New microsatellite markers for pigeonpea (cajanus cajan (L.) millsp.)

<p>Abstract</p> <p>Background</p> <p>Pigeonpea is a nutritious tropical legume with several desirable characteristics but has been relatively neglected in terms of research. More efficient improvement can be achieved in this crop through molecular breeding but adequate molecular markers are lacking and no linkage map has been developed so far. Microsatellites remain the markers of choice due to their high polymorphism and their transferability from closely related genera. The overall objective of this study was to develop microsatellite markers from an enriched library of pigeonpea as well as testing the transferability of soybean microsatellites in pigeonpea.</p> <p>Results</p> <p>Primers were designed for 113 pigeonpea genomic SSRs, 73 of which amplified interpretable bands. Thirty-five of the primers revealed polymorphism among 24 pigeonpea breeding lines. The number of alleles detected ranged from 2 to 6 with a total of 110 alleles and an average of 3.1 alleles per locus. GT/CA and GAA class of repeats were the most abundant di-nucleotide and tri-nucleotide repeats respectively. Additionally, 220 soybean primers were tested in pigeonpea, 39 of which amplified interpretable bands.</p> <p>Conclusion</p> <p>Despite the observed morphological diversity, there is little genetic diversity within cultivated pigeonpea as revealed by the developed microsatellites. Although some of the tested soybean microsatellites may be transferable to pigeonpea, lack of useful polymorphism may hinder their full use. A robust set of markers will still have to be developed for pigeonpea genome if molecular breeding is to be achieved.</p

Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies

Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity

Single feature polymorphisms (SFPs) for drought tolerance in pigeonpea (Cajanus spp.)

Single feature polymorphisms (SFPs) are microarray-based molecular markers that are detected by hybridization of DNA or cRNA to oligonucleotide probes. With an objective to identify the potential polymorphic markers for drought tolerance in pigeonpea [Cajanus cajan (L.) Millspaugh], an important legume crop for the semi-arid tropics but deficient in genomic resources, Affymetrix Genome Arrays of soybean (Glycine max), a closely related species of pigeonpea were used on cRNA of six parental genotypes of three mapping populations of pigeonpea segregating for agronomic traits like drought tolerance and pod borer (Helicoverpa armigiera) resistance. By using robustified projection pursuit method on 15 pair-wise comparisons for the six parental genotypes, 5,692 SFPs were identified. Number of SFPs varied from 780 (ICPL 8755 × ICPL 227) to 854 (ICPL 151 × ICPL 87) per parental combination of the mapping populations. Randomly selected 179 SFPs were used for validation by Sanger sequencing and good quality sequence data were obtained for 99 genes of which 75 genes showed sequence polymorphisms. While associating the sequence polymorphisms with SFPs detected, true positives were observed for 52.6% SFPs detected. In terms of parental combinations of the mapping populations, occurrence of true positives was 34.48% for ICPL 151 × ICPL 87, 41.86% for ICPL 8755 × ICPL 227, and 81.58% for ICP 28 × ICPW 94. In addition, a set of 139 candidate genes that may be associated with drought tolerance has been identified based on gene ontology analysis of the homologous pigeonpea genes to the soybean genes that detected SFPs between the parents of the mapping populations segregating for drought tolerance

Developing a mHealth intervention to promote uptake of HIV testing among African communities in the UK: a qualitative study

Background: HIV-related mHealth interventions have demonstrable efficacy in supporting treatment adherence, although the evidence base for promoting HIV testing is inconclusive. Progress is constrained by a limited understanding of processes used to develop interventions and weak theoretical underpinnings. This paper describes a research project that informed the development of a theory-based mHealth intervention to promote HIV testing amongst city-dwelling African communities in the UK. Methods: A community-based participatory social marketing design was adopted. Six focus groups (48 participants in total) were undertaken and analysed using a thematic framework approach, guided by constructs from the Health Belief Model. Key themes were incorporated into a set of text messages, which were pre-tested and refined. Results: The focus groups identified a relatively low perception of HIV risk, especially amongst men, and a range of social and structural barriers to HIV testing. In terms of self-efficacy around HIV testing, respondents highlighted a need for communities and professionals to work together to build a context of trust through co-location in, and co-involvement of, local communities which would in turn enhance confidence in, and support for, HIV testing activities of health professionals. Findings suggested that messages should: avoid an exclusive focus on HIV, be tailored and personalised, come from a trusted source, allay fears and focus on support and health benefits. Conclusions: HIV remains a stigmatized and de-prioritized issue within African migrant communities in the UK, posing barriers to HIV testing initiatives. A community-based participatory social marketing design can be successfully used to develop a culturally appropriate text messaging HIV intervention. Key challenges involved turning community research recommendations into brief text messages of only 160 characters. The intervention needs to be evaluated in a randomized control trial. Future research should explore the application of the processes and methodologies described in this paper within other communities

Genetic Patterns of Domestication in Pigeonpea (Cajanus cajan (L.) Millsp.) and Wild Cajanus Relatives

Pigeonpea (Cajanus cajan) is an annual or short-lived perennial food legume of acute regional importance, providing significant protein to the human diet in less developed regions of Asia and Africa. Due to its narrow genetic base, pigeonpea improvement is increasingly reliant on introgression of valuable traits from wild forms, a practice that would benefit from knowledge of its domestication history and relationships to wild species. Here we use 752 single nucleotide polymorphisms (SNPs) derived from 670 low copy orthologous genes to clarify the evolutionary history of pigeonpea (79 accessions) and its wild relatives (31 accessions). We identified three well-supported lineages that are geographically clustered and congruent with previous nuclear and plastid sequence-based phylogenies. Among all species analyzed Cajanus cajanifolius is the most probable progenitor of cultivated pigeonpea. Multiple lines of evidence suggest recent gene flow between cultivated and non-cultivated forms, as well as historical gene flow between diverged but sympatric species. Evidence supports that primary domestication occurred in India, with a second and more recent nested population bottleneck focused in tropical regions that is the likely consequence of pigeonpea breeding. We find abundant allelic variation and genetic diversity among the wild relatives, with the exception of wild species from Australia for which we report a third bottleneck unrelated to domestication within India. Domesticated C. cajan possess 75% less allelic diversity than the progenitor clade of wild Indian species, indicating a severe “domestication bottleneck” during pigeonpea domestication

The Transcriptome of Compatible and Incompatible Interactions of Potato (Solanum tuberosum) with Phytophthora infestans Revealed by DeepSAGE Analysis

Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of potato (Solanum tuberosum). Understanding the molecular basis of resistance and susceptibility to late blight is therefore highly relevant for developing resistant cultivars, either by marker-assissted selection or by transgenic approaches. Specific P. infestans races having the Avr1 effector gene trigger a hypersensitive resistance response in potato plants carrying the R1 resistance gene (incompatible interaction) and cause disease in plants lacking R1 (compatible interaction). The transcriptomes of the compatible and incompatible interaction were captured by DeepSAGE analysis of 44 biological samples comprising five genotypes, differing only by the presence or absence of the R1 transgene, three infection time points and three biological replicates. 30.859 unique 21 base pair sequence tags were obtained, one third of which did not match any known potato transcript sequence. Two third of the tags were expressed at low frequency (<10 tag counts/million). 20.470 unitags matched to approximately twelve thousand potato transcribed genes. Tag frequencies were compared between compatible and incompatible interactions over the infection time course and between compatible and incompatible genotypes. Transcriptional changes were more numerous in compatible than in incompatible interactions. In contrast to incompatible interactions, transcriptional changes in the compatible interaction were observed predominantly for multigene families encoding defense response genes and genes functional in photosynthesis and CO2 fixation. Numerous transcriptional differences were also observed between near isogenic genotypes prior to infection with P. infestans. Our DeepSAGE transcriptome analysis uncovered novel candidate genes for plant host pathogen interactions, examples of which are discussed with respect to possible function

Modern Genomic Tools for Pigeonpea Improvement: Status and Prospects

Pigeonpea owing to its ability to sustain harsh environment and limited input/water requirement remains an excellent remunerative crop in the face of increasing climatic adversities. With nearly 70% share in global pigeonpea production, India is the leading pigeonpea producing country. Since the mid-1900s, constant research efforts directed to improve yield and resistance levels of pigeonpea have resulted in the development and deployment of several commercially accepted cultivars in India, accommodating into diverse agro-climatic zones. However, the crop productivity needs incremental improvements in order to meet the growing nutritional demands, especially in developing countries like India where pigeonpea forms a dominant part of vegetarian diet. Empowering crop improvement strategies with genomic tool kit is imperative to attain the project gains in crop yield. In the context, adoption of next-generation sequencing (NGS) technology has helped establish a wide range of genomic resources to support pigeonpea breeding, and the existing molecular tool kit includes genome-wide genetic markers, transcriptome/genome assemblies, and candidate genes/QTLs for target traits. Similarly, availability of whole mitochondrial genome sequence and derived DNA markers is immensely relevant in order to furthering the understanding of cytoplasmic male sterility (CMS) system and hybrid breeding. This chapter covers the progress of developing modern genomic resources in pigeonpea and highlights their vital role in designing future crop breeding schemes

Pigeonpea

Pigeonpea was labeled as an orphan crop but is now a trendy and pacesetter, with ample genetic and genomic information becoming available in recent times. It is now possible to cross wild relatives not only from the Cajanus group placed in the secondary and tertiary gene pool but also the related genera placed in the quaternary gene pool. This is no small achievement for a legume which is an important crop of Asia and Africa and plays a major role in the diet of majority of the people of this region. The need of the hour is further committed research on wide crosses in pigeonpea

Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies

Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity