Microsatellite markers in Spanish lime (Melicoccus bijugatus Jacq., Sapindaceae), a neglected Neotropical fruit crop

Spanish lime (Melicoccus bijugatus Jacq.) is aNeotropical fruit tree cultivated, mainly, in orchards for self-consumption or local sale. The genus Melicoccus includes other nine species with edible fruits, some of these species are at risk of extinction. Like for the vast majority of tropical fruit trees, there is no information on the genetic diversity of Spanish lime and its related species, and this is mostly due to the lack of molecular markers. The objectives of this study were to present the first microsatellite markers developed for Spanish lime, testing its usefulness on a sample of cultivated accessions, as well as its transferability to Huaya India (M. oliviformis). To do this, we performed high-throughput sequencing of microsatellite-enriched libraries of Spanish lime using Roche 454, assembled 9567 DNA contig sequences and identified 10,117 microsatellites. After screening 384 of those microsatellites on four DNA samples, 31 polymorphic markers were used to screen 25 accessions of Spanish lime and five of Huaya India collected in Yucatan, Mexico. Genetic diversity was low in Spanish lime (A = 20.61, HE = 0.38) and similar for both sexes of this species. Neighbor-Joining and PCoA analyses clearly discriminated between the two Melicoccus species studied. Nine of the markers showed unique alleles for Huaya India. The set of microsatellite markers developed has a great potential to generate information in relation to conservation genetics, improvement of elite cultivars and breeding programs for Spanish lime and related species

Recent developments in primer design for DNA polymorphism and mRNA profiling in higher plants

Primer design is a critical step in the application of PCR-based technologies in gene expression and genetic diversity analysis. As more plant genomes have been sequenced in recent years, the emphasis of primer design strategy has shifted to genome-wide and high-throughput direction. This paper summarizes recent advances in primer design for profiling of DNA polymorphism and mRNA in higher plants, as well as new primer systems developed for animals that can be adapted for plants

Genomic resources for water yam (Dioscorea alata L.): analyses of EST sequences, De Novo sequencing and GBS libraries

Published online: 29 July 201

UPIC: Perl scripts to determine the number of SSR markers to run

We introduce here the concept of Unique Pattern Informative Combinations (UPIC), a decision tool for the cost-effective design of DNA fingerprinting/genotyping experiments using simple-sequence/tandem repeat (SSR/STR) markers. After the first screening of SSR-markers tested on a subset of DNA samples, the user can apply UPIC to find marker combinations that maximize the genetic information obtained by a minimum or desirable number of markers. This allows a cost-effective planning of future experiments. We have developed Perl scripts to calculate all possible subset combinations of SSR markers, and determine based on unique patterns or alleles, which combinations can discriminate among all DNA samples included in a test. This makes UPIC an essential tool for optimizing resources when working with microsatellites. An example using real data from eight markers and 12 genotypes shows that UPIC detected groups of as few as three markers sufficient to discriminate all 12- DNA samples. Should markers for future experiments be chosen based only on polymorphism-information content (PIC), the necessary number of markers for discrimination of all samples cannot be determined. We also show that choosing markers using UPIC, an informative combination of four markers can provide similar information as using a combination of six markers (23 vs. 25 patterns, respectively), granting a more efficient planning of experiments. Perl scripts with documentation are also included to calculate the percentage of heterozygous loci on the DNA samples tested and to calculate three PIC values depending on the type of fertilization and allele frequency of the organism

Comparison of whole-genome amplifications for microsatellite genotyping of \u3ci\u3eRotylenchulus reniformis\u3c/i\u3e

Currently, a large number of microsatellites are available for Rotylenchulus reniformis (reniform nematode); however, two barriers exist for genotyping samples from different geographical areas. The limited amount of nucleic acids obtained from single nematodes which would require their multiplication to obtain enough DNA for testing; and the strictly regulated transport of live samples and multiplication in greenhouse for being a plant pathogen. Whole-genome amplification (WGA) of samples consisting of one and five dead gravid females with their associated egg masses was successfully performed on disrupted tissue using three commercial kits. DNA yield after WGA ranged from 0.5 to 8 μg and was used to test 96 microsatellite markers we previously developed for the reniform nematode. The results were compared to those of fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in MSRR03. Using WGA of single gravid females with their associated egg masses, 86-93% of the alleles found on MSRR03 were detected, and 87-88% of the alleles found on MSRR03 when using WGA of samples composed of five gravid females with their associated egg masses as template. Our results indicate that reniform nematode samples as small as a single gravid female with her associated egg mass can be used in WGA and direct testing with microsatellites, giving consistent results when compared to the original population

Comparison of whole-genome amplifications for microsatellite genotyping of \u3ci\u3eRotylenchulus reniformis\u3c/i\u3e

Currently, a large number of microsatellites are available for Rotylenchulus reniformis (reniform nematode); however, two barriers exist for genotyping samples from different geographical areas. The limited amount of nucleic acids obtained from single nematodes which would require their multiplication to obtain enough DNA for testing; and the strictly regulated transport of live samples and multiplication in greenhouse for being a plant pathogen. Whole-genome amplification (WGA) of samples consisting of one and five dead gravid females with their associated egg masses was successfully performed on disrupted tissue using three commercial kits. DNA yield after WGA ranged from 0.5 to 8 μg and was used to test 96 microsatellite markers we previously developed for the reniform nematode. The results were compared to those of fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in MSRR03. Using WGA of single gravid females with their associated egg masses, 86-93% of the alleles found on MSRR03 were detected, and 87-88% of the alleles found on MSRR03 when using WGA of samples composed of five gravid females with their associated egg masses as template. Our results indicate that reniform nematode samples as small as a single gravid female with her associated egg mass can be used in WGA and direct testing with microsatellites, giving consistent results when compared to the original population

Identification of the family of aquaporin genes and their expression in upland cotton (Gossypium hirsutum L.)

<p>Abstract</p> <p>Background</p> <p>Cotton (<it>Gossypium spp</it>.) is produced in over 30 countries and represents the most important natural fiber in the world. One of the primary factors affecting both the quantity and quality of cotton production is water. A major facilitator of water movement through cell membranes of cotton and other plants are the aquaporin proteins. Aquaporin proteins are present as diverse forms in plants, where they function as transport systems for water and other small molecules. The plant aquaporins belong to the large major intrinsic protein (MIP) family. In higher plants, they consist of five subfamilies including plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), NOD26-like intrinsic proteins (NIP), small basic intrinsic proteins (SIP), and the recently discovered X intrinsic proteins (XIP). Although a great deal is known about aquaporins in plants, very little is known in cotton.</p> <p>Results</p> <p>From a molecular cloning effort, together with a bioinformatic homology search, 71 upland cotton (<it>G. hirsutum</it>) aquaporin genes were identified. The cotton aquaporins consist of 28 PIP and 23 TIP members with high sequence similarity. We also identified 12 NIP and 7 SIP members that showed more divergence. In addition, one XIP member was identified that formed a distinct 5^thsubfamily. To explore the physiological roles of these aquaporin genes in cotton, expression analyses were performed for a select set of aquaporin genes from each subfamily using semi-quantitative reverse transcription (RT)-PCR. Our results suggest that many cotton aquaporin genes have high sequence similarity and diverse roles as evidenced by analysis of sequences and their expression.</p> <p>Conclusion</p> <p>This study presents a comprehensive identification of 71 cotton aquaporin genes. Phylogenetic analysis of amino acid sequences divided the large and highly similar multi-gene family into the known 5 aquaporin subfamilies. Together with expression and bioinformatic analyses, our results support the idea that the genes identified in this study represent an important genetic resource providing potential targets to modify the water use properties of cotton.</p

Molecular evolution of herbicide resistance to phytoene desaturase inhibitors in \u3ci\u3eHydrilla verticillata\u3c/i\u3e and its potential use to generate herbicide-resistant crops

Hydrilla [Hydrilla verticillata (Lf) Royle] is one of the most serious invasive aquatic weed problems in the USA. This plant possesses numerous mechanisms of vegetative reproduction that enable it to spread very rapidly. Management of this weed has been achieved by the systemic treatment of water bodies with the herbicide fluridone. At least three dioecious fluridone-resistant biotypes of hydrilla with two- to fivefold higher resistance to the herbicide than the wild-type have been identified. Resistance is the result of one of three independent somatic mutations at the arginine 304 codon of the gene encoding phytoene desaturase, the molecular target site of fluridone. The specific activities of the three purified phytoene desaturase variants are similar to the wild-type enzyme. The appearance of these herbicideresistant biotypes may jeopardize the ability to control the spread of this non-indigenous species to other water bodies in the southern USA. The objective of this paper is to provide general information about the biology and physiology of this aquatic weed in relation to its recent development of resistance to the herbicide fluridone, and to discuss how this discovery might lead to a new generation of herbicide-resistant crops

Molecular evolution of herbicide resistance to phytoene desaturase inhibitors in \u3ci\u3eHydrilla verticillata\u3c/i\u3e and its potential use to generate herbicide-resistant crops

Hydrilla [Hydrilla verticillata (Lf) Royle] is one of the most serious invasive aquatic weed problems in the USA. This plant possesses numerous mechanisms of vegetative reproduction that enable it to spread very rapidly. Management of this weed has been achieved by the systemic treatment of water bodies with the herbicide fluridone. At least three dioecious fluridone-resistant biotypes of hydrilla with two- to fivefold higher resistance to the herbicide than the wild-type have been identified. Resistance is the result of one of three independent somatic mutations at the arginine 304 codon of the gene encoding phytoene desaturase, the molecular target site of fluridone. The specific activities of the three purified phytoene desaturase variants are similar to the wild-type enzyme. The appearance of these herbicideresistant biotypes may jeopardize the ability to control the spread of this non-indigenous species to other water bodies in the southern USA. The objective of this paper is to provide general information about the biology and physiology of this aquatic weed in relation to its recent development of resistance to the herbicide fluridone, and to discuss how this discovery might lead to a new generation of herbicide-resistant crops

Comparison of whole-genome amplifications for microsatellite genotyping of Rotylenchulus reniformis

Currently, a large number of microsatellites are available for Rotylenchulus reniformis (reniform nematode); however, two barriers exist for genotyping samples from different geographical areas. The limited amount of nucleic acids obtained from single nematodes which would require their multiplication to obtain enough DNA for testing; and the strictly regulated transport of live samples and multiplication in greenhouse for being a plant pathogen. Whole-genome amplification (WGA) of samples consisting of one and five dead gravid females with their associated egg masses was successfully performed on disrupted tissue using three commercial kits. DNA yield after WGA ranged from 0.5 to 8 \ub5g and was used to test 96 microsatellite markers we previously developed for the reniform nematode. The results were compared to those of fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in MSRR03. Using WGA of single gravid females with their associated egg masses, 86-93% of the alleles found on MSRR03 were detected, and 87-88% of the alleles found on MSRR03 when using WGA of samples composed of five gravid females with their associated egg masses as template. Our results indicate that reniform nematode samples as small as a single gravid female with her associated egg mass can be used in WGA and direct testing with microsatellites, giving consistent results when compared to the original population