Genetic linkage mapping and QTL identification for salinity tolerance in Indian mustard(Brassica juncea L.Czern and Coss.)using SSR markers

Soil salinity is one of the major environmental constraints that limits crop yield and nearly 7%of the total area worldwide is affected by salinity.Salinity-induced oxidative stress causes membrane damage during germination and seedling growth.Indian mustard is a major oilseed crop in India and its production and productivity are severely affected by salt stress.Breeding Brassica cultivars for salinity tolerance by conventional means is very difficult and time-consuming.Therefore,understanding the molecular components associated with salt tolerance is needed to facilitate breeding for salt tolerance in Brassica.In this investigation,quantitative trait loci(QTLs)associated with salt tolerance were identified using F_(2:3)mapping population developed from a cross between CS52(salinity tolerant)and RH30(salinity sensitive).Parents and F_(2:3)were evaluated under controlled and salinity stress conditions for 14 morpho-physiological traits for two consecutive generations(F2 and F_(2:3)),explaining proportion of the phenotypic variance under control condition.Simple sequence repeat(SSR)markers were used for mapping studies.A genetic linkage map based on 42 simple sequence repeats(SSRs)markers was constructed covering 2298.5 cM(Haldane)to identify the loci associated with salt tolerance in Brassica juncea.Forty-one SSRs showing polymorphism in the parents(CS52 and RH30)were mapped on 8 linkage groups(C1–C8).One marker(nga 129)did not map to any of the linkage group and was excluded from mapping.Linkage group 5(C5;317.9 cM)was longest and linkage group 1(C1,255.0 cM)was shortest.Further,we identified 15 QTLs controlling 8 traits using F_(2:3)population.These QTLs explained 12.44–60.63%of the phenotypic variation with a LOD score range of 3.62–5.97.Out of these QTLs,QMI4.1 related to membrane injury showed 51.28%phenotypic variance with a LOD score of 3.34.QTL QBYP8.1 related to biological yield per plant showed 60.63%phenotypic variance at a LOD score of 3.62.The highest LOD score of 5.97 was recorded for QTL related to seed yield per plant(QSYP4.1).Major QTLs were QTL for biological yield per plant(QBYP8.1),QTL for siliquae per plant(QSP4.1),QTL for primary branches(QPB4.1),QTLs for seed per siliqua(QSS4.1,QSS4.2),QTL for seed yield per plant(QSYP4.1),and QTL for membrane injury(QMI8.1)which showed more than 50%phenotypic variance.These QTLs identified in our study need to be confirmed in other populations as well so that these can be used in marker-assisted selection and breeding to enhance salt tolerance in Brassica juncea.

Selection of Submergence Tolerant Homozygous Line by STS Marker and Twice Submergence Stress

One sequence tagged site marker Subl-1 and twice submergence stress method were used in selection of submergence tolerant homozygous line from Sub-lBS, a submergence tolerant, bentazon sensitive and photoperiod-sensitive and/or thermo-sensitive genic male sterile line that developed by our laboratory. The results revealed that the original Sub-lBS was heterozygous in SublA-1 locus even though it was identical in almost all of agronomical traits and the segregation of SublA-1 was in accordance with Mendelian law based on chi-square test. And then the original Sub-IBS was divided into two groups： one was ofSublA-1 introgression and the other was not; and the two groups were tested by twice submergence stress method. After the first submergence stress that lasted for 12 d, the average plant heights were significant difference at the 1% level between the two groups. After recovery for 10 d, the second submergence stress sustained for 18 d was carried on; and the group with SublA-1 gene was found apparently tolerant than the other group in submergence tolerance.

Validation of EST-derived STS markers localized on Qfhs.ndsu-3BS for Fusarium head blight resistance in wheat using a‘Wangshuibai’derived population

A few EST-derived STS markers localized on Qfhs.ndsu-3BS, a major QTL for resistance to Fusarium head blight （FHB） in wheat, have been previously identified in the ＇Sumai 3＇/＇Stoa＇ population. In this study, we used a ＇Wangshuibai＇ （resistant）/＇Seri82＇ （susceptible） derived population, linkage group, QTL, and quantitative gene expression analysis to assess the genetic background dependence and stability of the EST-derived STS markers for use in marker aided selection to improve FHB resistance in wheat. Based on our results, a QTL in the map interval of Xsts3B-138_1-Xgwrn493 on chromosome 3BS was detected for FHB resistance, which accounted for up to 16% of the phenotypic variation. BLASTN analysis indicated that Xsts3B-138_1 sequence had significant similarity with the resistance gene analogue. Real-time quantitative PCR showed that the relative expression of Xsts3B-138_1 in ＇Wangshuibai＇ at 96 h after inoculation was 2.6 times higher than ＇Seri82＇. Our results underlined that EST-derived STS3B-138 markers could be predominantly used in marker aided selection to improve FHB resistance in wheat.

Identification of Introgressed Lines by the SSR Markers and Agronomic Traits

The wild cotton cultivars and species have abundant genetic polymorphisms,and they possess lots of excellent genes,such as drought resistance,insect resistance,fine and strong fiber,and so on.

Bulked Segregant Analysis to Detect QTL Related to Heat Tolerance in Rice(Oryza sativa L.) Using SSR Markers

The study was undertaken to assess the genetic effect of quantitative trait loci （QTLs） conferring heat tolerance at flowering stage in rice. A population consisting of 279 F2 individuals from the cross between 996, a heat tolerant cultivar and 4628, a heat-sensitive cultivar, was analyzed for their segregation pattern of the difference of seed set rate under optimal temperature condition and high temperature condition. The difference of seed set rate under optimal temperature condition and high temperature condition showed normal distribution, indicating the polygenic control over the trait. To identify main effect of QTL for heat tolerance, the parents were surveyed with 200 primer pairs of simple sequence repeats （SSR）. The parental survey revealed 30% polymorphism between parents. In order to detect the main QTL association with heat tolerance, a strategy of combining the DNA pooling from selected segregants and genotyping was adopted. The association of putative markers identified based on DNA pooling from selected segregants was established by single marker analysis （SMA）. The results of SMA revealed that SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The heat tolerance during flowering stage in rice was controlled by multiple gene. The SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The two genetic loci, especially for RM3735 on chromosome 4, can be used in marker-assistant-selected method in heat tolerance breeding in rice.

Genetic Analysis and Preliminary Mapping of a Highly Male-Sterile Gene in Foxtail Millet(Setaria italica L.Beauv.) Using SSR Markers

Breeding of male-sterile lines has become the mainstream for the heterosis utilization in foxtail millet,but the genetic basis of most male-sterile lines used for the hybrid is still an area to be elucidated.In this study,a highly male-sterile line Gao146A was investigated.Genetic analysis indicated that the highly male-sterile phenotype was controlled by a single recessive gene a single recessive gene.Using F 2 population derived from cross Gao146A/K103,one gene controlling the highly male- sterility,tentatively named as ms1,which linked to SSR marker b234 with genetic distance of 16.7 cM,was mapped on the chromosome VI.These results not only laid the foundation for fine mapping of this highly male-sterile gene,but also helped to accelerate the improvement of highly male-sterile lines by using molecular marker assisted breeding method.

Genetic Diversity of Maize Populations Developed by Two Kinds of Recurrent Selection Methods Investigated with SSR Markers

Two cycles of biparental mass selection （MS） and one cycle of half-sib-S3 family combining selection （HS-S3） for yield were carried out in 2 synthetic maize populations P4C0 and P5C0 synchronously. The genetic diversity of 8 maize populations, including both the basic populations and their developed populations, were evaluated by 30 SSR primers. On the 30 SSR loci, a total of 184 alleles had been detected in these populations. At each locus, the number of alleles varied from 2 to 14, with an average of 6.13. The number and ratio of polymorphic loci in both the basic populations were higher than those of their developed populations, respectively. There was nearly no difference after MS but decreased after HS-S3 in both the basic populations in the mean gene heterozygosity. The mean genetic distance changed slightly after MS but decreased in a bigger degree after HS-S3 in both the basic populations. Analyses on the distribution of genetic distances showed that the ranges of the genetic distance were wider after MS and most of the genetic distances in populations developed by HS-S3 were smaller than those in both the basic populations. The number of genotypes increased after MS but decreased after HS-S3 in both the basic populations. The genetic diversity of intra-population was much more than genetic diversity of inter-population in both the basic populations. All these indexes demonstrated that the genetic diversity of populations after MS was similar to their basic populations, and the genetic diversity was maintained during MS, whereas the genetic diversity of populations decreased after HS-S3. This result indicated that heterogeneity between some of the individuals in the developed populations increased after MS, whereas the populations become more homozygotic after HS-S3.

Transcriptome analysis of salt-responsive genes and SSR marker exploration in Carex rigescens using RNA-seq

Carex rigescens （Franch.） V. Krecz is a wild turfgrass perennial species in the Carex genus that is widely distributed in salinised areas of northern China. To investigate genome-wide salt-response gene networks in C. rigescens, transcriptome analysis using high-throughput RNA sequencing on C. rigescens exposed to a 0.4% salt treatment （Cr_Salt） was compared to a non-salt control （Cr_Ctrl）. In total, 57 742 546 and 47 063 488 clean reads were obtained from the Cr Ctrl and Cr Salt treatments, respectively. Additionally, 21 954 unigenes were found and annotated using multiple databases. Among these unigenes, 34 were found to respond to salt stress at a statistically significant level with 6 genes up-regulated and 28 downregulated. Specifically, genes encoding an EF-hand domain, ZFP and AP2 were responsive to salt stress, highlighting their roles in future research regarding salt tolerance in C. rigescens and other plants. According to our quantitative RT-PCR results, the expression pattern of all detected differentially expressed genes were consistent with the RNA-seq results. Furthermore, we identified 11 643 simple sequence repeats （SSRs） from the unigenes. A total of 144 amplified successfully in the C. rigescens cultivar LOping 1, and 69 of them reflected polymorphisms between the two genotypes tested. This is the first genome-wide transcriptome study of C. rigescens in both salt-responsive gene investigation and SSR marker exploration. Our results provide further insights into genome annotation, novel gene discovery, molecular breeding and comparative genomics in C. rigescens and related grass species.

Detection of Allelic Variation in Chinese Wheat (Triticum aestivum L.) Germplasm with Drought ToleranceUsing SSR Markers

Allelic variation in two domestic wheat landraces, Pingyaobaimai and Mazhamai, two cornerstone breeding materials and their derived cultivars with drought tolerance was detected by SSR (simple sequence repeat) markers. The clustering of 25 accessions showed that the similarity between Pingyaobaimai and Yandal817, the latter was developed from the former, was 0.71, the highest one of all accessions, but the similarities were very low between these two accessions and other accessions including their derived cultivars. A similar situation was revealed between Mazhamai and its derived cultivars. Pingyaobaimai and its three derived cultivars shared three alleles at loci Xgwm526, Xgwm538 and Xgwm126 on chromosome arms 2BL, 4BL and 5AL, respectively. There were six shared alleles in Mazhamai and its derived cultivars, in order of Xgwm157, Xgwm126, Xgwm212, Xgwm626, Xgwm471 and Xgwm44 on chromosome arms 2DL, 5AL, 5DL, 6BL, 7AS and 7DC, respectively. Only one shared allele was detected between the pedigrees of Pingyaobaimai and Mazhamai. The difference of shared alleles in two cornerstone breeding materials and their derived cultivars revealed the diversity in Chinese wheat germplasm with drought tolerance and the complication in genetic basis of drought tolerance in wheat.

Fingerprinting 146 Chinese chestnut(Castanea mollissima Blume)accessions and selecting a core collection using SSR markers

Chinese chestnut is an important nut tree around the world.Although the types of Chinese chestnut resources are abundant,resource utilization and protection of chestnut accessions are still very limited.Here,we fingerprinted and determined the genetic relationships and core collections of Chinese chestnuts using 18 fluorescently labeled SSR markers generated from 146 chestnut accessions.Our analyses showed that these markers from the tested accessions are highly polymorphic,with an average allele number(N_(a))and polymorphic information content(PIC)of 8.100 and 0.622 per locus,respectively.Using these strongly distinguishing markers,we successfully constructed unique fingerprints for 146 chestnut accessions and selected seven of the SSR markers as core markers to rapidly distinguish different accessions.Our exploration of the genetic relationships among the five cultivar groups indicated that Chinese chestnut accessions are divided into three regional type groups:group I(North China(NC)and Northwest China(NWC)cultivar groups),group II(middle and lower reaches of the Yangtze River(MLY)cultivar group)and group III(Southeast China(SEC)and Southwest China(SWC)cultivar groups).Finally,we selected 45 core collection members which represent the most genetic diversity of Chinese chestnut accessions.This study provides valuable information for identifying chestnut accessions and understanding the phylogenetic relationships among cultivar groups,which can serve as the basis for efficient breeding in the future.

Analysis of an Applied Core Collection of Adzuki Bean Germplasm by Using SSR Markers

Genetic diversity of 158 accessions of an applied core collection of adzuki bean （Vigna angularis） and 18 wild genotypes were assessed by using 85 microsatellite markers. With an average of 5.81 alleles per locus, 493 alleles were detected, and their distribution frequencies lower than 5% accounted for 73.02% of the total number. The distributions of alleles between the cultivated and the wild adzuki bean germplasm are different, with a higher allelic diversity in the wild germplasm than that of the cultivated ones. An obvious genetic differentiation was also observed between the wild and the cultivated adzuki beans, and SSR markers may be useful in study identification and classification of them. Among cultivated adzuki bean, the genetic similarity coefficient varied from 0.366 to 0.939. Genetic structure analysis can clearly separate the wild genotypes from the cultivated adzuki bean, and also can divide the cultivated ones into different populations, as these populations are closely agreeable with the ecological regions where they originally grow. The results of this study will be useful in arranging local breeding programs, especially in the aspect of parental combinations or identification of progenies. These SSR markers can also provide important information to explain the genetic relationship between the cultivated and wild adzuki beans, and to accelerate the wild gene resources in broadening the gene pool in breeding program.

Research in migration route of hatchery released Chinese shrimp(Fenneropenaeus chinensis) in the Bohai Bay using method of SSR marker

This study provides new insights for the hatchery released Chinese shrimp(Fenneropenaeus chinensis),including proportion,dynamic migration route,after they were released into nature for stock enhancement using a new strategy quite different than ever.Chinese shrimp were sampled at 22 survey stations during two investigation voyages acrossing 74 survey stations in the Bohai Sea from July 16 to August 9 in 2015.Among 289 sampled individuals during the second voyage,totally 155 shrimps were identified as hatchery shrimp released into the Laizhou Bay at mid-May in 2015 based on finger-print of eight SSR(simple sequence repeats)markers,and the proportion of hatchery released shrimp in recapture samples were from 41.30%–85.71%in each station with an average value 53.63%,which verified a previous view point that up to 90%of autumn season Chinese shrimp landing in the Bohai Sea were composed of hatchery released.Meanwhile,the dynamic migration route of hatchery released shrimp revealed that part of released shrimp migrated heading northwest along the west coast of the Bohai Sea up to the Bohai Bay but just remained at the Laizhou Bay until over-wintering migration at midOctober when they initiate over-wintering migration.Present unnatural spring season shrimp fishing model cut the throat of spawner shrimp chance to swim back to their respective spawning plants at each spring,it still no chance to clarify whether the hatchery released shrimp could replenish to the reproduce population and complete a whole life cycle as same as their natural relatives.

Construction of a Genetic Map and Mapping Quantitative Trait Loci for Salt Tolerance During the Vegetative Stage in Tomato by SSR Markers

Quantitative trait loci （QTLs） contributing to salt tolerance during the vegetative stage in tomato were investigated. A moderately salt-sensitive Lycopersicon esculentum Mill.‘XF 98-7’ was hybridized with a salt-tolerant Lycopersicon pimpinellifolium accession LA2184, and F2 and F3 populations were developed. The F2 population was used for SSR mapping and the F3 families were evaluated for salt tolerance in solution cultures containing 1% NaCl. A LOD score threshold of 2.0 was chosen to identify putative QTLs and to estimate their additive effect and phenotypic variation. Two genomic regions （LEtat003-SSR139, SSR119-SSR17） were detected on chromosome 4 beating significant QTLs for salt tolerance, respectively accounted for 6.03% and 8.01% of the phenotypic variation. The QTL in the marker interval of LEtat003-SSR139 showed significant negative effects, while the other QTL in the marker interval of SSR119-SSR17 showed significant positive effects. The identification of genomic locations with both positive and negative effects on this trait suggests the likelihood of recovering transgressive segregants in progeny derived from these parental lines. Results and its application in developing salt-tolerant tomatoes as molecular markers are discussed in this paper.

Evaluation of Genetic Diversity of Sichuan Common Wheat Landraces in China by SSR Markers

Genetic diversity of 62 Sichuan wheat landraces accessions of China was investigated by agronomic traits and SSR markers. The landrace population showed the characters of higher tiller capability and more kernels/spike, especially tiller no./plant of six accessions was over 40 and kernels/spike of three accessions was more than 70. A total of 547 alleles in 124 polymorphic loci were detected with an average of 4.76 alleles per locus by 114 SSR markers. Parameters analysis indicated that the genetic diversity ranked as genome A 〉 genome B 〉 genome D, and the homoeologous groups ranked as 5〉4〉3〉1〉2〉7〉6 based on genetic richness （Ri）. Furthermore, chromosomes 2A, 1B and 3D had more diversity than that of chromosomes 4A, 7A and 6B. The variation of SSR loci on chromosomes 1B, 2A, 2D, 3B, and 4B implied that, in the past, different selective pressures might have acted on different chromosome regions of these landraces. Our results suggested that Sichuan common wheat landraces is a useful genetic resource for genetic research and wheat improvement.

Detection of Distorted Segregation in Genotype of Pollen Calli Derived from Hybrid F_1 of Cultivated Rice (Oryza sativa L.) Using SSR Markers

S-a, S-b and S-c are three loci for F1 pollen sterility in cultivated rice （Oryza sativa L.）. Taichung 65 （T65） is all Sj/Sj at these three loci, while its F1 pollen sterile near-isogenic lines, TISL2 （S-b）, TISL4 （S-a） and TISL5 （S-c） is Sj/Sj according to their respective sterility locus. Using SSR molecular marker to detect the segregation of the allele Si and Sj in pollen calli population induced from different hybrid F1, which have different pollen sterility locus, showed that the segregation of allele Si and Sj was distorted. The distorted direction of pollen calli population in vitro was not the same as F2 population in vivo. The quantities of pollen callus carrying Sj were much more than that of carrying Siat S-a and S-c locus, the ratio of Si and Sj were 1：4.81 and 1：1.96 respectively. But the opposite tendency was observed at S-b locus, the ratio of Si and Sj being 1：0.35. At the same time, all these results were undisturbed by either culture medium or culture period.

Assessment of wheat variety distinctness using SSR markers

Assessment of variety distinctness is important for both the registration and the protection of particular variety. However, the current testing system, which assesses a range of morphological characters of each pair of varieties grown side-by-side, is time-consuming and is not suitable for the assessment of hundreds of samples. The objective of this study was to develop a procedure for the assessment of wheat variety distinctness using simple sequence repeat（SSR） markers. A comparison between the molecular and morphological profile of 797 varieties was made. On the basis of the comparison, pairs of varieties with a genetic similarity value（GSV） ≤90% were deemed to be distinct, accounting for ~85% of varieties assessed in wheat regional trials. For the remaining ~15% of varieties, GSVs between different varieties were 〉90%, among which ~35% were not distinct and the other ~65% were distinct. Therefore, if given a GSV〉90%, the pairs of varieties should be morphologically assessed in the field. To avoid any errors in the assessments, we proposed the elimination of contaminant plants from the sample before comparing the varietal genotypes, scoring of the genotype at each locus with a pair of allele numbers when constructing a molecular profile, and faithfully recording two alleles at a non-homozygous locus. To reduce the workload and cost, a three-grade markers comparison among varieties is suggested. In addition, 80 SSR markers and a technical procedure for assessment of wheat variety distinctness have been proposed. Based on the procedure, the distinctness assessment of ~85% of all wheat varieties is completed in our laboratory annually. Consequently, total field assessment has been reduced considerably.

Development of an STS Marker Linked to Powdery Mildew Resistance Genes PmLK906 and Pm4a by Gene Chip Hybridization

Wheat （Triticum aestivum L.） line Lankao 90（6） carries a recessive powdery mildew resistance gene temporarily named PmLK906 on chromosome 2AL. Near PmLK906 there is another known powdery mildew resistance gene locus Pm4. To track the two powdery mildew resistance genes in wheat breeding program by marker assisted selection （MAS）, a linked molecular marker was developed in this study. Wheat gene chip hybridization combined with bulked segregant analysis （BSA） was used to develop an sequence-tagged sites （STS） marker for PmLK906 and Pm4. A new 2 125 bp full-length cDNA clone （GenBank accession no. EU082094） similar to csAtPR5 ofAegilops tauschii was isolated from Lankao 90（6） 21-12, and temporarily named TaAetPR5. Specific products could be amplified from cultivars or lines possessing Pm4a, Pm4b and PmLK906 with primers p9-7pl and p9-7p2 derived from TaAetPR5. TaAetPR5 was linked to PmLK906 at a genetic distance of 7.62 cM, and cosegregated with Pm4a. The p9-7p1 and p9-7p2 could be used as an STS marker for these resistance genes in wheat breeding. Because this marker was cosegregated with Pm4a, it can be used in map-based cloning of the alleles at Pm4 locus also.

Construction of DNA Fingerprint Using SSR Marker for Hybrid Rice Cultivars Approved by Hunan Province

[Objective] This study aimed to construct DNA fingerprint for hybrid rice cultivars those have been approved by Hunan Province. [Method] The primers which produced polymorphic and bright DNA bands were selected to construct the DNA molecular fingerprint map for 77 major hybrid rice cultivars approved by Hunan Province. [ Result] A total of 48 SSR primers were selected. Every cultivar had its unique fingerprint map so that the obtained data could identify differ- ent hybrid rice cultivars. [ Conclusion] This study made great contributions to the perfection of hybrid rice germplasm identification.

Segregation Correlation of SSR Markers and Flower Traits in Aneuploid Tobacco

Few reports have been seen on the segregation of polyploids and on the relationship between the segregation of SSR loci and that of the morphological traits in polyploid progeny. This paper attempted to gain an insight into the segregation of the hexaploid tobacco progeny and to understand the correlation of the segregation of SSR loci with that of the flower morphological traits. The segregation was evaluated with both SSR markers and morphological traits of the flowers. Twenty pairs of SSR primers were screened and a total of 42 SSR loci were identified. Chi-square analysis showed that the segregation ratio of the SSR loci in the aneuploid progeny was between 1:1 and 2:1. Six morphological traits of flowers, including the lengths of the flower and the calyx, the widths of the calyx, the corolla tube and the corolla, and the coloring of the flower, from 180 progeny and 2 progenitor plants, were determined and Chi-square analyzed. All traits were in consistent also with a segregation ratio of 1:1 to 2:1 except the width of the calyx.

Association analysis of grain traits with SSR markers between Aegilops tauschii and hexaploid wheat(Triticum aestivum L.)

Seven important grain traits, including grain length（GL）, grain width（GW）, grain perimeter（GP）, grain area（GA）, grain length/width ratio（GLW）, roundness（GR）, and thousand-grain weight（TGW）, were analyzed using a set of 139 simple sequence repeat（SSR） markers in 130 hexaploid wheat varieties and 193 Aegilops tauschii accessions worldwide. In total, 1 612 alleles in Ae. tauschii and 1 360 alleles in hexaploid wheat（Triticum aestivum L.） were detected throughout the D genome. 197 marker-trait associations in Ae. tauschii were identified with 58 different SSR loci in 3 environments, and the average phenotypic variation value（R2） ranged from 0.68 to 15.12%. In contrast, 208 marker-trait associations were identified in wheat with 66 different SSR markers in 4 environments and the average phenotypic R2 ranged from 0.90 to 19.92%. Further analysis indicated that there are 6 common SSR loci present in both Ae. tauschii and hexaploid wheat, which are significantly associated with the 5 investigated grain traits（i.e., GA, GP, GR, GL, and TGW） and in total, 16 alleles derived from the 6 aforementioned SSR loci were shared by Ae. tauschii and hexaploid wheat. These preliminary data suggest the existence of common alleles may explain the evolutionary process and the selection between Ae. tauschii and hexaploid wheat. Furthermore, the genetic differentiation of grain shape and thousand-grain weight were observed in the evolutionary developmental process from Ae. tauschii to hexaploid wheat.