A Cleaved Amplified Polymorphic Sequence Marker to Detect Variation in Wx Locus Conditioning Translucent Endosperm in Rice

The translucent endosperm trait in a japonica rice variety ＇Kantou 194＇ is controlled by a Wx-mq gene which is allelic to Wx locus by genetic analysis and allelic test. The amylose content analysis showed that an intermediate amylose content between those of glutinous and non-glutinous rice existed in endosperm of homozygous Wx-mq genotype. The slight changes of amylose content in different varieties and F1 grains with an identical Wx-mq genotype might be influenced by dissimilar genetic background. To identify the Wx-mq genotype simply and rapidly, a cleaved amplified polymorphic sequence （CAPS） marker was designed. The result from the molecular detection indicated that it could be used for marker-assisted selection for low amylose content varieties in rice breeding.

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.

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.

Microsatellite Analysis of Expressed Sequence Tags and Development of EST-SSR Markers for Melampsora spp.

In this study,64 498 ESTs of Melampsoraspp.was assembled into 1 998 contigs,and 604SSR loci were detected on these contigs,with 736.6 bp containing one SSR on average.Among these SSRs,trinucleotide repeats were the most abundant repeats（44.70%）.As for the composition of microsatellites, AC and AT repeats were the richest motif in dinucleotide repeats,and AGT and AAG repeats were the most frequent motifs in trinucleotide repeats,whereas（AAAN） n and（AAAAN） n repeats were dominant in tetranucleotide and pentanucleotide repeats,respectively.All the dominant repeat motifs of different types of SSRs were rich in A and T.In EST sequences of Melampsoraspp.genome,microsatellites longer than 20 bp accounted for about 15.07%.It was noticeable that microsatellites were highly rich in the expressed sequences of Melampsoraspp.genome,which implied that SSRs played a significant role in triggering the gene mutation in Melampsoraspp.genome.A total of 455 SSR primers were designed according to the detected microsatellites using Primer 5.0 and Oligo 6. 0,and 30 primer pairs were randomly selected for amplification test.Among these primer pairs,27 primer pairs succeed in amplification,with a successful rate of 90%.Eight primer pairs generated polymorphic fingerprints in Melampsoraspp.collected from different poplar genotypes,accounting for 26.7% of the total primer pairs.The EST-SSR markers developed fromMelampsoraspp.EST sequences provided important marker resources for studying Melampsoraspp.from the aspects of pathogen identification and survey of genetic variation.

Genome-wide association with transcriptomics reveals a shade-tolerance gene network in soybean

Shade tolerance is essential for soybeans in inter/relay cropping systems.A genome-wide association study(GWAS)integrated with transcriptome sequencing was performed to identify genes and construct a genetic network governing the trait in a set of recombinant inbred lines derived from two soybean parents with contrasting shade tolerance.An improved GWAS procedure,restricted two-stage multi-locus genome-wide association study based on gene/allele sequence markers(GASM-RTM-GWAS),identified 140 genes and their alleles associated with shade-tolerance index(STI),146 with relative pith cell length(RCL),and nine with both.Annotation of these genes by biological categories allowed the construction of a protein–protein interaction network by 187 genes,of which half were differentially expressed under shading and non-shading conditions as well as at different growth stages.From the identified genes,three ones jointly identified for both traits by both GWAS and transcriptome and two genes with maximum links were chosen as beginners for entrance into the network.Altogether,both STI and RCL gene systems worked for shade-tolerance with genes interacted each other,this confirmed that shadetolerance is regulated by more than single group of interacted genes,involving multiple biological functions as a gene network.

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.

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.

Comparison of Cheng's Index-and SSR Marker-based Classification of Asian Cultivated Rice

A total of 100 cultivated rice accessions,with a clear isozyme-based classification,were analyzed based on Cheng＇s index and simple sequence repeat （SSR） marker.The results showed that the isozyme-based classification was in high accordance with that based on Cheng＇s index and SSR markers.Mantel-test revealed that the Euclidean distance of Cheng＇s index was significantly correlated with Nei＇s unbiased genetic distance of SSR markers （r=0.466,P ≤ 0.01）.According to the model-based group and cluster analysis,the Cheng＇s index-and SSR-based classification coincided with each other,with the goodness of fit of 82.1% and 84.7% in indica,97.4% and 95.1% in japonica,respectively,showing higher accordance than that within subspecies.Therefore,Cheng＇s index could be used to classify subspecies,while SSR marker could be more efficient to analyze the subgroups within subspecies.

Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

The DNAfragments about 1 600 bp were amplified using random amplified polymorphism DNA （RAPD） primer OPAl2 with the templates of mitochondrial DNA of Zhenshan 97A and Zhenshan 97B, and were sequenced. The nucleotide sequences and lengths of the fragments from Zhenshan 97A and Zhenshan 97B showed no difference. The precise length of the fragment was 1 588 bp. Sequence characterized amplification region （SCAR） primers were then developed to discriminate the cytoplasmic male sterile （CMS） lines and their maintainer lines. A specific 1 588 bp fragment could be amplified with SCAR primers, CHI19F2/CHI19R2 and CHI20F3/CHI23R3, in the mitochondrial DNA of Zhenshan 97A, but not Zhenshan 97B. Furthermore, the specific fragment could be also amplified from the total DNA from green leaf tissues of Zhenshan 97A with SCAR primers, but not Zhenshan 97B. With the corresponding primers, the specific fragment could also be amplified from the total DNA of green leaves of other two CMS lines with wild abortive type cytoplasm （CMS-WA）, namely Zhenpin A and Tianfeng A, but not in their maintainer lines. Moreover, using total DNA as template, each of the four pairs of SCAR primers could also be used to amplify the 1 588 bp fragment in CMS-ID （Indonesia paddy type） line 11-32A but not in 11-32B, and the specific fragment was amplified from the DNA of both F1 and F2 seedlings of Shanyou 63. The results of detecting the genetic purity of a man-made mixture of the seeds of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes. Taken together, these results indicated that the specific 1 588 bp-fragment amplified by CHI20F3/CHI23R3 was the unique amplification products of CMS mitochondrial DNA, and could be used to distinguish CMS-WA and CMS-ID lines from their corresponding maintainer lines at the seedling stage.

Genome-wide mining, characterization, and development of microsatellite markers in Marsupenaeus japonicus by genome survey sequencing

The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats(SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identified, among which dinucleotide repeats were the most frequent class(44.08%), followed by mononucleotides(29.67%), trinucleotides(18.96%), tetranucleotides(5.66%), hexanucleotides(1.07%), and pentanucleotides(0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci(90.0%) were successfully amplified with specific products and 24(80.0%) were polymorphic. For the amplified polymorphic loci, the alleles ranged from 5 to 17(with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had significant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identified SSRs significantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.

Optimization of SSR Reaction System for Identifying the Authenticity of Maize Hybrids

[Objective]The aim was to optimize SSR reaction system applied in the identification of authenticity of maize variety.[Method]The technical parameters of SSR including PCR reaction system,annealing temperature and electrophoresis time were optimized to identify 10 major maize varieties in Liaoning Province.[Result]The optimum PCR reaction system was：14.60 μl sterile ultrapure water,2.00 μl 10 × Buffer（Mg2＋）,1.20 μl dNTPs,0.20 μl Taq enzyme,0.50 μl each of the forward and reverse primers and1.00 μl DNA stock solution.Annealing temperature and electrophoresis time could greatly influence the results of PCR amplification.The optimal annealing temperature and electrophoresis time required for the ideal electrophoresis bands under the same conditions were different when different primers were used.[Conclusion]The system was feasible to be applied in rapid identification of authenticity of hybrid maize varieties.

ISSR Analysis on Genetic Diversity of the 34 Populations of Oryza meyeriana Distributing in Yunnan Province,China

The genetic diversity of the 34 populations of wild rice Oryza meyeriana Baill. distributed in Yunnan Province, China was analyzed using 13 inter-simple sequence repeat （ISSR） markers. A total of 168 bands were amplified, of which 135 polymorphic bands were discovered and the percentage of polymorphic bands （PPB） was 80.36%. A genetic diversity was revealed as Nei＇s gene diversity （H） = 0.2666 and Shannon information index （I） = 0.4028 at population level. The 34 populations were divided into different groups based on administrative regions, latitude and longitudes, river areas, altitudes of their origins, and their indexes such as Na （number of alleles）, Ne （effective number of alleles）, H, I and PPB were calculated. Richer genetic diversity was found in the wild rice populations distributed in Simao Prefecture than that in Lingcang Prefecture or Xishuangbanna Prefecture whereas the least genetic diversity was in Baoshan Prefecture or Dehong Prefecture. Rich genetic diversity was also discovered in the wild rice populations originated from higher than 710 m altitude around the middle and lower reaches of the Lancang River belonging to the Pacific Ocean drainage system. The 34 populations could be classified into two groups, one group covered the wild rice distributing in Simao Prefecture only while the other group covered ones in Lingcang, Xishuangbanna and Dehong Prefectures. The issue on how to effectively conserve the wild rice germplasm was discussed.

Genetic Analysis and Preliminary Mapping of Two Recessive Resistance Genes to Brown Planthopper, Nilaparvata lugens Stl in Rice

An F2 population derived from the cross of WB01, an introgression line resistant to brown planthopper (BPH) originated from Oryza rufipogon Griff. and a susceptible indica variety 9311, was developed for genetic analysis and gene mapping. The population with 303 F2:3 families was genotyped by 141 simple sequence repeat (SSR) markers and used for gene mapping. Two softwares, Mapmaker/Exp 3.0 and Windows QTL Cartographer V2.0 were applied to detect QTLs. Totally, two QTLs resistant to BPH, named temporarily as bph22(t) and bph23(t), were identified to locate on chromosomes 4 and 8, individually had LOD values of 2.92 and 3.15, and explained 11.3% and 14 .9% of the phenotypic variation, respectively.

Genetic Diversity of Rice Landraces from Lowland and Upland Accessions of China

Genetic diversity of rice landraces from lowland and upland accessions of China was investigated using 66 polymorphic simple sequence repeat （SSR） markers. The total number of alleles detected from all 324 tested accessions was 555 with an average allele number （Na） of 8.409 per locus, the average effective number of alleles （Ne） of 3.574 and the average Shannon＇s information index （I） of 1.378. The genetic diversity was higher for the indica landraces compared to the japonica landraces, and the upland landraces were more genetically diverse than the lowland landraces. The SSR markers, RM72, RM232, RM219, RM241, RM224 and RM3 showed the highest rates of polymorphism and these SSR markers were suitable to assess the genetic diversity of rice germplasm resources. A dendrogram of 324 accessions of lowland and upland landraces showed that all rice accessions were mainly subdivided into two groups, japonica and indica, with some being intermediate. The distribution of lowland and upland landraces among the japonica and indica rice groups was distinct, with obvious differentiation between the lowland and upland landraces in japonica rice, but no such clear distinction in indica rice.

Molecular Diversity and Association Analysis of Drought and Salt Tolerance in Gossypium hirsutum L. Germplasm

Association mapping is a useful tool for the detection of genes selected during plant domestication based on their linkage disequilibrium（LD）. This study was carried out to estimate genetic diversity, population structure and the extent of LD to develop an association framework in order to identify genetic variations associated with drought and salt tolerance traits. 106 microsatellite marker primer pairs were used in 323 Gossypium hirsutum germplasms which were grown in the drought shed and salt pond for evaluation. Polymorphism（PIC=0.53） was found, and three groups were detected（K=3） with the second likelihood ΔK using STRUCTURE software. LD decay rates were estimated to be 13-15 cM at r2 0.20. Significant associations between polymorphic markers and drought and salt tolerance traits were observed using the general linear model（GLM） and mixed linear model（MLM）（P 0.01）. The results also demonstrated that association mapping within the population structure as well as stratification existing in cotton germplasm resources could complement and enhance quantitative trait loci（QTLs） information for marker-assisted selection.

Characterization and Molecular Mapping of a Stripe Rust Resistance Gene in Synthetic Wheat CI110

Stripe rust,caused by Puccinia striiformis f.sp.tritici（Pst）,is one of the most destructive diseases of wheat（Triticum aestivum L.）.To diversify stripe rust-resistant resources for wheat breeding programs,a CIMMYT synthetic wheat line CI110 was identified to be resistant to 28 isolates of Pst,including 6 Chinese prevalent races CYR28-CYR33.Genetic analysis indicated that a single dominant gene was responsible for the stripe rust resistance in CI110,temporarily designated YrC110.A molecular map,harboring YrC110 and 9 linked SSR markers,was constructed through simple sequence repeat（SSR）,and bulked segregant analysis.These linked markers and YrC110 were assigned on the short arm of chromosome 1B using the Chinese Spring nullisomic-tetrasomic and ditelosomic stocks.Gene postulation based on seedling reaction patterns to 30 Pst isolates suggested that the resistance gene YrC110 seemed different from the other known resistance genes tested,such as Yr9,Yr10,Yr15,Yr24,and Yr26/YrCH42.Four SSR markers Xbarc187150,Xgwm18227,Xgwm11223,and Xbarc240292 distinguished YrC110 from Yr10,Yr15,Yr24,and Yr26/YrCH42,and could be used as diagnostic ones for YrC110 in wheat resistant breeding programs against stripe rust.

Assessment of genetic diversity by simple sequence repeat markers among forty elite varieties in the germplasm for malting barley breeding

The genetic diversity and relationship among 40 elite barley varieties were analyzed based on simple sequence repeat （SSR） genotyping data. The amplified fragments from SSR primers were highly polymorphic in the badey accessions investigated. A total of 85 alleles were detected at 35 SSR loci, and allelic variations existed at 29 SSR loci. The allele number per locus ranged from 1 to 5 with an average of 2.4 alleles per locus detected from the 40 badey accessions. A cluster analysis based on the genetic similarity coefficients was conducted and the 40 varieties were classified into two groups. Seven malting barley varieties from China fell into the same subgroup. It was found that the genetic diversity within the Chinese malting barley varieties was narrower than that in other barley germplasm sources, suggesting the importance and feasibility of introducing elite genotypes from different origins for malting barley breeding in China.

Molecular mapping of a stripe rust resistance gene in Chinese wheat cultivar Mianmai 41

Stripe rust, caused by Puccinia striiformis f. sp. tritici（Pst）, is one of the most damaging diseases of wheat. Chinese wheat cultivar Mianmai 41 showed high resistance against most of the prevailing Pst races in China. Genetic analysis of the F1, F2 and F2：3 populations from a cross between Mianmai 41 and a susceptible line Mingxian 169 indicated that resistance to Pst race CYR32 was conferred by a single dominant gene, temporarily designated as Yr MY41. Molecular marker analysis placed the gene on chromosome 1B near the centromere. Six co-dominant genomic SSR markers Xwmc329, Xwmc406, Xgwm18, Xgwm131, Xgwm413, and Xbarc312, and one STS marker Xwe173 linked with the resistance gene. The two closest flanking SSR markers were Xgwm18 and Xwmc406, with genetic distances of 2.0 and 4.9 c M, respectively. A seedling test with 29 Pst isolates indicated the reaction patterns of Mianmai 41 were different from those of lines carrying Yr3, Yr9, Yr10, Yr15, Yr26, and Yr CH42 on chromosome 1B. Allelic tests indicated that Yr MY41 is likely a new allele at Yr26 locus.

Conidia of one Fusarium solani isolate from a soybean-production field enable to be virulent to soybean and make soybean seedlings wilted

Fusarium is usually thought to cause soybean root rot, which results in a large quantity of annual yield loss in soybean production, by its secretions including Fusarium toxins and cell wall degrading enzymes, but not by the conidia themselves that do not underlie any virulence so far. Here we report that the conidia of one Fusarium solani isolate are able to be virulent to soybean and make soybean seedlings wilted alone. We isolated them from the wilted plants in a soybean-production field and molecularly identified 17 Fusarium isolates through phylogenetic analysis. Of them, except for one isolate that showed diversity of virulence to different soybeans （virulent to one soybean whereas avirulent to another soybean）, the others were all virulent to the two tested soybeans： both conidia cultures and secretions could make soybean seedlings wilted at 5 days post infection, and their virulence had dosage effects that only conidia cultures of at least 5×10^6 conidia mL-1 could show virulence to soybean; however, the sole conidia of the F. solani isolate #4 also exhibited virulence to soybean and could make soybean seedlings wilted. Finally, we developed the specific cleaved amplified polymorphic sequences （CAPS） markers to easily differentiate Fusarium isolates. The isolate #4 in this work will likely be used to investigate the new mechanism of virulence of Fusarium to soybean.