Haplotyping of Rice Genotypes Using Simple Sequence Repeat Markers Associated with Salt Tolerance

Salt stress is a major problem in most of the rice growing areas in the world. A major QTLSaltol associated with salt tolerance at the seedling stage has been mapped on chromosome 1 in rice.This study aimed to characterize the haplotype diversity at Saltol and additional QTLs associated withsalt tolerance. Salt tolerance at the seedling stage was assessed in 54 rice genotypes in the scale of 1to 9 score at EC = 10 dSm^-1 under controlled environmental conditions. Seven new breeding linesincluding three KMR3/O. rufipogon introgression lines showed similar salt tolerant ability as FL478 andcan be good sources of new genes/alleles for salt tolerance. Simple sequence repeat （SSR） markerRM289 showed only two alleles and RM8094 showed seven alleles. Polymorphic information contentvalue varied from 0.55 for RM289 to 0.99 for RM8094 and RM493. Based on 14 SSR markers, the 54lines were clearly separated into two major clusters. Fourteen haplotypes were identified based onSaltol linked markers with FL478 as the reference. Alleles of RM8094 and RM3412 can discriminatebetween the salt tolerant and susceptible genotypes clearly and hence can be useful in marker-assistedselection at the seedling stage. Other markers RM10720 on chromosome 1 and RM149 and RM264 onchromosome 8 can also distinguish tolerant and susceptible lines but with lesser stringency.

Genetic Diversity of Chinese and Swedish Rapeseed (Brassica napus L.) Analyzed by Inter-Simple Sequence Repeats (ISSRs)

We have compared genetic diversity of 24 Chinese weak-winter, Swedish winter and spring B. napus accessions by inter-simple sequence repeats (ISSRs). By cluster analysis (UPGMA) based on 125 polymorphism bands amplified with 20 primers, the 24 accessions were divided into three groups. Six Swedish winter lines and eight Chinese weak-winter lines were in the group I and the groupⅡwere two Chinese weak-winter lines XiangyoulS and Bao81. The third group contained eight Swedish spring lines. Principal co-ordinates analysis (PCO) showed similar groupings to cluster analysis. Results from cluster analysis and PCO analysis showed very clearly that Chinese weak-winter, Swedish spring and winter accessions were distinguished from each other and Chinese weak-winter accessions in this study were genetically closer to Swedish winter accessions than to Swedish spring accessions. The Chinese weak-winter accessions had larger diversity than Swedish spring or winter accessions did. This study indicated that ISSR is a suitable and effective tool to evaluate genetic diversity among rapeseed germplasm.

Confirmation of Pearl Millet-Napiergrass Hybrids Using EST-Derived Simple Sequence Repeat (SSR) Markers

Prospects for deploying perennial grasses that are currently considered leading candidates for dedicated energy crops over large acreages are debatable because of several limitations, including vegetative propagation or small seed size, low biomass production during the first growing season, and incomplete assessments of crop invasiveness risk. Pearl Millet-Napiergrass hybrids (“PMN”;Pennisetum glaucum [L.] R. Br. × P. purpureum Schumach.), in contrast, are large-seeded, sterile feedstocks capable of high biomass production during establishment year. Novel methods are warranted for confirmation of PMN hybrids, as traditional morphological observations can be inconclusive and chromosome number determination using cytological methods is laborious and time consuming. Six putative PMN lines were produced in this study, and 10 progeny from each line were evaluated using morphological traits, seed fertility, flow cytometry, and expressed sequence tag-simple sequence repeat (EST-SSR) markers. All putative hybrid lines were sterile and failed to produce seed. The PMN hybrids could not be distinguished from either parent using flow cytometry due to highly similar nuclear genome DNA contents. A number of paternal napiergrass-specific EST-SSRs were identified for each PMN line, and four paternal-specific EST-SSRs conserved across all napiergrass accessions were selected to screen the putative PMN hybrids. These EST-SSRs confirmed that all F1 individuals analyzed were PMN hybrids. The use of paternal-specific markers therefore provides a valuable tool in the development of both “Seeded-yet-Sterile” biofuel PMN feedstocks and additional PMN cultivar-and parental species-specific markers.

Full-length transcriptome sequence and SSR marker development for genetic diversity research in yellowfin seabream Acanthopagrus latus

Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificial breeding and reproduction.We performed full-length transcriptome sequencing and assembly of the genome of yellowfin seabream.A total of 68086 unigenes were obtained,with an N50 of 3391 bp on average length of 2933 bp.A total number of 50593 expressed sequence tags linked to simple sequence repeats(EST-SSR)were identified,among them dinucleotide repeats(40.6%)and AC/GT motifs(38.5%)were the most frequent.Of the 190 EST-SSRs for which PCR primer pairs were designed,150 primer pairs successfully amplified target loci and 15 SSRs showed high polymorphism.The alleles per locus ranged 6-50 on average of 25.3.The expected and observed heterozygosity varied from 0.632 to 0.969 and from 0.519 to 0.953,respectively.The polymorphic index content(PIC)values of each locus ranged 0.587-0.966 on average of 0.851.Among six yellowfin seabream population samples preliminarily tested for genetic diversity and differentiation,the Fangchenggang(FCG)population in Guangxi Province had the highest mean observed heterozygosity(H_(o))value(0.786),whereas the Zhangzhou(ZZ)population in Fujian Province had the lowest(0.678).The pairwise fixation index(Fst)values indicated significant population differentiation among six yellowfin seabream populations.This study provided evidence for the usefulness of the transcriptomic resource information and EST-SSR markers for natural resource conservation,population genetics,and breeding studies of yellowfin seabream in South China.

油菜简单重复序列SSR(simple sequence repeat)研究进展

简单重复序列(simple sequence repeat, SSR)是重复单元少于6个核苷酸重复序列,广泛分布于动植物基因组中,呈孟德尔遗传,已被作为一种理想的共显性标记应用于动植物遗传研究中。本文重点介绍了SSR分类、特点,及近几年来油菜SSR标记的开发和SSR技术在油菜基因定位、品种鉴定中的应用,并对SSR标记在油菜中的应用进行了探讨。

Development of Starfruit Simple Sequence Repeat (SSR) Using Next Generation Sequencing

Starfruit (Averrhoa carambola L.) is an important fruit for Malaysian export and great attention has been made to improve starfruit fruit quality at Malaysian Agricultural Research and Development Institute (MARDI). The current study used next generation sequencing (NGS) technologies to develop starfruit simple sequence repeat (SSR) from 2 varieties namely B11 and B17 using Illumina HiSeq. The pre-processed reads were de novo assembled to generate approximately 75,000 and 74,000 scaffolds respectively. Total genome size for B11 and B17 were around 345 Mbp and 342 Mbp based on K-mer distribution analysis. In-silico microsatellite mining of each variety has identified more than 17,000 SSR in B11 and B17 respectively. Dinucleotides were the most abundant, accounting for more than 70% of all SSR and repeat motif GA (49%) was most common. A total of 239 SSR primer pairs were designed from contigs larger than 350 nucleotides and tested for amplification. The 30 polymorphic SSRs were used to DNA fingerprint of 12 starfruit hybrids. Polymorphism information content (PIC) ranged from 0.1411 to 0.6838, with an average of 0.3919. The Unweighted Pair-Group Method for Arithmetic Averages (UPGMA) dendrogram clustered 12 starfruit accessions into 2 groups.

RAPD and ISSR Markers of Fertility Restoring Gene for Aegilops kotschyi Cytoplasmic Male Sterility in Wheat

LK783 was found to be a good fertility restorer for K-type male sterility of wheat (Triticum aestivum L.). RAPD and ISSR (inter-simple sequence repeat polymorphism) markers were employed to map the major restoring gene in LK783. Maintainer and restorer DNA pools were established using the extreme sterile and fertile plants among KJ5418A//911289/LK783 F 1 population, respectively. Four hundred and eighteen RAPD primers and 33 ISSR primers were used for screening polymorphisms between the two pools, and amplification bands using a RAPD primer of OPK18 and an ISSR primer of UBC-845 were found polymorphic between the two pools. Linkage analysis showed that OPK18 450 and UBC-845 800 were linked to the restoring gene in LK783. The distance between the restoring gene and OPK18 450 was (15.07±6.28) cM (centiMorgan), with the distance between the restoring gene and UBC-845 800 being (8.20±4.85) cM. The marker of UBC-845 800 was located on chromosome 1BS by amplifying nulli-tetrasomics and 1B ditelosomics of Chinese Spring with the primer of UBC-845, indicating that the restoring gene in LK783 was located on 1BS. The breeding for new fertility restorer lines of K-type cytoplasmic male sterility of wheat would be facilitated by using the two markers.

Genetic Diversity of Tropical Hybrid Rice Germplasm Measured by Molecular Markers

Investigation of genetic diversity and relationships among breeding lines is of great importance to facilitate parent selection in hybrid rice breeding programs.In this study,we characterized 168 hybrid rice parents from International Rice Research Institute with 207 simple sequence repeat （SSR） and 353 single nucleotide polymorphism （SNP） markers.A total of 1 267 SSR and 706 SNP alleles were detected with the averages of 6.1 （SSR） and 2.0 （SNP） alleles per locus respectively across all lines.Based on the genetic distances estimated from the SSR and SNP markers separately and combined,the unrooted neighbor-joining cluster and STRUCTURE analyses consistently separated the 168 hybrid rice parents into two major groups：B-line and R-line,which is consistent with known parent pedigree information.The genetic distance matrices derived from the SSR and SNP genotyping were highly correlated （r=0.81,P 0.001）,indicating that both of the SSR and SNP markers have distinguishable power to detect polymorphism and are appropriate for genetic diversity analysis among tropical hybrid rice parents.A subset of 60 SSR markers were also chosen by the Core Hunter with 368 alleles,and the cluster analysis based on the total and subset of SSR markers highly corresponded at r=0.91 （P 0.001）,suggesting that fewer SSR markers can be used to classify and evaluate genetic diversity among parental lines.

Development and Characterization of Microsatellite Markers in Brassica rapa ssp.chinensis and Transferability Among Related Species

Simple sequence repeat （SSR） or microsatellite marker is a valuable tool for several purposes, such as mapping, fingerprinting, and breeding. In the present study, an intersimple sequence repeat （ISSR）-PCR technique was applied for developing SSR markers in non-heading Chinese cabbage （Brassica rapa）. A total of 190 SSRs were obtained. Among these, AG or CT （54.7%） was the most frequent repeat, followed by AC or GT （31.6%） of the microsatellites. The average number of the SSRs length array was 16 and 10 times, respectively. Based on the determined SSR sequences, 143 SSR primer pairs were designed to evaluate their transferabilities among the related species of Brassica. The number of alleles produced per marker averaged 2.91, and the polymorphism information content （PIC） value ranged from 0 to 0.863 with an average of 0.540. Monomorphism was observed in 16 primer pairs. The transferability percentage in CC genome was higher than in BB genome. More loci occurred in the BBCC genome. This result supported the hypothesis that BB genome was divergent from A and C genomes, and AA and CC genomes were relatively close. The polymorphic primers can be exploited for further evolution, fingerprinting, and variety identification.

Cluster Analysis on Japonica Rice(Oryza sativa L.) with Good Eating Quality Based on SSR Markers and Phenotypic Traits

Diversity of 60 conventional japonica rice accessions with good eating quality at home and abroad was analyzed using SSR molecular markers, agronomic traits and taste characteristics. A total of 290 alleles were detected in the 60 accessions at 72 SSR loci with the high similarity coefficients varying between 0.600 and 0.924. The loci on chromosome 5 showed the greatest value in average allele number. Additionally, most of the SSR loci could detect 3 to 4 alleles. An UPGMA dendrogram based on the cluster analysis of the genetic similarity coefficients showed that the grouping trend of part of the rice accessions was geographic-related and most of the rice accessions in Jiangsu Province, China were clustered together. Furthermore, many domestic accessions from south and north origins in China were close to the foreign japonica rice varieties, as proved by their pedigree origin from the foreign high-quality sources. For taste characteristics, part of the accessions with excellent taste were clearly clustered into one category though they came from different geographical regions, which indicates that taste characteristics of some varieties were mainly genetically determined. In addition, the agronomic traits of japonica rice with good taste might be closely related with their geographical origins, but the relationship between superior taste characteristics and agronomic traits should be further clarified.

Identification of necrophagous fly species from 12 different cities in China using ISSR and SCAR markers

Objective:To identify necrophagous fly speies from different regions in China using inter simple sequenc repeat(ISSR) and sequence-characterized amplified region(SCAR) melocular markers and to analyze their gene difference and genetic relationship.Methods:Five carrion fly species were collected from 12 cities and regions in China,including Musca domestica(M.domestica), Lucilia sericata(L.sericata),Chrysomya megacephala(C.megacephala),Helicophagella melanura(H.melanura),Boethcherisca peregrina,and they were studied using ISSR and SCAR markers.Results:Eight ISSR primers were used for amplification of 121 samples.679 clear and stable bands were identified,of which 516 bands were polymorphic.Several species-specific ISSR fragment were cloned and sequenced as an initial effort to derive the SCAR markers.Using M.domestica SCAR specific primers,SCAR-PCR amplification was performed for 8 M.domestca population sample DNA from different regions in China as well as L sericata,C.megacephala, H.melanura and Lucillia cupirina.The result showed only M.domestica produced specificalty 600 bp fragment,but L sericata,C.megacephala,H.melanura and Lucillia cupirina did not produce the same specific fragment.Clustering analysis showed clustering of most flies of M. domestica,C.megacephala and L sericata.M.domestica samples from different regions in China yielded different banding patterns.Conclusions:Application of ISSR-PCR and SCAR markers to identify necrophagous fly species from 12 cities and regions in China is first reported.ISSR-PCR and SCAR markers provide a quick reliable molecular marker technique for the identification of different species of necrophagous fly.

Genetic analysis of selected Sargassum fusiforme (Harvey) Setchell (Sargassaceae, Phaeophyta) strains with RAPD and ISSR markers

Since the 1980s,Sargassum fusiforme has been cultivated in Zhejiang,South China,and nowadays it becomes one of the important commercial seaweeds in China.With traditions of eating habits in the East Asian countries,this brown alga is used as food,because it contained functional oligo/polysaccharides and chemical components,and was regarded playing roles in antioxidant activities and regulating immunology.Through over 15 years’selection,breeding and cultivation,we obtained three strains with good traits and testified their characters during the production,which included the cultivars with high yield and other two good characters,either all the selected strains were applied in the Sargassum production.To avoid confusion during the selection and nursery,it was preferred to establish one fingerprint for distinguishing the Sargassum cultivars from different strains.Random amplified polymorphic DNA(RAPD)and inter-simple sequence repeat(ISSR)methods were adopted to analyze the genetic diversities of the selected S.fusiforme strains.With that,one fingerprint with RAPD markers was constructed,and one sequence characterized amplifi ed region(SCAR)marker to S.fusiforme was obtained.It is indicated that the applied fingerprint could be valid in S.fusiforme genetic and germplasm justification,and will be positive to molecular marker assistance in its selection and cultivation.

The loss of genetic diversity during captive breeding of the endangered sculpin, Trachidermus fasciatus, based on ISSR markers: implications for its conservation

Abstract Inter-simple sequence repeat （ISSR） markers were used to determine the genetic variation and genetic differentiation of cultured and wild populations of Trachidermus fasciatus, an endangered catadromous fish species in China. Six selected primers were used to amplify DNA samples from 85 individuals, and 353 loci were detected. Relatively low genetic diversity was detected in the cultured population （the percentage of polymorphic loci PPL=73.80%, Nei＇s gene diversity h--0.178 2, Shannon information index I=0.276 9）. However, the genetic diversity at the species level was relatively high （PPL-91.78%; h = 0.258 3, I= 0.398 6）. The UPGMA tree grouped together the genotypes almost according to their cultured and wild origin, showing distinct differences in genetic structure between wild and cultured populations. The pairwise F^t values confirmed significant genetic differentiation between wild and cultured samples. The cultivated population seems to be low in genetic diversity as a result of detrimental genetic effects in the captive population. The results suggest that ISSR markers are effective for rapid assessment of the degree of diversity of a population, thus giving important topical information relevant to preserving endangered species.

Analysis of SSR in Citrus Sequences from EMBL Database

Abundance of simple sequence repeat (SSR) in Citrus sequences from EMBL database was investigated by usingcomputer program MISA (MIcroSAtellite), which aimed to provide useful information for the development of SSR markers.Among 32 896 sequences of Citrus, 4 987 SSRs were found in 4 167 sequences and the average distance between SSRs wasapproximately 3.5 kb. Mononucleotide repeats (50.6%) were the most abundant repeats. And di-, tri-, tetra-, penta- andhexa-nucleotide repeats were 22.8, 25.2, 1, 0.08, and 0.36%, respectively. The most abundant motif was A/T followed indescending order by AG/CT, AC/GT, AT/TA. AAT/ATT, AAG/CTT, AGC/CGT, ACG/CTG and C/G. They comprised about90% of all microsatellites. Ten primer pairs were designed, and three of them produced clear visible bands among Citrusand its related genera.

Identification of seeds of Pinus species by microsatellite markers

The 276 pair-primers （nuclear and chloroplast microsatellite） developed from seven species of Pinaceae were selected and identified for cross-species transferability to ten Pinus species （P massoniana, P kesiya, P tabulaeformis P densiflora, P thunbergii, P caribaea, P taeda, P yunnanensis, P densata, P sylvestris）belonging to Sect. Pinus by BSA （bulked segregate analysis） method. The results showed that 23 of 276 （8.0%） markers were successful to have amplification product in ten species, and 5 of 23 （21.7%） were polymorphic cross species and lack of polymorphic within species. Eight of 10 Pinus species were identified by using single primer, two and more combination of primers, but there were still no effective SSR primers for identifying other 2 species （P. kesiya and P. densata）.

Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

The discovery of thermo-sensitive genic male sterility(TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F_2 populations derived from crosses between IR68301 S, an indica TGMS rice line, and IR14632(tropical japonica), Supanburi 91062(indica) and IR67966-188-2-2-1(tropical japonica), respectively.Approximately 1:3 ratio between sterile and normal pollen of F_2 plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat(SSR) and insertion-deletion(InDel) markers were used to identify markers linked to the tms gene. The linkage analysis based on the three populations indicated that the tms locus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F_2 population, the results showed that the tms locus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from the tms gene to these two flanking markers were 1.10 and 0.82 cM, respectively.InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to the tms gene using F_2 populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S.

Genetic diversity of Oryza rufipogon Griff. in Hainan Province analyzed by ISSR and SSR markers

Assessment of genetic diversity is an essential component in germplasm characterization and conservation.There are three wild rice species in Hainan Province,including Oryza rufipogon Griff.In order to detect the genetic diversity of different populations of Oryza rufipogon in Hainan,ISSR(inter-simple sequence repeat)and SSR(simple sequence repeat)markers were used to investigate 180 accessions from six localities in Hainan.Fourteen ISSR primers amplified 185 alleles with 171(92.43%)polymorphic,the number of alleles ranged from 8 to 17,with an average of 13.14 alleles per locus.Thirty-eight pairs of SSR primers used in this study amplified 213 alleles with 190(89.20%)polymorphic,the number of alleles ranged from 2 to 14,with an average of 5.66 alleles per locus.Both ISSR and SSR analyses revealed a high level of genetic diversity in the wild populations.The population with the highest genetic diversity is Wanning(WN),and the population with lowest genetic diversity is Wenchang(WC).The results of a UPGMA cluster using the NTSYS program showed that each population has a low degree of genetic differentiation.Furthermore,the Mantel test revealed that the genetic similarities detected by ISSR and SSR were significantly correlated(r=0.8634,t=93.67)when detecting genetic diversity at the species level.The two molecular marker systems were able to determine the genetic diversity among Oryza rufipogon,and the two groups of indexes obtained by using the two markers have a high level of consistency.

Genome-wide development of interspecific microsatellite markers for Saccharum officinarum and Saccharum spontaneum

Sugarcane has a large,complex,polyploid genome that has hindered the progress of genomic research and molecular marker-assisted selection.The user-friendly SSR markers have attracted considerable attention owing to their ideal genetic attributes.However,these markers were not characterized and developed at the genome-wide scale due to the previously lacking high-quality chromosome-level assembled sugarcane genomes.In this present study,744305and 361638 candidate SSRs were identified from the genomes of S.officinarum and S.spontaneum,respectively.We verified the reliability of the predicted SSRs by using 1200 interspecific SSR primer pairs to detect polymorphisms among 11 representative accessions of Saccharum,including S.spontaneum,S.officinarum,S.robustum,and modern sugarcane hybrid.The results showed that 660 SSR markers displayed interspecific polymorphisms among these accessions.Furthermore,100 SSRs were randomly selected to detect the genetic diversity for 39 representative Saccharum accessions.A total of 320 alleles were generated using 100 polymorphic primers,with each marker ranging from two to seven alleles.The genetic diversity analysis revealed that these accessions were distributed in four main groups,including group I(14 S.spontaneum accessions),group II(two S.officinarum accessions),group III(18 modern sugarcane hybrid accessions),and group IV(five S.robustum accessions).Experimental verification supported the reliability of the SSR markers based on genome-wide predictions.The development of a large number of SSR markers based on wet experiments is valuable for genetic studies,including genetic linkage maps,comparative genome analysis,genome-wide association studies,and marker-assisted selection in Saccharum.

Large scale identification of SSR marker in perilla by next generation sequencing

Perilla frutescens (L.) is an edible, medicinal crop, and most popular in East Asia. Its molecular breeding and research are hampered by the paucity of molecular markers. Simple sequence repeat (SSR) markers are ubiquitous and widely used in eukaryotic genomes. EST-SSRs identification of perilla was performed in 116,387 reads generated by Illumina paired-end sequencing technology. In total 25,449 unigenes containing SSR and 33,867 SSR loci were identified, and 19,400 primer pairs were designed. Polymorphism of SSR primers was conducted by searching for insertions and deletions (INDELs), and 1,567 unique SSRs were predicted. Totally, 200 SSR primer pairs were selected for polymorphic validation among 23 perilla accessions. Results showed that 175 primer pairs produced amplicons, and 30 pairs exhibited polymorphism. Polymorphic ratio was higher by using INDEL method than using conventional primers. Phylogenetic analysis showed the 2 distinct groups: P. frutescens var. frutescens and P. frutescens var. crispa. Wrinkled leaf trait and seed trait were distinct between these 2 groups. However, no clear leaf color or geographic relationship was detected. The large scale development and identification of SSR marker in this research laid a foundation for genetic analysis and marker assisted breeding of cultivated perilla.

Assessment of Genetic Diversities of Selected Laminaria （Laminariales, Phaeophyta） Gametophytes by Inter-Simple Sequence Repeat Analysis

Abstract: Inter-simple sequence repeat (ISSR) analysis was used to assess genetic diversity among 10 pairs of male and female Laminaria gametophytes. A total of 58 amplification loci was obtained from 10 selected ISSR primers, of which 34 revealed polymorphism among the gametophytes. Genetic distances were calculated with the Dice coefficient ranging from 0.006 to 0.223. A dendrogram based on the unweighted pair-group method arithmetic (UPGMA) average showed that most male and female gametophytes of the same species were clustered together and that 10 pairs of gametophytes were divided into four groups. This was generally consistent with the taxonomic categories. The main group consisted of six pairs of gametophytes, which were selected from Laminaria japonica Aresch. by intensive inbreeding through artificial hybridization. One specific marker was cloned, but was not converted successfully into a sequence characterized amplified region (SCAR) marker. Our results demonstrate the feasibility of applying ISSR markers to evaluate Laminaria germplasm diversities.