Study on Marker-assisted Breeding of Soybean Vitamin E

Hefeng 25 variety with low vitamin E content in Heilongjiang Province and Bayfield variety with high vitamin E content in Canada were crossed.A total of 144 F_(2:7) recombinant inbred lines (RILs) were used as materials.The genetic linkage mapping of soybean vitamin E was constructed.Soybean varieties were marker-assisted selected in the interval of refined quantitative trait locus (QTLs).QTLs were identified in α-,γ-,δ-and the total tocopherol contents of soybean seeds.Fine QTLs of soybean vitamin E content were identified in the interval between Sat_239 and Satt022 on N linkage group.It was valuable to narrow the interval by marker-assisted selection (MAS).There were seven major QTLs of vitamin E content in soybean.MAS related to vitamin E content in soybean was carried out in the intervals between Sat_239 and Satt022.Considering all the kinds of agronomic traits,six strains with high yield and good quality of vitamin E were chosen,numbered 4,54,104,114,122 and 135.     

QTL Identification of the Insensitive Response to Photoperiod and Temperature in Soybean by Association Mapping

The insensitive response to photoperiod and temperature is an important quantitative trait for soybean in wide adaptation breeding. The natural variation in response to photoperiod and temperature was detected using 275 accessions of soybean [Glycine max (L.) Merrill] from China. Genome-wide association mapping, based on population structure analysis, was carried out using 118 SSR markers by the TASSEL GLM (general linear model) program. Nine SSR markers (P<0.01) were associated with the value of the response to photoperiod and temperature (VRPT) caused by days to flowering (DF), among which, Satt308 (LG M), Satt150 (LG M) and Satt440 (LG I), were identified in both 2006 and 2007. Twelve SSR markers (P<0.01) were associated with VRPT caused by days to maturity (DM), among which three markers, Satt387 (LG N), Satt307 (LG C2) and AW310961 (LG J), were detected in both 2006 and 2007. In addition, a total of 20 elite alleles were screened out over 2006 and 2007 for being associated with an insensitive response to photoperiod and temperature (IRPT) caused by DF and a total of seven different elite alleles were screened out for being associated with IRPT caused by DM. Among these elite alleles, five alleles, Satt150-244, Satt308-164, Satt308-206, Satt440-176, and Satt440-206, were associated with IRPT caused by DF and were identified in both years, but only one allele, Satt307-170, was identified as being associated with an IRPT caused by DM. Based on these elite alleles, a set of typical accessions were screened out. The result about the genetic basis of IRPT is meaningful for soybean wide adaption breeding.     

Isolation and Structural Analysis of the Seed-Specific Promoter from Soybean

The promoter region (BCSP666) of β-conglycinin α-subunit gene from the genomic DNA of soybean Jilin 43 was isolated by PCR method. Sequencing analysis showed that the cloned fragment BCSP666 had the similar structure to the soybean seed-specific promoter β-conglycinin α'-subunit gene promoter and β-conglycinin β-subunit gene promoter, and it also contains many motifs that contribute to the seed-specific promoter activity. Based on this sequencing analysis, we deduced that promoter fragment BCSP666 had the seed-sepecific promoter activity. And then we constructed the seedspecific expression vector pBMI666 with the promoter fragment BCSP666 and △6-fatty acid desaturase gene from Mortierella isabellina. The △6-fatty acid desaturase is the rate-limiting enzyme of the desaturation of linoleic acid in the production of a human essential fatty acid, γ-linolenic acid(GLA). The production of γ-linolenic acid(GLA) was observed in soybean callus cells, which were transformed with this vector. This confirmed the activity of the activity fragment BCSP666.     

Genetic Analysis and Molecular Mapping of Two SMV-Resistance Traits in Soybean:Adult-Plant Resistance and Resistance to Seed Coat Mottling

Soybean mosaic virus(SMV) could lead to adult-plant system diseases,and cause mottling of soybean seeds.Genetic analysis and molecular mapping were conducted using an F2 population and derived F3 families from two crosses of Dongnong 3C624(susceptible) × Dongnong 8143(resistant) and Dongnong 3C628(susceptible) × Tie 6915(resistant).Simple sequence repeat(SSR) markers with bulked segregation analysis(BSA) were used to conduct genetic mapping of the resistance to SMV1 in the segregating populations.The results indicated that resistance to SMV1 in adultplants and the resistance to seed coat mottling in Dongnong 8143 and Tie 6915 was separately controlled by one single dominant gene.The two dominant genes were identified to be linked on the MLG F by Mendel's genetics and SSR genetic mapping.The order and distance of markers DPRSMV1 and DSRSMV1 were Sat_229-6.9 cM-DSRSMV1-4.6 cM-Sat_317-3.6 cM-DPRSMV1-5.2 cM-Satt335.The order and distance of markers TPRSMV1 and TSRSMV1 was Satt160-16.1 cM-TPRSMV1-7.3 cM-Satt516-2.0 cM-TSRSMV1-4.5 cM-Sat_133.This research provides the useful information for breeders to select the two types of SMV resistance simultaneously in soybean breeding through molecular marker-assisted selection(MAS).     

Genetics and Molecular Mapping of a High-Temperature Resistance Gene to Stripe Rust in Seeding-Stage in Winter Wheat Cultivar Lantian 1

Stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici (Pst), is a severe foliar disease of common wheat (Triticum aestivum L.) in the world. Resistance is the best approach to control the disease. The winter wheat cultivar Lantian 1 has high-temperature resistance to stripe rust. To determing the gene(s) for the stripe rust resistance, Lantian 1 was crossed with Mingxian 169 (M169). Seedlings of the parents, and F 1 , F 2 and F 2-3 progenies were tested with races CYR32 of Pst under controlled greenhouse conditions. Lantian 1 has a single partially dominant gene conferred resistance to race CYR32, designated as YrLT1. Simple sequence repeat (SSR) techniques were used to identify molecular markers linked to YrLT1. A linkage group of five SSR markers was constructed for YrLT1 using 166 F 2 plants. Based on the SSR marker consensus map and the position on wheat chromosome, the resistance gene was assigned on chromosome 2DL. Amplification of a set of nulli-tetrasomic Chinese Spring lines with SSR marker Xwmc797 confirmed that the resistance gene was located on the long arm of chromosome 2D. Because of its chromosomal location and the high-temperature resistance, this gene is different from previously described genes. The molecular map spanned 29.9 cM, and the genetic distance of two close markers Xbarc228 and Xcfd16 to resistance gene locus was 4.0 and 5.7 cM, respectively. The polymorphism rates of the flanking markers in 46 wheat lines were 2.1 and 2.1%, respectively; and the two markers in combination could distinguish the alleles at the resistance locus in 97.9% of tested genotypes. This new gene and flanking markers should be useful in developing wheat cultivars with high level and possible durable resistance to stripe rust.     

Inheritance of Late Maturity α-amylase （LMA） in a Recombinant Inbreed Lines of Chuanmai 42 Crossed with Chuannong 16

[Objective] To study the inheritance and possible molecular marker of α-LMA and their relations to falling number. [Method] Recombined inbreed lines (RILS) of Chuanmai 42 (LMA genotype) crossed with Chuannong 16 (no LMA) were employed to test the late maturity α-amylase (LMA) by EL ISA kits,the Falling Number (FN),and three QTL,SSR markers (Xgwm 577,Xwmc276,Xwmc273) of LMA gene. [Result] Results suggested that LMA was controlled by a single,and the three SSR markers seemed to be not efficient to test LMA gene in this RILS. LMA was an important factor on FN,meanwhile FN was also affected by other factors. [Conclusion] The study could provide references for the breed of quality wheat.     

Heredity Analysis and Gene Mapping of Bruchid Resistance of a Mungbean Cultivar V2709

Bruchid is a serious insect pest of mungbean [Vigna radiata （L.） Wilczek] and can inflict serious loss. A resistant variety from India, V2709, was crossed with a susceptible variety, Zhonglti 1, from the World Vegetable Center, Asian Vegetable Research and Development Center （AVRDC）. Segregation of the F2, BC1F1, and F3 populations showed that bruchid resistance of V2709 is controlled by a single dominant locus. To find molecular marker linked with the resistant locus, 63 randomly amplified polymorphic DNA markers and 113 sets of SSR/STS primers were used in a bulked segregant analysis. Two of the markers, OPC-06 and STSbr2, were found to be linked with the locus （named as Br2）. Further analysis suggested that the genetic distances between these two markers and Br2 were 11.0 and 5.8 cM, respectively.     

Identification of SSR Marker Linked to a Major Dwarfing Gene in Common Wheat

A segregating population with 410 F 2 individuals from the cross MERCIA(Rht-B1a) ×Dwarf 123 was made to identify a new major dwarfing gene carrying by novel wheat germplasm Dwarf 123.Combination of bulk segerant analysis method was used.A total of 145 SSR markers were tested for polymorphisms among parental lines and DNA bulks of F 2 population.Out of 145 primer pairs only three markers revealed corresponding polymorphism among parental lines and F 2 DNA bulks.The marker Barc20 was close to the dwarfing gene with a genetic distance of 1.8 cM,and markers Gwm513 and Gwm495 were linked to the gene with genetic distance of 6.7 and 13 cM,respectively.Linkage analysis mapped the dwarfing gene to the long arm of chromosome 4B with the order of Barc20-dwarfing gene-Gwm513-Gwm495.The Comparision between the new gene and the known Rht-B1 alleles showed that dwarfing gene Rht-Ai123 was different from the others.The identification of the new dwarfing gene and its linked markers will greatly facilitate its utilization in wheat high yield breeding for reducing plant height.     

Genetic Analysis and Molecular Mapping of Two SMV-Resistance Traits in Soybean： Adult-Plant Resistance and Resistance to Seed Coat Mottling

Soybean mosaic virus （SMV） could lead to adult-plant system diseases, and cause mottling of soybean seeds. Genetic analysis and molecular mapping were conducted using an F2 population and derived F3 families from two crosses of Dongnong 3C624 （susceptible）x Dongnong 8143 （resistant） and Dongnong 3C628 （susceptible）× Tie 6915 （resistant）. Simple sequence repeat （SSR） markers with bulked segregation analysis （BSA） were used to conduct genetic mapping of the resistance to SMV1 in the segregating populations. The results indicated that resistance to SMV1 in adultplants and the resistance to seed coat mottling in Dongnong 8143 and Tie 6915 was separately controlled by one single dominant gene. The two dominant genes were identified to be linked on the MLG F by Mendel＇s genetics and SSR genetic mapping. The order and distance of markers DPRSMV1 and DSRSMV1 were Sat 229-6.9 cM-DSRSMV1-4.6 cM-Sat_317-3.6 cM-DPRSMV1-5.2 cM-Satt335. The order and distance of markers TPRSMV1 and TSRSMV1 was Satt160-16.1 cM-TPRSMV1-7.3 cM-Satt516-2.0 cM-TSRSMV1-4.5 cM-Sat_133. This research provides the useful information for breeders to select the two types of SMV resistance simultaneously in soybean breeding through molecular marker-assisted selection （MAS）.     

Development of Soybean EST-SSR Markers and Their Use to Assess Genetic Diversity in the Subgenus Soja

Developing expressed sequence tag-derived SSR （EST-SSR） markers is imperative in genetic research. In this paper, we reported 37 EST-SSR markers which were developed from 286 unigenes obtained from soybean cDNA library. Among the 286 markers designed for the 4 accessions of Glycine max and 6 of its wild progenitor （G. soja） within the subgenus Soja, 209 markers amplified DNA fragments, taking 73.1% and 37 markers appeared to be polymorphic, which was 12.9% of the total. The 37 loci detected a total of 142 alleles, while the PIC values varied from 0.194 to 0.794. Both the number of alleles per locus and PIC value were significantly related to the SSR motif. Six EST-SSR loci may be fixed for different alleles between G. max and G. soja since they were particularly polymorphic among the 6 G. soja accessions. A neighbor-joining tree placed the G. max accessions together as a group within the G. soja, though the average genetic distance among G. soja accessions was much higher. These new EST-SSRs markers will be useful for genetic diversity analysis, genetic mapping construction and gene discovery in Soja subgenus.     

Analysis of SSR Fingerprints in Introduced Silkworm Germplasm Resources

Thirty-five SSR markers were used to construct 96 silkworm races fingerprint. All the SSR markers were polymorphic and unambiguously separated silkworm strains from each other. A total of 467 alleles were detected with a mean value of 13.34 alleles/locus （range 3-28）. The mean polymorphism index content （PIC） was 0.71 （range 0.299-0.919）. UPGMA cluster analysis of Nei＇s genetic distance grouped silkworm strains on the basis of their origin. The results indicated that SSR markers are efficient tools for fingerprinting cultivars and conducting genetic diversity studies in the silkworm.     

Genetic Analysis and Molecular Mapping of a Stripe Rust Resistance Gene YrH9014 in Wheat Line H9014-14-4-6-1

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most widespread and destructive wheat diseases in many wheat-growing regions of the world. The winter wheat translocation line H9014-14-4-6-1 has all stage resistance. To identify stripe rust resistance genes, the segregating populations were developed from the cross between H9014-14-4-6-1 and Mingxian 169 (a wheat cultivar susceptible to all Pst races identified in China). The seedlings of the parents and F 1 plants, F2 , F3 and BC 1 generations were tested with Pst races under controlled greenhouse conditions. Two genes for resistance to stripe rust were identified, one dominant gene conferred resistance to SUN11-4, temporarily designated YrH9014 and the other recessive gene conferred resistance to CYR33. The bulked segregant analysis and simple sequence repeat (SSR) markers were used to identify polymorphic markers associated with YrH9014. Seven polymorphic SSR markers were used to genotype the F2 population inoculated with SUN11-4. A linkage map was constructed according to the genotypes of seven SSR markers and resistance gene. The molecular map spanned 24.3 cM, and the genetic distance of the two closest markers Xbarc13 and Xbarc55 to gene locus was 1.4 and 3.6 cM, respectively. Based on the position of SSR marker, the resistance gene YrH9014 was located on chromosome arm 2BS. Amplification of a set of nulli-tetrasomic Chinese Spring lines with SSR marker Xbarc13 indicated that YrH9014 was located on chromosome 2B. Based on chromosomal location, the reaction patterns and pedigree analysis, YrH9014 should be a novel resistance gene to stripe rust. This new gene and flanking markers got from this study should be useful for marker-assisted selection (MAS) in breeding programs for stripe rust.     

Blast-Resistance Inheritance of Space-Induced Rice Lines and Their Genomic Polymorphism by Microsatellite Markers

To understand the resistance inheritance basis of space-induced rice lines to blast, and to probe mutants＇ genomic DNA polymorphism compared with ground control by microsatellite markers, three space-induced lines were crossed with a highly susceptible variety LTH, and their F1 and F2 populations were inoculated by two representative blast isolates with broad pathogenicity to analyze their resistance inheritance basis. Meanwhile three mutant lines and the ground control were analyzed by 225 rice SSR （simple sequence repeat） primer pairs selected throughout the 12 chromosomes of whole rice genome, to scan the mutagenesis in genome of the mutant lines. The results indicated the blast-resistant genes harbored in these mutant lines were dominant. It was demonstrated that the resistance of mutant H1 to isolate GD0193 and GD3286 was controlled by a single gene, respectively; while mutants H2 and H3 were controlled by two pairs of major genes against isolate GD3286 and H2 showed complicated genetic mechanism to isolate GD0193. H3＇s resistance to isolate GD0193 was verified to be controlled by a single gene. According to the results of SSR analysis, three mutant lines showed different mutant rates as compared with the ground control, and the mutant rates also varied. Resistance genes can be induced from rice by space mutation, and different genomic variations were detected in blast-resistant lines.     

Resistance identification of bivalent fungi-resistant genes transformed soybean to Phytophthora sojae

Soybean is one of the most important sources of edible oil and proteins in the world.However,it suffers from many kinds of fungal diseases which is a major limiting factor in soybean production.The fungal disease can be effectively controlled by breeding plant cultivars with genetic transformation.In this study,the resistance to Phytophthora sojae of five bivalent transgenic soybean lines was identified using the hypocotyls inoculation technique.The lines were the T,of the transgenic soybean which were transformed with kidney bean chitinase gene and barley ribosome inactivating protein gene,and were positive by Southern Blot analysis.The resistance difference was studied through comparing the death percentage of transgenic soybean with the control.The results showed that four lines were more resistant to P.sojae,whereas other one had no significant difference in comparison with the control.These transgenic soybean lines with enhanced resistance to P.sojae will be useful in soybean resistance breeding.     

Fine Mapping and Candidate Gene Analysis of Resistance Gene RSC3Q to Soybean mosaic virus in Qihuang 1

Soybean mosaic virus （SMV） disease is one of the most destructive viral diseases in soybean （Glycine max （L.） Merr.）. SMV strain SC3 is the major prevalent strain in huang-huai and Yangtze valleys, China. The soybean cultivar Qihuang 1 is of a rich resistance spectrum and has a wide range of application in breeding programs in China. In this study, F1, F2 and F2：3 from Qihuang 1×nannong 1138-2 were used to study inheritance and linkage mapping of the SC3 resistance gene in Qihuang 1. The secondary F2 population and near isogenic lines （nILs） derived from residual heterozygous lines （RhLs） of Qihuang 1×nannong 1138-2 were separatively used in the ifne mapping and candidate gene analysis of the resistance gene. Results indicated that a single dominant gene （designated RSC3Q） controls resistance, which was located on chromosome 13. Two genomic-simple sequence repeat （SSR） markers BARCSOYSSR_13_1114 and BARCSOYSSR_13_1136 were found lfanking the two sides of the RSC3Q. The interval between the two markers was 651 kb. Quantitative real-time PCR analysis of the candidate genes showed that ifve genes （Glyma13g25730, 25750, 25950, 25970 and 26000） were likely involved in soybean SMV resistance. These results would have utility in cloning of RSC3Q resistance candidate gene and marker-assisted selection （MaS） in resistance breeding to SMV.     

Development of a Highly Informative Microsatellite （SSR） Marker Framework for Rice （Oryza sativa L.） Genotyping

To select highly informative microsatellite markers （SSRs） and establish a useful genetic SSR framework for rice genotyping, 15 rice （Oryza sativa L.） cultivars including six indica varieties and nine japonica varieties were used to analyze the polymorphism information content （PIC） value of 489 SSR markers. A total of 1 296 alleles were detected by 405 polymorphic markers with an average of 3.2 per locus. The PIC value of each chromosome was ranged from 0.4039 （chromosome 2） to 0.5840 （chromosome 11）. Among the two rice subspecies, indica （0.3685-0.4952） gave a higher PIC value than japonica （0.1326-0.3164） and displayed a higher genetic diversity. Genetic diversity of indica was high on chromosome 12 （0.4952） and low on chromosome 8 （0.3685）, while that for japonica was high on chromosome 11 （0.3164） and low on chromosome 2 （0.1326）. A SSR framework including 141 highly informative markers for genotyping was selected from 199 SSR markers （PIC〉0.50）. Ninety-three SSR markers distributed on 12 chromosomes were found to be related to indica-japonica differentiation. Of these 93 pairs of SSR primers, 17 pairs were considered as core primers （all the japonica varieties have the same specific alleles, while the indica varieties have another specific alleles）, 48 pairs as the second classic primers （all the japonica or indica varieties have the same specific alleles, while the indica or japanica varieties have two or more other specific alleles ） and 28 pairs as the third classic primers （all the japonica and indica varieties have two or more alleles, but the specific alleles are different between japonica and indica）. Thirty-two SSR markers were selected to be highly informative and useful for genetic diversity analysis of japonica varieties. This work provides a lot of useful information of SSR markers for rice breeding programs, especially for genotyping, diversity analysis and genetic mapping.     

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.     

QTL Analysis of Major Agronomic Traits in Soybean

Soybean is a main crop, and most agronomic traits of soybean are quantitative; therefore, there is vely important studying and applying value to locating these traits. A F2：10 RIL population containing 154 lines, derived from the cross between Charleston as female and Dongnong 594 as male parent, were used in this experiment. A genetic linkage map was constructed with 164 SSR primers, which were screened with the two parents and amplified on the 154 lines. 12 agronomic traits different between the two parents were investigated, and QTLs of all the traits were analyzed using the software Windows QTL Cartographer V2.0. The agronomic traits included quality traits： protein content, oil content, and content of protein and oil; yield traits： pods per plant, seed weight per plant, and 100 seeds weight; and other agronomic traits： plant height, days to maturity, branches, nod number in main stem, average leaf length, and average leaf width. The results showed that 68 QTLs in total were found for the 12 agronomic traits. The number of QTLs per trait varied from 3 for the average leaf width to 11 for 100 seeds weight and plant height, and was 5.8 on average. Good accordance was seen in many QTLs between the results of this study and the results obtained by other similar studies; therefore, these QTLs may be valuable for molecular marker assistant selection in soybean. In this study, 68 major QTLs of 12 important traits of soybean were analyzed.     

QTL for grain yield and yield parameters in winter wheat(Triticum aestivum L.)

Field trials with a set of 108 doubled haploid lines(DHs) derived from a cross between the Chinese winter wheat cvs.CA9613 and H1488 were run at Beijing(China).Phenotypic data were recorded for major agronomic yield traits,i.e.grain weight per ear,grain number per ear and thousand grain weight(Tgw) in two field trials at Beijing.Based on the phenotypic data and a genetic map comprising 168 SSR markers,an analysis of quantitative trait loci(QTL) was carried out for yield and yield parameters using the composite interval mapping(CIM) approach.A total of 14 QTL were detected for these traits across two environments.Four of these QTL located on chromosomes 1A and 2B,respectively,exhibited pleiotropic effects.Loci showing pleiotropic effects will be very useful for understanding the homeologous relationships of QTL and designing an appropriate marker-assisted selection programme by multi-trait selection in order to accumulate("pyramide") favorable alleles at different loci.     

Tagging Salt Tolerant Gene Using PCR Markers in Soybean

The purpose of this study was to screen and identify PCR markers associated with salt tolerant gene in soybean( Glycine soja L. ) so that salt tolerance can be identified efficiently and accurately. Between these tolerant and sensitivity to salt and three crosses were tested in this experiment. By BSA method, two codominant PCR markers were identified through the salt tolerant (sensitive) cuitivars bulks and the salt tolerant (sensitive) individual bulks of a F2 population. There was a 600bp band in the sensitive individuals and a 700bp band or two 700bp/600bp bands in the tolerant individuals. The markers were closely linked with salt tolerant/sensitive alleles. Moreover the markers were tested in the other two F2 populations from “salt tolerant cultivar &#215; sensitive cuitivar“ and confirmed by 12 salt tolerance cultivars and 13 salt sensitive cultivars with different genetic background. It indicated that the markers (700bp and 600bp) could be applied in salt tolerant identification of the soybean germplasm resources, and markers-assisted selection in salt tolerant breeding of soybean. The markers, its obtained method and application were patented for invention in 1998.