Density enhancement of a faba bean genetic linkage map (Vicia faba) based on simple sequence repeats markers

Genetic mapping for faba bean lags far behind other major crops. Density enhancement of the faba bean genetic linkage map was carried out by screening 5,325 genomic SSR primers and 2033 expressed sequence tag (EST)‐SSR primers on the parental cultivars '91825' and 'K1563'. Two hundred and fifteen genomic SSR and 133 EST‐SSR primer pairs that detected polymorphisms in the parents were used to screen 129 F₂ individuals. This study added 337 more SSR markers and extended the previous linkage map by 2928.45 cM to a total of 4516.75 cM. The number of SSR markers in the linkage groups varied from 12 to 136 while the length of each linkage group ranged from 129.35 to 1180.21 cM. The average distance between adjacent loci in the enhanced genetic linkage map was 9.71 cM, which is 2.79 cM shorter than the first linkage map of faba bean. The density‐enhanced genetic map of faba bean will be useful for marker‐assisted selection and breeding in this important legume crop.     

Characterization of novel sugarcane expressed sequence tag microsatellites and their comparison with genomic SSRs

Microsatellites or simple sequence repeats (SSRs) are one of the most suitable markers for genome analysis as they have great potential to aid breeders to develop new improved sugarcane varieties. The development of SSR derived from expressed sequence tags (EST) opens new opportunities for genetic investigations at a functional level. In the present work, the polymorphism obtained with a subset of 51 EST–SSRs derived from sucest was compared with those generated by 50 genomic SSRs (gSSR) in terms of number of alleles, polymorphism information content, discrimination power and their ability to establish genetic relationships among 18 sugarcane clones including three Saccharum species (S. officinarum, S. barberi, S. sinense). The majority of EST–SSRs loci had four to six alleles in contrast to the seven to nine observed for the gSSRs loci. Approximately, 35% of the gSSRs had PIC values around 0.90 in contrast to 15% of the EST–SSRs. However, the mean discrimination power of the two types of SSR did not differ significantly as much as the average genetic similarity (GS) based on Dice coefficient. The correlation between GS of the two types of SSRs was high (r = 0.71/P = 0.99) and significant. Although differences were observed between dendrograms obtained with each SSR type, both were in good agreement with pedigree information. The S. officinarum clone IJ76‐314 was grouped apart from the other clones evaluated. The results here demonstrate that EST–SSRs can be successfully used for genetic relationship analysis, extending the knowledge of genetic diversity of sugarcane to a functional level.     

Development of genomic simple sequence repeat markers from an enriched genomic library of grass pea (Lathyrus sativus L.)

Simple sequence repeat (SSR) or microsatellite markers are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker‐assisted selection and breeding. In this study, a SSR genomic enriched library was developed in Lathyrus sativus (grass pea) by affinity capture of restriction fragments to biotinylated microsatellite oligonucleotides. About 400 randomly selected clones were sequenced, and SSRs were present in approximately 30% of them. Clones contained 75%, 9% and 16% of simple, interrupted and compound SSRs, respectively. Of the 10 SSRs tested, 7 primer pairs produced clearly distinguishable DNA banding patterns. Successively, SSR primer pairs were successfully tested to reveal polymorphism in a set of four different grass pea germplasm accessions. The transferability of SSR markers was high among three related species of Lathyrus, namely Lathyrus cicera, Lathyrus ochrus and Lathyrus tingitanus, and the legume crop, Pisum sativum. These results indicate that the novel SSR markers are informative and will be useful and convenient for genetic analysis in grass pea and related species.     

Highly variable ‘Arhar’ simple sequence repeat markers for molecular diversity and phylogenetic studies in pigeonpea [Cajanus cajan (L.) Millisp.]

Pigeonpea is an important tropical grain legume widely cultivated in South and South‐East Asia for versatile end usage as food, feed, fodder and fuel. Recent publication of draft genome sequence of pigeonpea (‘Arhar’) has allowed mining of large numbers of genomic simple sequence repeat (SSR) markers most of which are either not validated or show very little polymorphism in simple agarose gel–based assays. Here, we describe a special category of 370 validated highly variable ‘Arhar’ genomic SSR (HASSR) markers, which provide much higher level of polymorphism than a random set of SSR markers. These markers were validated for consistent amplification in a set of eight pigeonpea varieties and 152 of these (41%) showed allelic polymorphism on agarose gel electrophoresis. Twenty‐four highest polymorphic HASSR markers were used on a larger set of forty pigeonpea varieties and eight wild Cajanus species for the analysis of genetic diversity and phylogenetic relationship. Genomic HASSR markers described here are highly suitable for genetic diversity and phylogenic studies in pigeonpea.     

利用2个F_2群体整合中国豌豆高密度SSR遗传连锁图谱

豌豆(Pisum sativum L.)是一种重要的食用豆类作物,在全世界范围内广泛种植,既可作为人类食物,也可作为牲畜饲料。用SSR标记构建的遗传连锁图谱在豌豆和其他作物的标记辅助育种中发挥着重要的作用。尽管对豌豆遗传连锁作图的研究已有悠久历史,但公众可获得且可转移的SSR标记以及基于遗传独特的中国豌豆种质的高密度遗传连锁图谱仍然有限。为了获得更多可转移的SSR标记和中国豌豆的高密度遗传连锁图谱,本研究首先从自主开发和文献获取的12,491个全基因组SSR标记中筛选了617个多态性SSR标记,并用于G0003973×G0005527 F_2群体遗传连锁图谱的加密。加密后的图谱全长扩展到5330.6 cM,包含603个SSR标记,标记平均间距离8.8 cM,相比之前的图谱有明显改善。基于上述结果,我们又筛选了119个具有多态性的SSR标记,用于构建大样本W6-22600×W6-15174 F_2群体的遗传连锁图谱,新图谱累积长度为1127.1 cM,包含118个SSR标记,装配在7条连锁群上。最后,将来自以上2个遗传图谱的数据进行整合,得到了一张覆盖范围6592.6 cM的整合图谱,包含668个SSR标记,由509个基因组SSR、134个EST-SSR和25个锚定标记组成,分布在7条连锁群上。这些SSR标记和遗传连锁图谱将为豌豆的遗传研究和标记辅助育种提供有力工具。     

An SSR-based molecular genetic map of cassava

Summary Microsatellites or simple sequence repeats (SSR) are the markers of choice for molecular genetic mapping and marker-assisted selection in many crop species. A microsatellite-based linkage map of cassava was drawn using SSR markers and a F₂ population consisting of 268 individuals. The F₂ population was derived from selfing the genotype K150, an early yielding genotype from an F₁ progeny from a cross between two non-inbred elite cassava varieties, TMS 30572 and CM 2177-2 from IITA and CIAT respectively. A set of 472 SSR markers, previously developed from cassava genomic and cDNA libraries, were screened for polymorphism in K150 and its parents TMS 30572 and CM 2177-2. One hundred and twenty two polymorphic SSR markers were identified and utilized for linkage analysis. The map has 100 markers spanning 1236.7 cM, distributed on 22 linkage groups with an average marker distance of 17.92 cM. Marker density across the genome was uniform. This is the first SSR based linkage map of cassava and represents an important step towards quantitative trait loci mapping and genetic analysis of complex traits in M. esculenta species in national research program and other institutes with minimal laboratory facilities. SSR markers reduce the time and cost of mapping quantitative trait loci (QTL) controlling traits of agronomic interest, and are of potential use for marker-assisted selection (MAS).     

Converting restriction fragment length polymorphism to single‐strand conformation polymorphism markers and its application in the fine mapping of a trichome gene in cotton

A well‐characterized and systematically organized collection of genetic markers is crucial in the study of any crop species. It is the basis of map‐based gene cloning and crop improvements through marker‐assisted selections. Single‐strand conformation polymorphism (SSCP) has been a robust way of discovering new polymorphisms in marker development without the requirement of sequencing. Here, we report the first approach of applying SSCP marker discovery methods in the genetic map construction and gene mapping of cotton species. A total of 80 restriction fragment length polymorphism (RFLP) markers were selected from a region on published cotton genetic maps around the T1 gene related to cotton trichome. Among the 80 RFLPs, 28 showed polymorphisms through SSCP, showing a polymorphic rate of approximately 35%, which is much higher than that of simple sequence repeat (SSR) markers in the same region (7.8%). By integrating these newly generated SSCP markers, a detailed genetic map was reconstructed around this region using an F₂ population derived from a cross between Gossypium arboreum and G. herboceum. The reconstructed region comprises 22 SSCP markers, eight SSR markers and the T1 gene, spanning 21.6 cM. The marker order of the new map agrees well with published reference RFLP maps. The above results suggest that SSCP method can be applied very efficiently and reliably to the marker development of cotton genomes. It will prove to be even more valuable and robust after the public release of cotton whole‐genome sequences.     

Breeding and genomics status in faba bean (Vicia faba)

Faba bean is an important legume crop because of its high‐yield potential and nutrition‐dense grains. There have been significant achievements in faba bean improvement in the last four decades, which led to the doubling of the global yield average. This study reviews the genetic diversity, the breeding methodologies, major achievement on biotic and abiotic traits, and the recent molecular approaches. The high genetic diversity among faba bean accessions has been useful for increasing yield potential of the crop. Substantial increase in yield potential can be gained through the development of cultivars in open pollinated conditions. In the past, many faba bean varieties that are tolerant to abiotic and biotic stresses were released worldwide. The average yield gains varied from 1.65% per year in Syria to 4.17% per year in Ethiopia. The recent advances in molecular technologies will be used to develop more coherent genetic maps, which would also facilitate assembling and ordering genomic scaffolds in a future genome‐sequencing effort and molecular‐breeding approach.     

Comparative assessment of genetic diversity between wild and cultivated barley using gSSR and EST‐SSR markers

Barley is an economically important cereal crop especially for feed and malt production, but its value as food is increasing due to various health benefits. Wild barley is the progenitor of modern day barley cultivars possessing a rich source of genetic variation for various biotic and abiotic stresses. Species‐specific molecular markers have great potential for efficient introgression of these important traits from wild to cultivated barley. In the present study, 140 microsatellite markers were screened to assess the genetic variation and species‐specific markers between wild and cultivated germplasm. Of these 140, a polymorphic set of 48 genomic (gSSR) and 16 EST‐SSRs amplified a total of 685 alleles. Cluster analysis discriminated all 47 accessions and classified wild and cultivated genotypes into two distinct groups, according to their geographic origin. Our analysis indicated that gSSRs were more informative than EST‐based SSRs. Results from PCoA analysis for species‐specific alleles clearly suggest that wild barley genotypes contain a higher number of unique alleles.     

芝麻EST-SSR标记的开发和初步研究

为了加速分子标记在芝麻研究中的应用,利用网上现有的芝麻EST(expressed sequence tags)数据信息,开展了芝麻EST-SSR功能性标记的开发和利用研究。 在所有的3 328条芝麻EST序列中共确认得到1 785条非冗余EST序列。其中,在含有微卫星重复的148条序列中共检测有155个EST-SSR。非冗余EST序列总长为774.266 kb,平均每4.99 kb含有一个EST-SSR。EST-SSR的分布频率和特征分析表明,以AG/TC为重复基元(motif)的SSR出现最多,占总SSR的37.42%。利用这些序列,设计开发了50对EST-SSR引物,并分别选用36个芝麻、2个棉花、2个大豆和2个油葵进行多态性和通用性研究。其中44对引物在供试芝麻材料中扩增出条带,共产生108个位点,平均每对引物产生2.45个位点,多态信息含量(polymorphism information content, PIC)平均值为0.390。根据遗传相似性系数进行聚类,有26个芝麻材料聚类在两个大的亚类(III和IV)中,聚类结果表明芝麻的基因型与地理来源之间没有必然的联系。此外,分别有2对、3对和4对引物可以在棉花、大豆和油葵中进行通用性扩增。本研究证实这种全新开发的芝麻EST-SSR标记在芝麻遗传多样性分析、遗传图谱构建以及比较基因组等研究方面有广阔的利用前景。     

Molecular mapping of greenbug (Schizaphis graminum) resistance gene Rsg1 in barley

The greenbug, Schizaphis graminum (Rondani) is an extremely damaging aphid pest of barley (Hordeum vulgare L.) particularly in the southern Great Plains of the USA. The simply inherited, dominant resistance gene Rsg1 is in all greenbug‐resistant US barley cultivars. In this study, we conducted molecular mapping of Rsg1 using an F_2:3 population derived from a cross between the greenbug‐resistant Post 90*4/R015 and susceptible CI2260 inbred lines. Segregation of host responses to greenbug biotype E infestation confirmed that a single dominant gene is responsible for greenbug resistance in Post 90*4/R015. Simple sequence repeat (SSR) markers evenly distributed along the seven barley chromosomes were employed for the construction of a framework genetic map. Linkage analysis placed the Rsg1 locus in the long arm of chromosome 3H (3HL) flanked by SSR markers Bmag0877 and GBM1420 that were 35 cM apart. Polymorphic single‐nucleotide polymorphism (SNP) markers in 3HL were identified from an Illumina GoldenGate SNP assay and used for targeted mapping to locate Rsg1 to an 8.4‐cM interval. Comparative analysis identified syntenic genomic regions in Brachypodium distachyon chromosome 2, in which 37 putative genes were annotated including a NB‐LRR‐type resistance gene homologue that may be a potential candidate gene for the Rsg1 locus of barley. Results from this study offer a starting point for fine mapping and cloning of this aphid resistance gene in barley.     

Genetic diversity in groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genotypes and detection of marker trait associations for plant habit and seed size using genomic and genic SSRs

Groundnut (Arachis hypogaea L.) an important oilseed crop in India is known to have narrow genetic base. Therefore, the assessment of genetic diversity and detection of marker-trait association are important objectives for the genetic improvement of groundnut. The present study involved the development of 192 SSR markers from Arachis genomic survey sequences. From these, seven polymorphic SSRs along with 15 other genomic SSRs, 19 genic SSRs, and three STS markers were used to detect genetic diversity among 44 groundnut genotypes. These polymorphic SSR markers amplified 155 bands (76 genomic and 79 genic), of these 128 bands (67 genomic and 61 genic) were polymorphic. The genomic SSR exhibited 88.1% and genic SSRs displayed 77.2% allelic polymorphism. The polymorphic information content (PIC) of the markers ranged from 0.04 to 0.95. The pair-wise genetic similarity ranged from 24.2 to 90.7% for genomic SSR and 32.9 to 97.9% for genic SSR markers. Cluster analysis based on the pooled data from both genomic and genic SSRs revealed a dendrogram which could distinguish all the genotypes. Further, the AMOVA analysis detected 16.7% genetic variation due to differences in seed size and 13.0% due to plant habit. Based on locus-by-locus AMOVA and Kruskal-Wallis ANOVA and further confirmation by discriminant analysis and general linear model, six markers were found to be associated with plant habit and four markers with seed size.     

Integration of AFLPs,SSRs and SNPs markers into a new genetic map of industrial chicory (Cichorium intybus L. var. sativum)

A chicory genetic map of 1208 cM has been created using 247 F₂ plants and 237 markers (170 AFLP, 28 SSR, 27 EST‐SNP and 12 EST‐SSR). This map covers 84% of the chicory genome. The chicory‐genic‐markers‐associated sequences were used to find potential orthologs in mapped lettuce ESTs from the Compositae Genome Project Database. Twenty‐seven putative orthologous pairs were retained, pinpointing seven putative blocks of synteny that covered 11% of the chicory genome and 13% of the lettuce genome, opening new perspectives for the analysis of these two species.     

Integrated breeding approaches for improving drought and heat adaptation in chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is a dry season food legume largely grown on residual soil moisture after the rainy season. The crop often experiences moisture stress towards end of the crop season (terminal drought). The crop may also face heat stress at the reproductive stage if sowing is delayed. The breeding approaches for improving adaptation to these stresses include the development of varieties with early maturity and enhanced abiotic stress tolerance. Several varieties with improved drought tolerance have been developed by selecting for grain yield under moisture stress conditions. Similarly, selection for pod set in the crop subjected to heat stress during reproductive stage has helped in the development of heat‐tolerant varieties. A genomic region, called QTL‐hotspot, controlling several drought tolerance‐related traits has been introgressed into several popular cultivars using marker‐assisted backcrossing (MABC), and introgression lines giving significantly higher yield than the popular cultivars have been identified. Multiparent advanced generation intercross (MAGIC) approach has been found promising in enhancing genetic recombination and developing lines with enhanced tolerance to terminal drought and heat stresses.     

Genetic diversity of garlic (Allium sativum L.) germplasm from China by fluorescent‐based AFLP,SSR and InDel markers

Garlic (Allium sativum L.), an asexually propagated crop, is an important vegetable and medicinal plant. China is the biggest garlic producer in the world; however, the genetic background of garlic from China is not well understood. In this study, population structure and clustering analysis of garlic germplasm was performed using amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) and insertion–deletion (InDel) markers. Among 212 accessions of garlic, genetic diversity analysis identified 546 alleles amplified by AFLP, SSR and InDel primers, and 492 of these were polymorphic. All accessions were divided into five groups by structure analysis and neighbor‐joining clustering. Most traits, including allicin content, were only slightly affected by population structure, which indicated that this germplasm can be used as populations for association mapping. The results provide a molecular basis for understanding the genetic diversity of the garlic germplasm preserved in China.     

Development and utilization of genomic and genic microsatellite markers in Assam tea (Camellia assamica ssp. assamica) and related Camellia species

Microsatellite or simple sequence repeat (SSR) markers are valuable tools for many purposes, such as phylogenetic, fingerprinting and molecular breeding studies. However, such marker resources are unavailable in Assam tea (Camellia assamica ssp. assamica; Masters). With an objective to enrich the repertoire of microsatellite markers in traditional tea, 185 novel microsatellite (150 genomic and 35 genic) markers were identified from (GA)n‐enriched genomic libraries and public expressed sequence data in Assam tea. High‐quality 0.412‐Mb non‐redundant (NR) genomic data set derived from nucleotide sequencing of 1297 (GA)n‐enriched genomic positive clones and 2723 unigenes (1.33 Mb) predicted from 10 803 random public expressed sequence tags (ESTs) in C. assamica ssp. assamica were utilized for identification of genomic and genic microsatellite markers, respectively. The average number of alleles and polymorphic information content (PIC) recorded for the newly developed SSR markers were 6.17 and 0.398, respectively. The average observed (H_o) and expected (H_e) heterozygosity varied from 0.626 to 0.697, respectively. These markers were found to be highly transferable (74.5–100%) to cultivated (C. sinensis, C. assamica ssp. lasiocalyx) and five wild Camellia species. Genetic diversity coefficient detected a high level of divergence in 24 cultivated tea accessions (69.3%). Phylogenetic analysis revealed that major groupings were broadly in accordance with taxonomic classification of tea, and all the wild Camellia species remained as an out‐group. The high polymorphic content coupled with high rate of cross‐transferability demonstrates wider applicability of novel microsatellite markers in genotyping, genetic diversity, genome mapping and evolutionary studies in various Camellia species.     

Development,characterization and mapping of microsatellite markers for lentil (Lens culinaris Medik.)

Lentil is the sixth most important pulse crop terms of production in the world, but the number of available and mapped SSR markers are limited. To develop SSR markers in lentil, four genomic libraries for (CA)n, (GA)n, (AAC)n and (ATG)n repeats were constructed. A total of 360 SSR primers were designed and validated using 15 Turkish lentil cultivars and genotypes. The most polymorphic repeat motifs were GA and CT, with a mean number of alleles per locus of 7.80 and 6.55, respectively. Seventy‐eight SSR primers amplified a total of 400 polymorphic alleles, whereas 71 SSR primers produced markers within the expected size range. For 78 polymorphic SSR primers, the average number of alleles per locus was 5.1 and PIC value ranged from 0.07 to 0.89, with an average of 0.58. A linkage map was constructed using 92 individual F₂ plants derived from a cross between Karacada? × Silvan, with 47 SSR markers. The SSR markers developed in this study could be used for germplasm classification and identification and mapping of QTL in lentil.     

Genetic linkage map of Chinese native variety faba bean (Vicia faba L.) based on simple sequence repeat markers

Simple sequence repeat (SSR) marker is a powerful tool for construction of genetic linkage map which can be applied for quantitative trait loci (QTL) and marker‐assisted selection (MAS). In this study, a genetic map of faba bean was constructed with SSR markers using a 129 F₂ individuals population derived from the cross of Chinese native variety 91825 (large seed) and K1563 (small seed). By screening 11 551 SSR primers between two parents, 149 primer pairs were detected polymorphic and used for F₂ population analysis. This SSR‐based genetic linkage map consisted of 15 linkage groups with 128 SSR. The map encompassed 1587 cM with an average genetic distance of 12.4 cM. The genetic map generated in this study will be beneficial for genetic studies of faba bean for identification of marker‐locus‐trait associations as well as comparative mapping among faba bean, pea and grasspea.