Statistical Databases

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a diffi cult and costly process. NGS technologies allow the effi cient identifi cation of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the effi cient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry ( Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

Economically, legumes (Fabaceae) represent the second most important family of crop plants after the grass family, Poaceae. Grain legumes account for 27% of world crop production and provide 33% of the dietary protein consumed by humans, while pasture and forage legumes provide vital part of animal feed. Fabaceae, the third largest family of flowering plants, has traditionally been divided into the following three subfamilies: Caesalpinioideae, Mimosoideae, and Papilionoideae, all together with 800 genera and 20,000 species. The latter subfamily contains most of the major cultivated food and feed crops. Among the grain legumes are some of mankind's earliest crop plants, whose domestication parallelled that of cereals: Soybean in China; faba bean, lentil, chickpea and pea in the Fertile Crescent of the Near East; cowpeas and bambara groundnut in Africa; soybean and mungbeans in East Asia; pigeonpea and the grams in South Asia; and common bean, lima bean, scarlet runner bean, tepary bean and lupin in Central and South America. The importance of legumes is evidenced by their high representation in ex situ germplasm collections, with more than 1,000,000 accessions worldwide. A detailed knowledge of the phylogenetic relationships of the Fabaceae is essential for understanding the origin and diversification of this economically and ecologically important family of angiosperms. This review aims to combine the phylogenetic and genetic diversity approaches to better illustrate the origin, domestication history and preserved germplasm of major legume crops from 13 genera of six tribes and to indicate further potential both for science and agriculture.

/pdf/legume-crops-phylogeny-and-genetic-diversity-for-science-and-1lvkdujjsr.pdf

Legume Crops Phylogeny and Genetic Diversity for Science and Breeding

Molecular markers, due to their stability, cost-effectiveness and ease of use provide an immensely popular tool for a variety of applications including genome mapping, gene tagging, genetic diversity diversity, phylogenetic analysis and forensic investigations In the last three decades, a number of molecular marker techniques have been developed and exploited worldwide in different systems However, only a handful of these techniques, namely RFLPs, RAPDs, AFLPs, ISSRs, SSRs and SNPs have received global acceptance A recent revolution in DNA sequencing techniques has taken the discovery and application of molecular markers to high-throughput and ultrahigh-throughput levels Although, the choice of marker will obviously depend on the targeted use, microsatellites, SNPs and genotyping by sequencing (GBS) largely fulfill most of the user requirements Further, modern transcriptomic and functional markers will lead the ventures onto high-density genetic map construction, identification of QTLs, breeding and conservation strategies in times to come in combination with other high throughput techniques This review presents an overview of different marker technologies and their variants with a comparative account of their characteristic features and applications

Development and use of molecular markers: past and present.

Genetic Data Analysis: Methods for Discrete Population Genetic Data

Faba bean (Vicia faba L.) is an important food legume crop, grown for human consumption globally including in China, Turkey, Egypt and Ethiopia. Although genetic gain has been made through conventional selection and breeding efforts, this could be substantially improved through the application of molecular methods. For this, a set of reliable molecular markers representative of the entire genome is required. A library with 125,559 putative SSR sequences was constructed and characterized for repeat type and length from a mixed genome of 247 spring and winter sown faba bean genotypes using 454 sequencing. A suit of 28,503 primer pair sequences were designed and 150 were randomly selected for validation. Of these, 94 produced reproducible amplicons that were polymorphic among 32 faba bean genotypes selected from diverse geographical locations. The number of alleles per locus ranged from 2 to 8, the expected heterozygocities ranged from 0.0000 to 1.0000, and the observed heterozygosities ranged from 0.0908 to 0.8410. The validation by UPGMA cluster analysis of 32 genotypes based on Nei's genetic distance, showed high quality and effectiveness of those novel SSR markers developed via next generation sequencing technology. Large scale SSR marker development was successfully achieved using next generation sequencing of the V. faba genome. These novel markers are valuable for constructing genetic linkage maps, future QTL mapping, and marker-assisted trait selection in faba bean breeding efforts.

/pdf/high-throughput-novel-microsatellite-marker-of-faba-bean-via-u5radvv03n.pdf

High-throughput novel microsatellite marker of faba bean via next generation sequencing.

Food legumes comprise all legumes grown for human food in China as either dry grains or vegetables, except for soybean and groundnut. China has a vast territory with complex ecological conditions. Rotation, intercropping, and mixed cropping involving pulses are normal cropping systems in China. Whether indigenous or introduced crops, pulses have played an important role in Chinese cropping systems and made an important contribution to food resources for humans since ancient times. The six major food legume species (pea, faba bean, common bean, mung bean, adzuki bean, and cowpea) are the most well-known pulses in China, as well as those with more local distributions; runner bean, lima bean, chickpea, lentil, grass pea, lupine, rice bean, black gram, hyacinth bean, pigeon pea, velvet bean, winged bean, guar bean, sword bean, and jack bean. China has remained the world's leading producer of peas, faba beans, mung beans, and adzuki beans in recent decades, as documented by FAO statistics and China Agriculture Statistical Reports. The demand for food legumes as a healthy food will markedly increase with the improvement of living standards in China. Since China officially joined the World Trade Organization (WTO) in 2001, imports of pea from Canada and Australia have rapidly increased, resulting in reduced prices for dry pea and other food legumes. With reduced profits for food legume crops, their sowing area and total production has decreased within China. At the same time, the rising consumer demand for vegetable food legumes as a healthy food has led to attractive market prices and sharp production increases in China. Vegetable food legumes have reduced growing duration and enable flexibility in cropping systems. In the future, production of dry food legumes will range from stable to slowly decreasing, while production of vegetable food legumes will continue to increase.

Food legume production in China.

Genetic diversity and relationships of 802 faba bean (Vicia faba L.) landraces and varieties from different geographical locations of China and abroad were examined using ISSR markers. A total of 212 repeatable amplified bands were generated with 11 ISSR primers, of which 209 were polymorphic. Accessions from North China showed highest genetic diversity, while accessions from central China showed low level of diversity. Chinese spring faba bean germplasm was clearly separated from Chinese winter faba bean, based on principal component analysis and UPGMA clustering analysis. Winter accessions from Zhejiang (East China), Jiangxi (East China), Sichuan (Southwest China) and Guizhou (Southwest China) were quite distinct to that from other provinces in China. Great differentiation between Chinese accessions and those from rest of the world was shown with a UPGMA dendrogram. AMOVA analyses demonstrated large variation and differentiation within and among groups of accessions from China. As a continental geographic group, accessions from Europe were genetically closer to those from North Africa. Based on ISSR data, grouping results of accessions from Asia, Europe and Africa were obviously associated with their geographical origin. The overall results indicated that the genetic relationship of faba bean germplasm was closely associated with their geographical origin and their ecological habit.

Genetic diversity and relationship of global faba bean ( Vicia faba L.) germplasm revealed by ISSR markers

Grasspea (Lathyrus sativus L., 2n = 14), a member of the family Leguminosae, holds great agronomic potential as grain and forage legume crop in the arid areas for its superb resilience to abiotic stresses such as drought, flood and salinity. The crop could not make much progress through conventional breeding in the past, and there are hardly any detailed molecular biology studies due to paucity of reliable molecular markers representative of the entire genome. Using the 454 FLX Titanium pyrosequencing technique, 651,827 simple sequence repeat (SSR) loci were identified and 50,144 nonredundant primer pairs were successfully designed, of which 288 were randomly selected for validation among 23 L. sativus and one L. cicera accessions of diverse provenance. 74 were polymorphic, 70 monomorphic, and 144 with no PCR product. The number of observed alleles ranged from two to five, the observed heterozygosity from 0 to 0.9545, and Shannon’s information index ranged from 0.1013 to 1.0980, respectively. The dendrogram constructed by using unweighted pair group method with arithmetic mean (UPGMA) based on Nei's genetic distance, showed obvious distinctions and understandable relationships among the 24 accessions. The large number of SSR primer pairs developed in this study would make a significant contribution to genomics enabled improvement of grasspea.

/pdf/large-scale-microsatellite-development-in-grasspea-lathyrus-2mt2gw6s92.pdf

Large-scale microsatellite development in grasspea ( Lathyrus sativus L.), an orphan legume of the arid areas

Pea (Pisum sativum L.) is an important food legume globally, and is the plant species that J.G. Mendel used to lay the foundation of modern genetics. However, genomics resources of pea are limited comparing to other crop species. Application of marker assisted selection (MAS) in pea breeding has lagged behind many other crops. Development of a large number of novel and reliable SSR (simple sequence repeat) or microsatellite markers will help both basic and applied genomics research of this crop. The Illumina HiSeq 2500 System was used to uncover 8,899 putative SSR containing sequences, and 3,275 non-redundant primers were designed to amplify these SSRs. Among the 1,644 SSRs that were randomly selected for primer validation, 841 yielded reliable amplifications of detectable polymorphisms among 24 genotypes of cultivated pea (Pisum sativum L.) and wild relatives (P. fulvum Sm.) originated from diverse geographical locations. The dataset indicated that the allele number per locus ranged from 2 to 10, and that the polymorphism information content (PIC) ranged from 0.08 to 0.82 with an average of 0.38. These 1,644 novel SSR markers were also tested for polymorphism between genotypes G0003973 and G0005527. Finally, 33 polymorphic SSR markers were anchored on the genetic linkage map of G0003973 × G0005527 F2 population.

/pdf/high-throughput-development-of-ssr-markers-from-pea-pisum-317091eg6q.pdf

Tao Yang

Papers

High-throughput novel microsatellite marker of faba bean via next generation sequencing.

Food legume production in China.

Genetic diversity and relationship of global faba bean ( Vicia faba L.) germplasm revealed by ISSR markers

Large-scale microsatellite development in grasspea ( Lathyrus sativus L.), an orphan legume of the arid areas

High-Throughput Development of SSR Markers from Pea (Pisum sativum L.) Based on Next Generation Sequencing of a Purified Chinese Commercial Variety