A review of geographical distribution of marama bean [Tylosema esculentum (Burchell) Schreiber] and genetic diversity in the Namibian germplasm

Marama bean [Tylosema esculentum (Burchell) Schreiber] occurs naturally in arid and dry parts of Southern Africa, including Botswana, Namibia and South Africa. Due to the high nutrient value of the seeds and tubers, rich in protein, oil and starch, it is a potential crop for arid areas where few conventional crops can survive. The objectives of this study were to design microsatellite primers based on microsatellite repeats identified in Marama bean and to determine their ability to detect polymorphisms for use in diversity characterization. DNA fragments of T. esculentum containing microsatellite loci were isolated by enrichment of genomic DNA by a modified FIASCO technique and sequenced. Nine Marama bean microsatellite libraries enriched for (AAG)7, (GTT)7, (AGG)7, (GAG)7, (CA)10, (CT)10, (TCC)7, (CA)15 and (CAC)7 were created and thirty microsatellite primers based on the microsatellite regions of marama bean were designed and screened for polymorphism. Polymorphic microsatellites that were identified can be used as markers in future breeding programs. The screening for the polymorphic SSRs is being carried out to reach a target of 50 or more polymorphic SSR markers. It appears that the marama bean germplasm has abundant polymorphic microsatellites as 77% of the 30 loci screened initially were polymorphic. Key words: Marama bean, microsatellite markers, primers, polymorphism, maker assisted selection (MAS).

With 1 figure and 1 tableAbstractMolecular markers and genetic maps are useful tools for genetic research and molecular breeding. Although significant progress has been made recently on molecular marker development and genetic map construction in tobacco (Nicotiana tabacum), more efforts are still required to meet the needs of genetic research and breeding in this allotetraploid species. In this study, based on the expressed sequence tag (EST) data and genome sequence data of N. tabacum, we developed a total of 4886 simple sequence repeat (SSR) markers (including 1365 genomic SSRs and 3521 EST‐SSRs), which were functional in a set of eight tobacco varieties of four different types and were basically novel. Using these newly developed SSR markers as well as published SSR markers and a population of 207 double haploid (DH) lines derived from a cross between two flue‐cured tobacco varieties ‘Honghua Dajinyuan’ and ‘Hicks Broad Leaf’, we constructed a genetic map consisting of 611 SSR loci distributed on 24 tentative linkage groups and covering a total length of 1882.1 cM with an average distance of 3.1 cM between adjacent markers.

Tylosema esculentum (marama) has long been identified as a candidate crop for arid and semi-arid environments due to its success in these environments and the high nutritional value of the seed. Molecular markers are essential for the assessment of the levels of genetic variation present within and between populations of marama as well for future marker-assisted breeding efforts. Microsatellites were isolated using a modified FIASCO enrichment technique. Eighty pairs of primers were designed to amplify across a selected set of perfect microsatellite repeats with greater than 5 repeat units. Of the 80 primer pairs screened, 76% were able to detect polymorphism and 21% gave monomorphic bands while the other 3% gave inconsistent results. Four of the polymorphic SSR’s were used for genetic variation analysis and have proved to be useful and informative markers for assessing intra-specific and inter-specific variability of marama bean. Heterozygosity (H) within and between populations of marama bean in the Namibian germplasm ranged from 0.30 to 0.74. Some of the populations had low genetic variation while others had high genetic variation. Key words: Tylosema esculentum, microsatellites, FIASCO, polymorphism, genetic variation, heterozygosity.

ABSTRACTThe competitiveness of peanuts in domestic and global markets has been threatened by losses in productivity and quality that are attributed to diseases, pests, environmental stresses and allergy or food safety issues. Narrow genetic diversity and a deficiency of polymorphic DNA markers severely hindered construction of dense genetic maps and quantitative trait loci (QTL) mapping in order to deploy linked markers in marker-assisted peanut improvement. The U.S. Peanut Genome Initiative (PGI) was launched in 2004, and expanded to a global effort in 2006 to address these issues through coordination of international efforts in genome research beginning with molecular marker development and improvement of map resolution and coverage. Ultimately, a peanut genome sequencing project was launched in 2012 by the Peanut Genome Consortium (PGC). We reviewed the progress for accelerated development of peanut genomic resources in peanut, such as generation of expressed sequenced tags (ESTs) (252,832 ESTs as December 2012 in the public NCBI EST database), development of molecular markers (over 15,518 SSRs), and construction of peanut genetic linkage maps, in particular for cultivated peanut. Several consensus genetic maps have been constructed, and there are examples of recent international efforts to develop high density maps. An international reference consensus genetic map was developed recently with 897 marker loci based on 11 published mapping populations. Furthermore, a high-density integrated consensus map of cultivated peanut and wild diploid relatives also has been developed, which was enriched further with 3693 marker loci on a single map by adding information from five new genetic mapping populations to the published reference consensus map.

The dominance of a few staple crops (maize, rice, and wheat) in most agricultural systems hampers the application of interventions to improve food security and nutrition. Research and development attention has focused on improving the production and utilization of these crops, leaving other crops under‐researched and underutilized. Subsequently, there have been high malnutrition rates due to poor diet diversity, yet there are “opportunity crops” that remain under researched. The opportunity crops can unlock solutions to food insecurity, malnutrition, a lack of biodiversity, and indeed poor climate adaptation. The study explored diversification in agricultural systems to analyze whether reorientation of research investment to include under‐researched crops can increase nutrient gain and enhance dietary diversity. Research outputs benchmarked as the number of publications from three leading African universities, Nairobi, Pretoria, and Ghana, were related to crop diversity and nutrition of crops in five clusters: cereals, vegetables, legumes, roots and tubers, and nuts. The findings show that maize was the predominantly researched crop across the three institutions. Low research outputs were observed for pearl millet, finger millet, and yam across the three institutions: amaranth and nightshade (Pretoria), sweet potatoes (Pretoria and Ghana), Marama bean (Nairobi), and soya bean (Nairobi and Ghana). There was nutrient gain across all five clusters, particularly from under‐researched indigenous crops such as finger millet, amaranth, nightshade, yam, sweet potatoes, Marama bean, and soybean. Nutrient gain was contributed more by cereals and root and tuber crops from Pretoria, vegetables and nuts (Ghana), as well as legumes (Nairobi). The findings demonstrate that incorporating research on the least researched crops with successful integration of other research and development initiatives (policy and dissemination) can increase nutrition and improve dietary diversity. The nutrient gain will positively affect food security and nutrition, contributing to the achievement of Africa Agenda 2063, the United Nation's Sustainable Development Goals, and reducing food imports. The findings can inform research investment and decision across different institutions within the African continent. Research investment targeting crops such as finger millet, amaranthus, sweet potatoes, soya beans, and cashew nuts is needed considering the nutritional contribution, climate change adaptability, market potential, and biodiversity contribution. Further analysis should explore production, socio‐economic (marketability and income generation), and environmental gains (adaptive ability to climate change) for specific crops. The development of frameworks to guide the analysis of the nature and scope of factors affecting the contribution of these crops to food security and nutrition, as well as research on specific crops considering geographic distribution and institutional involvement, is also needed.

Sugarcane brown rust induced by Puccinia melanocephala is an important global disease. Exploring novel resistance genes and breeding varieties with durable resistance is the most economical and effective way of controlling brown rust. The establishment of complete and high-density molecular genetics maps is a key prerequisite for the discovery and location of sugarcane brown rust resistance genes. To obtain more polymorphic simple sequence repeat (SSR) markers for the construction of genetic linkage maps, six highly resistant varieties and six highly susceptible varieties were used as parents to screen the primers with clear bands, obvious polymorphism, and good repeatability. The highest numbers of polymorphic primers were obtained from the parental group of Liucheng 03-1137 × Dezhe 93-88 (52.38%). Ten pairs of primers including mSSCIR34 exhibited high polymorphism in the parental group of Liucheng 03-1137 × Dezhe 93-88. Here, we recommend ten SSR pairs of polymorphic primers including mSSCIR34 to use in the mapping populations of Liucheng 03-1137 × Dezhe 93-88. The polymorphic primers could facilitate the construction of genetic maps and lay an appropriate foundation for the localization of brown rust resistance genes and development of molecular genetics linkage markers.

Development and validation of molecular markers for double flower of Prunus mume

Food legumes, mainly comprising dry beans, dry peas, soybean, chickpea, pigeonpea, groundnut, greengram, blackgram, cowpea, lentil and lathyrus, have considerable area under cultivation globally and these are important constituents of cereal-based vegetarian diets. Keeping in view their tremendous importance for diversification and intensification of contemporary agriculture, systematic efforts towards their genetic improvement have been undertaken with classical breeding tools, lately complemented by the use of genomic tools. These genomic tools provide comprehensive information on genes involved in biochemical pathways leading upto nutritional compounds and can be used to understand the genetics of traits of interest and consequently, helping in marker assisted breeding. During the last two decades powerful genetic and genomic tools such as establishment of genetic and physical maps, expressed sequence tags, bioinformatic tools, genome-wide sequence data, genomic and metabolomic platforms, etc. have been developed for many legume species. These efforts have led to development of large scale molecular markers, identification of various marker trait associations, construction of genetic and linkage maps, expressed sequence tags database, partial or whole genome sequences, physical and molecular maps, DNA chips and bacterial artificial chromosome (BAC) libraries. After the genome sequencing of three model species, Medicago, Lotus and Glycine, draft genome sequences have recently been made available in agronomically important food legumes, pigeonpea and chickpea while similar efforts are underway in groundnut and greengram. The new generation sequencing (NGS) and genotyping platforms such as 454/FLX sequencing and Illumina GoldenGate/Solexa have revolutionized plant genomic research as these generate millions of ESTs per run. With the increased amount of genomic resources, there are now tremendous opportunities to integrate these with the genetic resources for their widespread use in routine legume improvement programmes by integrating them with conventional breeding tools. As a result, the genomics assisted breeding (GAB) can now be successfully used in legume improvement and development of improved genotypes having improved agronomic and quality traits and resistance to biotic and abiotic stresses. This chapter discusses the developments made in development of legume genomics and their role in overall improvement of food legumes.

Kiwifruit is a new fruit crop that was commercialized in the late 1970s. Recently, its cultivation and consumption have increased rapidly worldwide. Kiwifruit is a dioecious, deciduous, and climbing plant having fruit with hairs and various flesh colors and a variation in ploidy level; however, the industry consists of very simple cultivars or genotypes. The need for efficient cultivar improvement together with the evolutional and biological perspectives based on unique plant characteristics, have recently encouraged genome analysis and bioinformatics application. The draft genome sequence and chloroplast genome sequence of kiwifruit were released in 2013 and 2015, respectively; and gene annotation has been in progress. Recently, transcriptome analysis has shifted from previous ESTs analysis to the RNA-seq platform for intensive exploration of controlled genetic expression and gene discovery involved in fruit ascorbic acid biosynthesis, flesh coloration, maturation, and vine bacterial canker tolerance. For improving conventional breeding efficiency, molecular marker development and genetic linkage map construction have advanced from basic approaches using RFLP, RAPD, and AFLP to the development of NGS-based SSR and SNP markers linked to agronomically important traits and the construction of highly saturated linkage maps. However, genome and transcriptome studies have been limited in Korea. In the near future, kiwifruit genome and transcriptome studies are expected to translate to the practical application of molecular breeding.

Sugarcane brown rust, caused by Puccinia melanocephala, is one of the main diseases of sugarcane in China. The identification and discovery of new resistance genes have important theoretical and practical significance for preventing outbreaks of brown rust and ensuring the sustainable production of sugarcane. To screen for polymorphic simple-sequence repeat (SSR) molecular markers for localization of brown rust resistance genes, we used two populations that are suitable for genetic linkage map construction and mapping of new resistance genes to construct resistant and susceptible genetic pools. We then screened 449 pairs of primers to identify polymorphic SSR markers in the parental lines and the resistant/susceptible genetic pools. The results showed that 25 pairs of primers directed amplification of polymorphic DNA fragments between the parents of the cross combination ‘Yuetang 03-393’ × ‘ROC 24’, and 16 pairs of primers amplified polymorphic fragments between the parents of the cross combination ‘Liucheng 03-1137’ × ‘Dezhe 93-88’. Four pairs of primers (SMC236CG, SCESSR0928, SCESSR0636 and SCESSR2551) amplified polymorphic DNA fragments between the parental lines and the resistant/susceptible genetic pools in ‘Yuetang 03-393’ × ‘ROC 24’. The results of this study will establish a solid foundation for the mapping of new brown rust resistance genes, genetic linkage map construction and the development of closely-associated molecular markers in sugarcane.

The development of molecular markers is one of the most important challenges in phylogenetic and genome wide population genetics studies, especially in studies with non-model organisms. A highly promising approach for obtaining suitable markers is the utilization of genomic partitioning strategies for the simultaneous discovery and genotyping of a large number of markers. Unfortunately, not all markers obtained from these strategies provide enough information for solving multiple evolutionary questions at a reasonable taxonomic resolution. We have developed Development Of Molecular markers In Non-model Organisms (DOMINO), a bioinformatics tool for informative marker development from both next generation sequencing (NGS) data and pre-computed sequence alignments. The application implements popular NGS tools with new utilities in a highly versatile pipeline specifically designed to discover or select personalized markers at different levels of taxonomic resolution. These markers can be directly used to study the taxa surveyed for their design, utilized for further downstream PCR amplification in a broader set taxonomic scope, or exploited as suitable templates to bait design for target DNA enrichment techniques. We conducted an exhaustive evaluation of the performance of DOMINO via computer simulations and illustrate its utility to find informative markers in an empirical dataset. DOMINO is freely available from www.ub.edu/softevol/domino CONTACT: elsanchez@ub.edu or jrozas@ub.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Pineapple is the most important economic plant in the family Bromeliaceae and the third-most economically important tropical fruit in the world. It has become an important tropical fruit in Guangdong, Hainan, and Guangxi, which are suitable areas for its cultivation. However, modern and well-organized breeding systems have not yet been established for pineapple. In this review, we describe the current status of the geographical distribution, industrial development, and breeding of pineapple in China. The current status of pineapple breeding is introduced, including traditional breeding methods, such as crossbreeding, mutagenesis breeding, and biotechnology breeding, combining cell engineering and gene engineering. In addition, the research progress on assisted breeding technology based on genetic map construction and molecular marker development is presented. New challenges and perspectives for obtaining high fruit quality are discussed in the context of breeding programs for pineapple.

Specific length amplified fragment sequencing (SLAF-seq) technology is a simplified genome sequencing technology based on next-generation sequencing. SLAF-seq technology has several distinguishing characteristics: 1. Deep sequencing to ensure accuracy of genotyping; 2. Effectively reduce sequencing costs; 3. Pre-designed simplified representation scheme to optimize marker efficiency; 4. Doubled barcode system for large populations. The advantages and technical process of SLAF-seq are described briefly with summarized results for the application of SLAF-seq in development of molecular markers, construction of high-density genetic map and gene mapping in ornamental plants. Finally, the difficulties and prospects of this method are discussed in application.

BackgroundHouttuynia cordata Thunb. is an important traditional medical herb in China and other Asian countries, with high medicinal and economic value. However, a lack of available genomic information has become a limitation for research on this species. Thus, we carried out high-throughput transcriptomic sequencing of H. cordata to generate an enormous transcriptome sequence dataset for gene discovery and molecular marker development.Principal FindingsIllumina paired-end sequencing technology produced over 56 million sequencing reads from H. cordata mRNA. Subsequent de novo assembly yielded 63,954 unigenes, 39,982 (62.52%) and 26,122 (40.84%) of which had significant similarity to proteins in the NCBI nonredundant protein and Swiss-Prot databases (E-value <10−5), respectively. Of these annotated unigenes, 30,131 and 15,363 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. In addition, 24,434 (38.21%) unigenes were mapped onto 128 pathways using the KEGG pathway database and 17,964 (44.93%) unigenes showed homology to Vitis vinifera (Vitaceae) genes in BLASTx analysis. Furthermore, 4,800 cDNA SSRs were identified as potential molecular markers. Fifty primer pairs were randomly selected to detect polymorphism among 30 samples of H. cordata; 43 (86%) produced fragments of expected size, suggesting that the unigenes were suitable for specific primer design and of high quality, and the SSR marker could be widely used in marker-assisted selection and molecular breeding of H. cordata in the future.ConclusionsThis is the first application of Illumina paired-end sequencing technology to investigate the whole transcriptome of H. cordata and to assemble RNA-seq reads without a reference genome. These data should help researchers investigating the evolution and biological processes of this species. The SSR markers developed can be used for construction of high-resolution genetic linkage maps and for gene-based association analyses in H. cordata. This work will enable future functional genomic research and research into the distinctive active constituents of this genus.

During the past few years, disease symptoms in many Acacia trees in the Windhoek Mu‐ nicipality area and the rest of Namibia have been observed. This observation triggered the investigation of phytopathological aspects that are reported in this chapter. The im‐ portance of indigenous trees of the Namibia flora is apparent considering that Namibia has two old deserts within its borders: the Namib Desert and the Kalahari Desert. Nam‐ bia’s tourism and meat industry are dependent on the indigenous trees of Nambia flora. The trees are the primary source of vegetation land cover (attracting tourists), and they provide browsing food matter to domestic livestock and wild animals (sources of meat). Hence, it is important to ensure that a healthy vegetation is maintained in this area.. This survey is the first dedicated step to find ways of protecting them from disease-causing agents. The aim of this survey is to investigate the possible causes of disease symptoms in trees. It is important to understand the biology of the pathogenic agents to propose a pos‐ sible method to control the diseases. The survey involved sampling leaves, stems and roots from dying trees that show symptoms such as branch girdling, gum oozing and de‐ foliation, suspicious general twig wilting and die-back. The survey was carried out in pla‐ ces where symptoms were observed. The tree surveys were done on Aloe zebrina, Tylosema esculentum, Syzygium and Acacia species. Primary isolations from plant material and then single-spore pure cultures were made for identification. In this chapter, we re‐ port isolation and identification of Microsphearopsis sp., Dreschelra sp., Botryosphearia spp., Acremonium spp., Coniothyrium sp., Phellinus spp., Cytospora sp., Fusarium sp., Scytalidium sp., Phoma spp., Gliomastix sp., Trichoderma koningii, Peacilomyces variotii, Alternaria citri and Curvularia palescens from the diseased trees. This work is still ongoing. This study paves way for proper designing of control methods to protect crops, trees and their biodi‐ versity. The protection of plant biodiversity ensures better reaping of food products and other ecosystem services and products. Without knowledge of the identity of these dis‐ ease-causing agents, it is not possible to accurately identify and manage threats to food production and threats to the native botanical biodiversity of Namibia.