Simple Sequence Repeat Primers Research Articles

Development of novel SSR markers derived from genomic and transcriptomic data of Moringa oleifera L. var. PKM1 and their applicability

ABSTRACT Limited genomic resources, especially on breeder-friendly simple sequence repeats (SSR), are available in Moringa, a multi-purpose miracle tree. This study predicted a total of 29913 genomic-SSRs and 1364 genic-SSRs from the omics data obtained from a Moringa cultivar, PKM1. Among the genomic SSR motifs, 1, 414, 2183, 25600, 1106 and 609 SSRs were Mono, Di, Tri, Tetra, Penta, and Hexa repeats, respectively. Higher frequency for AGAG (24.01%) followed by GAGA (16.68) and AAAA (13.18%) was observed in genic-SSRs sequences. Primer pairs for 22,336 genomic- and genic-SSR markers were developed and 117 SSR primer pairs were randomly selected and assayed in four representative Moringa cultivars and ecotypes. Consequently, 94.44% of them have showed high level of DNA polymorphism by generating 1 to 4 alleles per SSR marker (with an average of 1.75) among the investigated Moringa accessions with a maximum polymorphic information content (PIC) of 0.95. Besides using as simple and affordable indicator to differentiate morphologically similar Moringa ecotypes, the SSRs reported in this study will also have several other potential applications such as seed purity analysis, intellectual property protection, identification of introgressions in interspecific crosses, evolutionary studies, germplasm management, gene and/or QTL identification and marker-assisted selection.

Population genetics of Hyphaene thebaica Mart., in Rabigh province, Saudi Arabia: Implications for conservation

Hyphaene thebaica is a perennial plant distributed in desert and subtropical regions of the world. In Rabigh Province, western Saudi Arabia, the few persisting populations of this species are exposed to many threats, including overcutting and, recently, human habitation. These threats are predicted to be exacerbated with the advancement of aridification caused by climate change. The conservation and revival of the diminished populations of H. thebaica requires an assessment of their genetic diversity and genetic differentiation. To accomplish this objective, we applied 10 simple sequence repeat (SSR) primer pairs, with which all are polymorphic loci. These polymorphic loci were used to determine the population genetics of 60 plant accessions sampled from 5 populations of H. thebaica located in five sites in Rabigh Province: Wadi EL Khaneg, Wadi Al Johfa, Wadi Al Hakak and Wadi Khurieba and Wadi Kuliayah . Low to moderate levels of genetic diversity were found in all populations (the values of the PPL% ranged between 52.5% and 22.5%) along with a decreased value of HT (0.388) and a considerable inbreeding value (F= 0.4552), which verified an obvious shortage of heterozygotes. High genetic differentiation among the populations and a low level of gene flow suggest isolation among the H. thebaica populations, which caused a severe deficiency in gene migration. The data obtained herein will inspire several recommendations for conservation the existing populations, including seed preservation, and management of human activities. All of these actions are urgently needed to prevent imminent extinction.

​SSR Based Genetic Diversity Analysis in Fenugreek (Trigonella foenum-graecum L.) Genotypes

Background: Fenugreek (Trigonella foenum-graecum L.) is an important seed spice crop widely grown all over the world. In India, the state of Rajasthan is known for fenugreek production and productivity in the world. A concerted assessment of genetic variability among the germplasm accession is essential for breeding new superior varieties. Molecular markers such as AFLP, RAPD, ISSR, SSR, SCAR, SCoT, SRAP have become for the characterization of the germplasm rapidly and accurately. The present study aimed to characterize 20 elite fenugreek genotypes using simple sequence repeat (SSR) markers to assess the existing genetic diversity of this medicinal crop. Methods: The present study was carried out at the Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan, India. Total genomic DNA was isolated from old leaves using the CTAB method (Doyle and Doyle, 1990). Further, PCR based genetic diversity was analyzed with using 50 SSR primer pairs. Dendrogram was constructed using NTSYSpc version 2.2 and clustering of the genotypes was done. Result: Twenty genotypes of fenugreek were assessed for genetic diversity analysis using SSR markers. Out of 50 markers 43 primer pairs produced 130 alleles with an average of 84.60% polymorphism. Jaccard’s similarity coefficient lied between and 0.39 to 0.82. Based on UPGMA clustering, a dendrogram consisting of five main clusters was generated with wide variability among the studied genotypes. These diverse genotypes so identified could be gainfully utilized in the fenugreek breeding programme.

Quantitative trait loci analysis for flower‐related traits in almond (Prunus dulcis)

AbstractAlmond is one of the most important nut crops in Turkey. Simple sequence repeat (SSR)‐based linkage maps are very reliable in constructing linkage maps due to the co‐dominant and highly reproducible nature of the SSR markers. The objective of this study was to construct well‐saturated, SSR‐based genetic linkage maps using ‘Gulcan‐2 × Lauranne’ and ‘Guara × Nurlu’ F1 populations and to identify QTLs related to the flower colour, stigma–anther position and full blooming date in almond. A total of 275 SSR primer pairs selected from the previous studies of various Prunus species were tested in both populations. A total of 175 SSR loci segregated in ‘Gulcan‐2 × Lauranne’ population and 162 SSR loci were mapped into eight linkage groups. The total map length was 514.6 cM, with an average marker density of 3.2 cM. In ‘Guara × Nurlu’ population, 159 markers segregated, and 153 SSR loci were mapped. The total map length was 493.9 cM with an average marker distance of 3.2 cM. Five and four major QTLs were detected in ‘Gulcan‐2 × Lauranne’ and ‘Guara × Nurlu’ populations, respectively. The linkage maps constructed in this study can be used in QTL analysis of important phenotypic traits in almond, whereas identified QTL regions and closely related markers can be used for marker assisted selection in future almond breeding programmes.

Genome-Wide Analysis of Simple Sequence Repeats in Cabbage (Brassica oleracea L.).

Cabbage (Brassica oleracea L. var. capitata) accounts for a critical vegetable crop belonging to Brassicaceae family, and it has been extensively planted worldwide. Simple sequence repeats (SSRs), the markers with high polymorphism and co-dominance degrees, offer a crucial genetic research resource. The current work identified totally 64,546 perfect and 93,724 imperfect SSR motifs in the genome of the cabbage ‘TO1000.’ Then, we divided SSRs based on the respective overall length and repeat number into different linkage groups. Later, we characterized cabbage genomes from the perspectives of motif length, motif-type classified and SSR level, and compared them across cruciferous genomes. Furthermore, a large set of 64,546 primer pairs were successfully identified, which generated altogether 1,113 SSR primers, including 916 (82.3%) exhibiting repeated and stable amplification. In addition, there were 32 informative SSR markers screened, which might decide 32 cabbage genotypes for their genetic diversity, with level of polymorphism information of 0.14–0.88. Cultivars were efficiently identified by the new strategy designating manual diagram for identifying cultivars. Lastly, 32 cabbage accessions were clearly separately by five Bol-SSR markers. Besides, we verified whether such SSRs were available and transferable in 10 Brassicaceae relatives. Based on the above findings, those genomic SSR markers identified in the present work may facilitate cabbage research, which lay a certain foundation for further gene tagging and genetic linkage analyses, like marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Molecular Dissection for Boll Shedding in Upland Cotton Using Microsatellites

DNA markers application in marker-assisted breeding of cotton is handicapped due to low genetic diversity in cotton germplasm. The present study was designed to identify DNA markers, predominately simple sequence repeats (SSRs), associated with tolerance/resistance to heat stress as a consequence of boll shedding. To find out the genetic diversity a total of 24 cotton genotypes and 50 SSR primers were used. Total 288 alleles were produced with an average of 5.7 alleles per primer. Bootstrap cluster analysis used to generate a dendrogram that cluster the 24 accessions into two main clusters. Eleven out of 24 genotypes fall in a single cluster. Phenotypically H-4074 gives more diversity, while genotypically H-4074 sheared the same genetic background as H-4070, H-4091 and H-4090. Low genetic diversity was observed among both genotypic and phenotypic as maximum varieties fall in single group. This study helps for selecting diverse accessions with multiple phenotypic traits, which were drought to boll shedding. It suggests further elaborating the molecular genetic diversity by using new SSR marker to improve the yield of cotton cultivars. These preliminary results set the stage for initiating in depth marker-trait association studies, which will be instrumental for initiating marker-assisted breeding in cotton.

Genetic diversity determined by agronomic traits and SSR markers in two South American orange‐fleshed sweetpotato breeding populations with potential for population hybrid breeding

AbstractOrange‐fleshed sweetpotato [Ipomoea batatas (L.) Lam.] (OFSP) breeding populations have gained importance for food security and health reasons. This study's main objectives were to determine genetic diversity in parental material of two OFSP populations developed in Peru (Jewel [PJ] and Zapallo [PZ]) relative to mega‐clones (MCs) using agronomic traits and simple sequence repeat (SSR) markers and to determine whether PJ and PZ are mutually heterotic by developing a PJ × PZ hybrid population (H0). Field trials were performed with clones for PJ (n = 49), PZ (n = 31), MC (n = 21), and H0 (n = 6,898) in Peru. Traits recorded were storage root yield (RYTHA), number of commercial roots per plant, foliage yield, biomass, harvest index, and dry matter (RDM), β‐carotene (RBC), protein, starch, sucrose, iron, zinc, and calcium content of storage roots. Sixty‐six pairs of SSR primers were used to determine molecular diversity. Statistics used were linear mixed models, principal component analysis, and standard procedures for molecular data. New genetic variation was found in PJ and PZ (e.g., RDM ≥29% with RBC ≥25 mg 100 g−1 dry weight basis). For most traits, genetic variance in PJ and PZ was as large as in MC. The SSR marker data clearly separated PJ and PZ into two gene‐pools, together covering nearly the entire MC molecular diversity. Average RYTHA in H0 was high (40.7 t ha−1) with average heterosis increment of 21.8% and range −30.6 to 139.4%. The PJ and PZ lend themselves to study of the efficiency of reciprocal recurrent selection in sweetpotato population hybrid breeding.

Genetic diversity and population structure analysis in a large collection of Vicia amoena in China with newly developed SSR markers

Vicia amoena is a high-nutritional quality forage similar to alfalfa. However, studies on the genetic background of V. amoena are scarce. In the present study, the genetic variation of 24 V. amoena populations was assessed with newly developed simple sequence repeat (SSR) markers. A total of 8799 SSRs were identified in the V. amoena genomic-enriched sequences, and the most abundant repeat number was four. A total of 569 sampled individuals were assayed to evaluate the genetic diversity of the V. amoena populations based on 21 polymorphic SSR primers. The polymorphism information content (PIC) ranged from 0.896 to 0.968, with an average of 0.931, which indicated that the markers were highly informative. Based on analysis of molecular variance, 88% of the variance occurred within populations, and the remaining 12% of the variance occurred among populations. The high degree of gene flow (Nm= 4.958) also showed slight differentiation among the V. amoena populations. The V. amoena populations were mainly clustered by steppe and mountain habitats based on principal coordinate analysis (PCoA) and STRUCTURE analysis. This indicated that the elevation and special habitat of geographical origins may be important factors affecting the clustered pattern of V. amoena populations. Neighbour-joining (NJ) analysis did not separate the populations well by geographical origin, which indicated that the genetic structure of V. amoena was complex and needs further study. Overall, our results showed that the newly developed SSR markers could benefit the V. amoena research community by providing genetic background information to help establish a foundation for breeding improvement and germplasm resource conservation.

Genome survey sequencing of common vetch (Vicia sativa L.) and genetic diversity analysis of Chinese germplasm with genomic SSR markers

BackgroundCommon vetch (Vicia sativa L.) is an annual legume with excellent suitability in cold and dry regions. Despite its great applied potential, the genomic information regarding common vetch currently remains unavailable.Methods and resultsIn the present study, the whole genome survey of common vetch was performed using the next-generation sequencing (NGS). A total of 79.84 Gbp high quality sequence data were obtained and assembled into 3,754,145 scaffolds with an N50 length of 3556 bp. According to the K-mer analyses, the genome size, heterozygosity rate and GC content of common vetch genome were estimated to be 1568 Mbp, 0.4345 and 35%, respectively. In addition, a total of 76,810 putative simple sequence repeats (SSRs) were identified. Among them, dinucleotide was the most abundant SSR type (44.94%), followed by Tri- (35.82%), Tetra- (13.22%), Penta- (4.47%) and Hexanucleotide (1.54%). Furthermore, a total of 58,175 SSR primer pairs were designed and ten of them were validated in Chinese common vetch. Further analysis showed that Chinese common vetch harbored high genetic diversity and could be clustered into two main subgroups.ConclusionThis is the first report about the genome features of common vetch, and the information will help to design whole genome sequencing strategies. The newly identified SSRs in this study provide basic molecular markers for germplasm characterization, genetic diversity and QTL mapping studies for common vetch.

Identification and Fine Mapping of a Locus Related to Leaf Up-Curling Trait (Bnuc3) in Brassica napus.

Leaf trait is an important target trait in crop breeding programs. Moderate leaf curling may be a help for improving crop yield by minimizing the shadowing by leaves. Mining locus for leaf curling trait is of significance for plant genetics and breeding researches. The present study identified a novel rapeseed accession with up-curling leaf, analyzed the up-curling leaf trait inheritance, and fine mapped the locus for up-curling leaf property (Bnuc3) in Brassica napus. Genetic analysis revealed that the up-curling leaf trait is controlled by a single dominant locus, named BnUC3. We performed an association study of BnUC3 with single nucleotide polymorphism (SNP) markers using a backcross population derived from the homozygous up-curling leaf line NJAU-M1295 and the canola variety ‘zhongshuang11’ with typical flat leaves, and mapped the BnUC3 locus in a 1.92 Mb interval of chromosome A02 of B. napus. To further map BnUC3, 232 simple sequence repeat (SSR) primers and four pairs of Insertion/Deletion (InDel) primers were developed for the mapping interval. Among them, five SSR markers and two InDel markers were polymorphic. By these markers, the mapping interval was narrowed to 92.0 kb using another F2 population. This fine mapping interval has 11 annotated genes among which BnaA02T0157000ZS were inferred to be candidate casual genes for up-curling leaf based on the cloned sequence analysis, gene functionality, and gene expression analysis. The current study laid a foundational basis for further elucidating the mechanism of BnUC3 and breeding of variety with up-curling leaf.

Genetic Structure and Geographical Differentiation of Larix sibirica Ledeb. in the Urals

The Ural Mountains and the West Eurasian Taiga forests are one of the most important centers of genetic diversity for Larix sibirica Ledeb. Forest fragmentation negatively impacts forest ecosystems, especially due to the impact of their intensive use on the effects of climate change. For the preservation and rational use of forest genetic resources, it is necessary to carefully investigate the genetic diversity of the main forest-forming plant species. The Larix genus species are among the most widespread woody plants in the world. The Siberian larch (Larix sibirica, Pinaceae) is found in the forest, forest-tundra, tundra (Southern part), and forest-steppe zones of the North, Northeast, and partly East of the European part of Russia and in Western and Eastern Siberia; in the Urals, the Siberian larch is distributed fragmentarily. In this study, eight pairs of simple sequence repeat (SSR) primers were used to analyse the genetic diversity and population structure of 15 Siberian larch populations in the Urals. Natural populations in the Urals exhibit indicators of genetic diversity comparable to those of Siberia populations (expected heterozygosity, He = 0.623; expected number of alleles, Ne = 4017; observed heterozygosity, Ho = 0.461). Genetic structure analysis revealed that the examined populations are relatively highly differentiated (Fst = 0.089). Using various algorithms for determining the spatial genetic structure, the examined populations formed three groups according to geographical location. The data obtained are required for the development of species conservation and restoration programs, which are especially important in the Middle Urals, which is the region with strong forest fragmentation.

Morphological and genetic diversity of Senecio vulgaris L. (Asteraceae) in Iran

Senecio vulgaris L., an annual herb belonging to the Asteraceae, is widely distributed in different regions of the world. There is no information on the intraspecific variations of the morphological and molecular features of this species. In the present investigation, we studied the morphological and genetic diversity of 81 accessions of S. vulgaris collected from 10 geographical populations. Eleven inter simple sequence repeat (ISSR) primers were used for the examination of genetic variations among the populations. Analysis of molecular variance (AMOVA) and GST analyses revealed significant differences among the investigated populations. A significant correlation between genetic distance and geographical distance was revealed by the Mantel test. However, reticulation analysis indicated the occurrence of gene flow among most of the populations studied. Principal component analysis (PCA) plot showed that the number of capitula, length of the cauline leaf and plant height were the most variable morphological characters. Principal coordinates analysis (PCoA) plot revealed two groups of populations, according to molecular and morphological data. The results suggested the existence of possible intraspecific taxonomic ranks within this species.

Agro-morphological and genetic diversity studies in Rice (Oryza sativa L.) germplasm using microsatellite markers.

Knowledge of the genetic diversity and population structure of germplasm collections is an important foundation for crop improvement. Rice production across a broad range of rice-growing environments results in a diverse array of local rice varieties. Many rice varieties have been lost as a result of biodiversity loss and are now grown in Pakistan. To protect the biodiversity of rice varieties, an experiment was carried out to check the genetic and morphological variations between 8 exotic and 7 local rice genotypes, using 5 different SSR markers, i.e., RM3, RM259, RM341, RM520, and RM11943. The analysis of morphological and quality traits of rice observed significant variation across genotypes. The results revealed that genotype Irri-Pak attained the highest plant height and primary branch plant-1, while genotype Mushkan produced a higher number of productive tillers and obtained a higher fertility factor (%). Similarly, the highest value for panicle length was observed for genotype Faker-e-Malakand, 1000-grains weight in genotype Calmochi, and maximum days to maturity was noticed in genotype Swati-2014. Moreover, the genotype Brio attained the highest value of stem diameter, while maximum seed length was noted in the genotype Sug Dasi. The highest number of primary branches plant-1 in genotype Ibge-I and secondary branches plant-1 in genotype Calmochi were noticed. A higher concentration of sodium and potassium was observed for the genotype Marte, while the genotype Muskan attained the maximum content of copper. Moreover, the highest concentration of iron in genotype Originario, zinc in genotype JP-5, and cadmium content were noticed in genotype Ibge. Similarly, the dendrogram analysis for quantitative parameters showed three clusters at 74.13% similarities. Whereas all the genotypes of European origin formed a separate cluster. A set of 5 simple sequence repeat primers, covering four chromosomes, amplified a total of 14 alleles and showed 100% polymorphism with an average PIC value ranging from 0.39 to 0.91. The UPGMA cluster analysis separated the 15 rice genotypes into 3 main groups based on 32.5% similarities and the highest genetic distance (45.1%) was observed between two genotypes (Fakher-e-malakand and Musa), having different geographical origins. There was no genetic distance between the genotypes Marte and Brio, irrespective of having the same origin. The maximum genetic distances were noted for genotype, Fakhre-e-Malakand and Musa having a different origin, while the minimum genetic distance was shown by genotypes, Marte and Onice, from the same origin.

Development and preliminary application of novel genomewide SSR markers for genetic diversity analysis of an economically important bio-control agent Platygaster robiniae (Hymenoptera: Platygastridae)

Platygaster robiniae Buhl and Duso (Hymenoptera: Platygastridae) is an egg-larvae parasitoid of the black locust gall midge (Obolodiplosis robiniae) (Haldeman) (Diptera: Cecidomyiidae) which is a serious invasive pest in China, where it attacks an important hardwood species, the black locust tree, Robini pseudoacacia L. (Fabales: Fabaceae). Despite the use of P. robiniae as an effective biocontrol agent, the absence of sequence data and other molecular markers have limited its genetic applications for pest management in forests. Simple-sequence repeats (SSRs) are valuable molecular markers for population genetic structure studies. In the present study, we identified 14,123 SSRs, of which 7799 SSR primer pairs were successfully designed. Subsequently, 240 SSR were chosen and tested with 48 P. robiniae accessions from two geographically separated populations in north and south China. Of these, 34 were polymorphic, with an average of three alleles (Na) and four genotypes (NG) each. The average values of observed heterozygosity (Ho) was 0.3514, expected heterozygosity (He) 0.4167, Shannon's information index (I) 0.7143, and polymorphism information content (PIC) 0.3558, respectively. Neighbour joining analysis (bootstrap 1000) revealed that Chengdu (CD) and Dangdong (DD) popluations clustered into two main divisions, and some individuals from two popluations clustered together as the third devision, which indicated the gene flow and genetic differentiation were present between two populations. Our finding indicates that these SSR markers will be useful for further studies on the genotype identification and genetic mapping of the genus Platygaster.

Aloe vera L. (Asphodelaceae): Supplementation of in-vitro culture medium with Aloe vera gel for production of genetically stable plants

As in vitro culture environments can be mutagenic, it's challenging to produce true-to-true type plants from meristems. Therefore, the present study aimed to devlop an efficient protocol for micropropagation from shoot tip explants of medicinally important Aloe vera using different concentrations of A. vera leaf gel (AvG) as organic supplement to the culture media. Assessment of the genetic stability of A. vera in vitro multiplied plants compared to the donor plant is one of the most important pre-requisites especially before its use for commercial and medicinal purposes. For shoot multiplication, the highest number of axillary shoots recorded was 13.27 ±5.11 with an average length of 2.21 ±0.84 cm on M3 medium (MS + 0.2 mg L−1 of β-indole butyric acid (IBA) + 3 mg L−1 benzylamino-purine (BA) + 5% AvG). All three strength of MS medium (1, 1/2, 1/4) with different concentrations of AvG resulted in 100% root induction after 4 weeks of culture. All the rooted microshoots (100%) were successfully acclimatized. To provide a reliable conclusion on its nutritive supply to MS medium, the total phenolic (TPC), condensed tannins (TTC), flavonoid (TFC), and flavonol (TFlC) content as well as the antioxydant activity of AvG were investigated. TPC and TTC values were found to be 31.02 ± 0.46 μg gallic acid equivalent mg−1 of extract and 4.00 ± 0.96 μg catechin equivalent mg−1 of extract, respectively while TFC and TFlC were 13.58 ± 0.84 and 9.75 ± 0.58 μg rutin equivalent mg−1 of extract, respectively. The results of antioxydant activity of AvG following DPPH, FRAP and ABTS assays were equal to 12.50 ± 0.89; 49.59 ± 2.07 and 64.93 ± 3.23 µmol Trolox equivalent mg−1 of extract, respectively which may explain the positive role of AvG as supplement.The genetic stability of in vitro propagated A. vera plants was evaluated using 10 inter simple sequence repeat (ISSR), 10 start codon targeted (SCoT) and 10 simple sequence repeat (SSR) markers. Out of these, 5 ISSR, 3 SCoT and 6 SSR primers produced resolvable, reproducible and scorable bands. ISSR primers generated 32 amplification products ranging from 200 to 1040 bp in size with a mean number of 6.4 bands while SCoT primers generated 33 amplicons with an average of 11 bands in the range of 160–2410 bp. In the SSR products, 6 bands were observed, each primer produced one band ranging from 410 to 720 bp. All banding profiles were monomorphic proving that the stability of the micropropagated A. vera plants was maintained. This study proved the suitability of supplementing in vitro culture media with AvG to enhance direct regeneration through shoot tip explants of A. vera for large scale micropropagation applications.

Genetic analysis of Lepidium spp. by SSR and ISSR molecular markers

Simple sequence repeat (SSR) and inter simple sequence repeat (ISSR) markers were employed to evaluate the genetic diversity and population structure of 22 accessions belonging to three Lepidium species. Results revealed a high level of genetic diversity in the Lepidium accessions. A total of 58 bands were amplified by 11 ISSR primers, of which all were polymorphic, while 11 SSR primers produced 30 bands, of which 27 (90.0%) were polymorphic. The average values of polymorphism information content (PIC), marker index (MI), effective multiplex ratio (EMR) and percentage of polymorphic loci (PPL) in the ISSR markers were higher than those in SSRs, whereas higher values of number of effective alleles (Ne) and Shannon's information index (I) were observed by SSR markers. Based on the number of observed alleles (Na), Ne, I, expected heterozygosity (He) and PPL, the highest genetic diversity was found in L.draba followed by L.sativum by both markers. The analysis of molecular variance showed that higher allelic diversity within species than among species. The 22 Lepidium accessions grouped into three main clusters based on neighbor-joining (NJ) clustering and principal coordinate analysis (PCoA), and three (ISSR) or four (SSR and pooled data) sub-populations based on STRUCTURE analysis independent of their taxonomy. This is the first report of genetic diversity and relationships among accessions of L.sativum, L.draba and L. latifolium using SSR and ISSR markers. Therefore, these results can be utilized to improve these Lepidium species for various desired traits through molecular and hybridization breeding techniques.

Development of Genome-wide Simple Sequence Repeat Markers from Whole-genome Sequence of Mungbean (Vigna radiata)

Background: Mungbean is an important pulse crop and it is mainly cultivated in Asia for human consumption. Its small genome and diploid nature make it a well-suited model organism among legume crops. Thus, cost-effective, reliable and highly polymorphic molecular markers distributing the whole genome are needed for diversity, mapping and functional genomics studies in this model species. Methods: The whole-genome sequence of mungbean was obtained and used as a source of identification of simple sequence repeats (SSR). The sequence reads were aligned and SSRs detection was performed using the Phobos plugin tandem repeat finder in the Geneious software program. A total of 12 mungbean genotypes were selected to validate the newly developed SSR markers. Result: In the present study, a total of 12, 49,774 and 11, 86, 386 perfect and imperfect SSR repeats were identified from the mungbean genome. The tri-repeats were the most abundant (26.10%), followed by hexa (20.82%), penta (20.45%), tetra (17.65%) and di-repeats (14.95%). We designed 1330 SSR primers based on the genomic sequence of flanking perfect SSRs (Di and tri-repeats). Among them, 50 SSR primers uniformly distributed across the 11 mungbean chromosomes were selected and used to validate 12 mungbean genotypes. The newly developed genomic SSR markers generated in the present study are a valuable genomic resource for the mungbean breeding programs.

Genetic Variation of Native Perilla Germplasms Collected from South Korea Using Simple Sequence Repeat (SSR) Markers and Morphological Characteristics.

Using morphological characteristics and simple sequence repeat (SSR) markers, we evaluated the morphological variation and genetic diversity of 200 Perilla accessions collected from the five regions of South Korea and another region. In morphological characteristics analysis, particularly leaf color, stem color, degree of pubescence, and leaf size have been found to help distinguish the morphological features of native Perilla accessions cultivated in South Korea. Twenty SSR primer sets confirmed a total of 137 alleles in the 200 Perilla accessions. The number of alleles per locus ranged from 3 to 13, with an average number of alleles per locus of 6.85. The average genetic diversity (GD) was 0.649, with a range of 0.290–0.828. From analysis of SSR markers, accessions from the Jeolla-do and Gyeongsang-do regions showed comparatively high genetic diversity values compared with those from other regions in South Korea. In the unweighted pair group method with arithmetic mean (UPGMA) analysis, the 200 Perilla accessions were found to cluster into three main groups and an outgroup with 42% genetic similarity, and did not show a clear geographic structure from the five regions of South Korea. Therefore, it is believed that landrace Perilla seeds are frequently exchanged by farmers through various routes between the five regions of South Korea. The results of this study are expected to provide interesting information on the conservation of these genetic resources and selection of useful resources for the development of varieties for seeds and leafy vegetables of cultivated Perilla frutescens var. frutescens in South Korea.

High-Frequency Homologous Recombination Occurred Preferentially in Populus.

Homologous recombination (HR), the most significant event in meiosis, has important implications for genetic diversity and evolution in organisms. Heteroduplex DNA (hDNA), the product of HR, can be captured by artificially induced chromosome doubling during the development of the embryo sac to inhibit postmeiotic segregation, subsequently, and hDNAs are directly detected using codominant simple sequence repeat (SSR) markers. In the present study, two hybrid triploid populations derived from doubling the chromosomes of the embryo sac induced by high temperature in Populus tomentosa served as starting materials. Eighty-seven, 62, and 79 SSR markers on chromosomes 01, 04, and 19, respectively, that were heterozygous in the maternal parent and different from the paternal parent were screened to detect and characterize the hDNA in P. tomentosa. The results showed that the hDNA frequency patterns on chromosomes changed slightly when the number of SSR primers increased. The highest hDNA frequency occurred at the adjacent terminal on chromosomes, which was slightly higher than those at the terminals in the two genotypic individuals, and the hDNA frequency gradually decreased as the locus-centromere distance decreased. With the increase in the number of SSR markers employed for detection, the number of recombination events (REs) detected significantly increased. In regions with high methylation or long terminal repeat (LTR) retrotransposon enrichment, the frequency of hDNA was low, and high frequencies were observed in regions with low sequence complexity and high gene density. High-frequency recombination occurring at high gene density regions strongly affected the association between molecular markers and quantitative trait loci (QTLs), which was an important factor contributing to the difficulty encountered by MAS in achieving the expected breeding results.

Fine mapping of the BnaC04.BIL1 gene controlling plant height in Brassica napus L

BackgroundPlant height is an important architecture trait which is a fundamental yield-determining trait in crops. Variety with dwarf or semi-dwarf phenotype is a major objective in the breeding because dwarfing architecture can help to increase harvest index, increase planting density, enhance lodging resistance, and thus be suitable for mechanization harvest. Although some germplasm or genes associated with dwarfing plant type have been carried out. The molecular mechanisms underlying dwarfism in oilseed rape (Brassica napus L.) are poorly understood, restricting the progress of breeding dwarf varieties in this species. Here, we report a new dwarf mutant Bndwarf2 from our B. napus germplasm. We studied its inheritance and mapped the dwarf locus BnDWARF2.ResultsThe inheritance analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped in an interval of 787.88 kb on the C04 chromosome of B. napus by Illumina Brassica 60 K Bead Chip Array. To fine-map BnDWARF2, 318 simple sequence repeat (SSR) primers were designed to uniformly cover the mapping interval. Among them, 15 polymorphic primers that narrowed down the BnDWARF2 locus to 34.62 kb were detected using a F2:3 family population with 889 individuals. Protein sequence analysis showed that only BnaC04.BIL1 (BnaC04g41660D) had two amino acid residues substitutions (Thr187Ser and Gln399His) between ZS11 and Bndwarf2, which encoding a GLYCOGEN SYNTHASE KINASE 3 (GSK3-like). The quantitative real-time PCR (qRT-PCR) analysis showed that the BnaC04.BIL1 gene expressed in all tissues of oilseed rape. Subcellular localization experiment showed that BnaC04.BIL1 was localized in the nucleus in tobacco leaf cells. Genetic transformation experiments confirmed that the BnaC04.BIL1 is responsible for the plant dwarf phenotype in the Bndwarf2 mutants. Overexpression of BnaC04.BIL1 reduced plant height, but also resulted in compact plant architecture.ConclusionsA dominant dwarfing gene, BnaC04.BIL1, encodes an GSK3-like that negatively regulates plant height, was mapped and isolated. Our identification of a distinct gene locus may help to improve lodging resistance in oilseed rape.