115 bp (gaps included). Comparatively, the P. rubens chloroplast genome reported here may be considered a near-complete draft. Global genome alignment and phylogenetic analysis based on the whole chloroplast genome sequences of Picea rubens and 10 other Picea species revealed high sequence synteny and conservation among 11 Picea species and phylogenetic relationships consistent with their known classical interrelationships and published molecular phylogeny. The P. rubens chloroplast genome sequence showed the highest similarity with that of P. mariana and the lowest with that of P. sitchensis. We have annotated 107 genes including 69 protein-coding genes, 28 tRNAs, 4 rRNAs, few pseudogenes, identified 42 SSRs, and successfully designed primers for 26 SSRs. Mononucleotide A/T repeats were the most common followed by dinucleotide AT repeats. A similar pattern of microsatellite repeats occurrence was found in the chloroplast genomes of 11 Picea species

Fodder Productivity of Different Genotypes of \u3cem\u3eCenchrus ciliaris\u3c/em\u3e under Hot Arid Climate of Thar Desert

Indian arid zone occupies about 31.7 million ha of land, of which 62% lies in western Rajasthan. The climate of the area is typically arid, characterized by hot dry summers, sub-humid monsoon and cold dry winters. The soils are coarse loamy sand with low level of nutrients. These factors render cropping an undependable proposition, while animal husbandry remains the main stake of the local people. Lasiurus sindicus, Cenchrus ciliaris, C. setigerus, Panicum antidotale, P. turgidum and Cymbopogon spp., are the main perennial grasses grow in this area. These grasses had three folds advantages in the arid agricultural economy, i.e., the cheapest livestock feed, soil builders and aid in soil conservation. Due to frequent droughts and overgrazing the productivity of the natural grasslands in the region has declined to \u3c 300 kg/ha per year. The ever existing gap between demand and supply of the fodder can be bridged by improving the rangelands by adopting improved grassland management techniques including genetically improved genotypes for their productivity and quality. Buffel grass (C. ciliaris L.) is one of the dominant grasses of Dichanthium-Cenchrus-Lasiurus grass cover of India (Dabadghao and Shankarnarayan, 1973). It is well distributed in hotter and drier parts of India, Mediterranean region, tropical and southern Africa. It is adapted to a wide range of soils and climatic conditions and can be cultivated in areas receiving rainfall from 150 to 1250 mm annually. It grows well on sandy to sandy-loam soils in semiarid and arid regions, forming mats or tussocks (Mansoor et al., 2002). The forage of this grass is highly palatable and rich in protein (Sawal et al., 2009). It has 6 to 10% crude protein, 34% crude fibre, 13% ash, 1.5% ether extract and 44% nitrogen free extract of dry matter at flowering. In earlier efforts at CAZRI, Jodhpur, germplasm was collected from different areas of arid zone and some accessions were identified for their fodder productivity and quality. Five genotypes were selected to assess their response for fodder production over the years under hot dry conditions

Molecular Diversity in Sewan Grass (\u3cem\u3eLasiurus sindicus\u3c/em\u3e Henr.): A Natural Inhabitant of Hot Arid Ecosystem of Thar Desert

Lasiurus sindicus Henr., locally known as “Sewan”, a member of family poaceae, is a tufted perennial, forming a more or less oblique and woody rhizomatous rootstock with many shoots arising from the base, often appearing almost bushy. This grass has developed a number of morphological, anatomical and biochemical strategies to withstand the extreme climatic conditions. The leaves show characteristic C4 NADP-ME type of anatomy and have developed sclerenchyma to impart mechanical strength during drought and high wind. Sewan is a dominating grass species of Dichanthium-Cenchrus–Lasiurus type grass lands of hot arid ecosystem of Great Indian Desert, covering western Rajasthan and parts of Pakistan. It grows naturally in wide range of dry areas covering North Africa, Sudano-Sahelian Africa, East Africa and Asia. It thrives well in dry climate receiving annual rainfall below 250 mm prevailing between 25-27°N latitude on well aerated alluvial soils or light sandy soils with a pH of 8.5, rocky ground and gravelly soils. Though this grass tolerates prolonged droughts but has not been found growing in higher rainfall zones and faces a serious threat of becoming an endangered due to changes in the land use pattern, increase in soil moisture regime and overgrazing. The Sewan grass, considered as the “King of Desert Grasses”, is quite palatable and nutritious for the livestock. Crude protein in young leaves varies from 7 to 14% and remains high even at maturity leading to its better suitability for efficient utilization in the animal based agri-horti-pastoral production system prevalent in hyper arid regions of western Rajasthan. In the three districts of western Rajasthan viz. Bikaner, Barmer and Jaisalmer the sustainability and productivity of livestock mainly depends on the sewan based pasture system. The present study was undertaken to analyze the extent of genetic variability existing among the L. sindicus germplasm, collected from Bikaner, Barmer and Jaisalmer, the diversity rich districts of hyper-arid Rajasthan, using ISSR and RAPD markers, for its importance in determining survival under changing climate