Rice molecular markers and genetic mapping:Current status and prospects

Dramatic changes in climatic conditions that supplement the biotic and abiotic stresses pose severe threat to the sustainable rice production and have made it a difficult task for rice molecular breeders to enhance production and productivity under these stress factors. The main focus of rice molecular breeders is to understand the fundamentals of molecular pathways involved in complex agronomic traits to increase the yield. The availability of complete rice genome sequence and recent improvements in rice genomics research has made it possible to detect and map accurately a large number of genes by using linkage to DNA markers. Linkage mapping is an effective approach to identify the genetic markers which are co-segregating with target traits within the family. The ideas of genetic diversity, quantitative trait locus（QTL） mapping, and marker-assisted selection（MAS） are evolving into more efficient concepts of linkage disequilibrium（LD） also called association mapping and genomic selection（GS）, respectively. The use of cost-effective DNA markers derived from the fine mapped position of the genes for important agronomic traits will provide opportunities for breeders to develop high-yielding, stress-resistant, and better quality rice cultivars. Here we focus on the progress of molecular marker technologies, their application in genetic mapping and evolution of association mapping techniques in rice.

Advances and prospects of genetic mapping of Verticillium wilt resistance in cotton

Verticillium wilt is one of the most important diseases affecting cotton production in China.The fungus,Verticillium dahliae,has a wide host range and a high degree of genetic variability.No resistance resources have been found in the available planting resources,thus presenting difficulties and challenges for our study.The long-term production practice shows that selection of disease-resistant varieties is the most economical and effective measure to control Verticillium wilt of cotton to reduce the yield loss and quality decline of cotton.In this paper,we summarized the genetic mapping population,the analysis method of genetic localization,the discovery,mining and cloning of disease-resistant quantitative trait loci/markers,and the analysis of their genetic functions,so as to provide information for the molecular breeding approach of disease-resistant cotton.

High-density genetic mapping identified a major locus for environmental sex expression in pumpkin(Cucurbita moschata Duch.)

Long-day length and high temperature inhibit sex expression in pumpkin(Cucurbita moschata Duch.),and therefore directly impact the production potential.In this study,female flowering patterns in photoperiod-insensitive(PPIS)and photoperiod-sensitive(PPS)germplasms differed significantly in a moderately long day and high temperature environment.However,both germplasms exhibited a similar response in short day with either low temperature or high temperature environment.Photoperiod sensitivity led to this difference in sex expression between the germplasms.For the traits of 1st female flowering node(FFFN)and number of female flowers(NFF),high-density linkage map construction and quantitative trait locus(QTL)mapping were performed using SLAF-seq technology and 162 F_(2) individuals generated from PPIS and PPS.In total,4655 SLAFs were selected and mapped on 20 linkage groups(LGs).The total map length was 2502.01 cM with an average interval distance of 0.75 cM.Major QTLs for both FFFN and NFF were detected on LG6 with intervals of 7.89 and 17.67 cM and PVE values of 30.5%and 22.9%,respectively.Further analyses of the major locus for FFFN revealed 73 protein-coding genes.Among them,4 were related to sex expression,photoperiod flowering,and hormone response.An InDel(insertion and deletion)marker partially correlated with FFFN of the F_(2) population was also developed.Our study identified the QTL for the sex expression response to environmental factors using the high-density linkage map.The identified candidate genes and markers will provide useful information about the molecular interaction between the environment and sex expression and for marker-assisted selection of pumpkin environment-insensitive resources.

QTL mapping for berry shape based on a high-density genetic map constructed by whole-genome resequencing in grape

Grape berry shape is an important agricultural trait.Clarifying its genetic basis is significant for cultivating grape varieties that meet market demands.However,the current study by forward genetics has not achieved in-depth results.Here,a high-density map was constructed to identify quantitative trait loci(QTLs)for berry shape.A total of 358709 polymorphic SNPs were obtained using whole-genome resequencing(WGS)based on 208 F2 individuals derived from round grape‘E42-6’and oblong grape‘Rizamat’.The 1635.65 cM high-density map was divided into 19 linkage groups with an average distance of 0.37 cM.Using this map,three significant QTLs for fruit shape index(ShI:ratio of berry length to berry width)identified over three years were mapped onto LG4 and LG5,including one stable QTL on Chr5 with the genomic region of 0.47–1.94 Mb.Combining with gene annotation and expression patterns based on RNA-seq data from two contrasting F2 individuals with round and oblong berry(their average ShI was 1.89 and 1.10,respectively)at four developmental stages,four candidate genes were selected from the above QTLs.They were mainly involved in DNA replication,cell wall modification,and phytohormone biosynthesis.Further analysis of RNA-seq data revealed that several important phytohormone synthesis and metabolic pathways were enriched based on differentially expressed genes(DEGs),which was consistent with the results of QTL mapping for genes related to plant hormone biosynthesis in the F2 population.Furthermore,a comparison of plant hormone content showed that there were significant differences in IAA and tZ content between the two contrasting F2 individuals at different developmental stages.Our findings provide molecular insights into the genetic variation in grape berry shape.Stable QTLs and their tightly linked markers offer the possibility of marker-assisted selection to accelerate berry shape breeding.

Construction of SNP genetic maps based on targeted next-generation sequencing and QTL mapping of vital agronomic traits in faba bean(Vicia faba L.)

Owing to the limitation of a large genome size(~13 Gb),the genetic and gene mapping studies on faba bean(Vicia faba L.)are lagging far behind those for other legumes.In this study,we selected three purified faba bean lines(Yundou 8137,H0003712,and H000572)as parents and constructed two F2 populations.These two F2 populations,namely 167 F2 plants in Pop1(Yundou 8137×H0003712)and 204 F2 plants in Pop2(H000572×Yundou 8137),were genotyped using a targeted next-generation sequencing(TNGS)genotyping platform,and two high-density single nucleotide polymorphisms(SNP)genetic linkage maps of faba bean were constructed.The map constructed from Pop1 contained 5103 SNPs with a length of 1333.31 cM and an average marker density of 0.26 cM.The map constructed from Pop2 contained 1904 SNPs with a greater length of 1610.61 cM.In these two F2 populations,QTL mapping identified 98 QTLs for 14 agronomic traits related to the flowers,pods,plant types and grains.The two maps were then merged into an integrated genetic linkage map containing 6895 SNPs,with a length of 3324.48 cM.These results not only lay the foundation for fine mapping and map-based cloning of related genes,but can also accelerate the molecular marker-assisted breeding of faba bean.

Rapid Constructing a Genetic Linkage Map by AFLP Technique and Mapping a New Gene tms5

In this study, we reported the repaid construction of a molecular marker linkage map of rice (Oryza sativa L.). An F-2 population from the cross between Annong S-1 and Nanjing 11 was used to construct a genetic linkage map of rice. Total of 142 newly screened AFLP markers and 30 anchor markers (25 SSR markers and 5 RFLP markers) were mapped on the 12 chromosomes covering 1537.4 cM of rice genome. The average interval between these markers was 9.0 cM. The total work which usually was finished in more than one year was finished within only 3 months by one person. This is the first plant AFLP map developed in China. A new thermosensitive genic male sterile gene in rice, tms5, was Egged and mapped onto chromosome 2 during the development of the linkage map.

Multiple phenotypes in genome-wide genetic mapping studies

For many psychiatric and other traits,diagnoses are based on a number of different criteria or phenotypes.Rather than carrying out genetic analyses on the final diagnosis,it has been suggested that relevant phenotypes should be analyzed directly.We provide an overview of statistical methods for the joint analysis of multiple phenotypes in case-control association studies.

Identification and fine mapping of PmNJ3946 for powdery mildew resistance in einkorn wheat

Powdery mildew caused by Blumeria graminis f.sp.tritici(Bgt)is a destructive wheat disease.Although it can be easily overcome by deployment of resistance genes,the resistance is often quickly compromised by pathogen virulence.Thus,exploration and characterization of new resistance genes is always ongoing.Line NJ3946 derived from a cross of einkorn wheat accessions TA2032 and M389 showed resistance to powdery mildew.Inheritance analysis of an F2 population derived from a cross of NJ3946 and M389 suggested that the resistance was conferred by a dominant allele.With polymorphic markers identified through bulked segregant analysis(BSA),this gene was mapped to a novel locus on chromosome 3A,and was designated as PmNJ3946.Bulked segregant RNA-seq analysis(BSR-seq)was conducted to obtain more closely linked markers,which allowed delimitation of the PMNJ3946 locus to a 0.9 cM interval covering a physical distance of less than 1 Mb.PMNJ3946 was flanked by Xwgrc5153 and SNP-derived marker CHS21_3A008915069,and co-segregated with SNP-derived markers CHS21_3A008939814 and CHS21_3A008943175.The PmNJ3946 discovery expands the diversity of powdery mildew resistance genes and is useful for wheat breeding.

Genetic Mapping of Laminaria japonica and L. Iongissima Using Amplified Fragment Length Polymorphism Markers in a ＂Two-Way Pseudo- Testcross＂ Strategy

With a ＂two-way pseudo-testcross＂ mapping strategy, we applied the amplified fragment length polymorphism （AFLP） markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family （Laminaria iongissima Aresch. × L. Japonica Miyabe） with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1：1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3：1 Mendelian segregation ratio. Among the loci with 1：1 segregating ratios, 79 loci were ordered in 14 linkage groups （648.6 cM） of the paternal map, and 72 loci were ordered in 14 linkage groups （601.9 cM） of the maternal map. The average density of loci was approximately 1 per 8 cM. To Investigate the homologies between two parental maps, we used 58 loci segregated 3：1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

QTL mapping of general combining abilities of four traits in maize using a high-density genetic map

General combining abilities （GCAs） are very important in utilization of heterosis in maize breeding. However, its genetic basis is unclear. In the present study, a set of 118 doubled haploid （DH） lines were induced from F1 generations produced from the cross between the inbred line Zheng 58 and the inbred line W499 belonging to the Reid subgroup. Using the MaizeSNP50 BeadChip, a high-density genetic map was constructed based on the DH population which included 1 147 bin markers with an average interval length of 2.00 cM. Meanwhile, the DH population was crossed with three testers including W16-5, HD568, and W556, which belong to the Sipingtou subgroup. The GCAs of the ear height （EH）, the kernel moisture content （KMC）, the kernel ratio （KR）, and the yield per plant （YPP） were estimated using these hybrids in three environments. Combining the high-density genetic map and the GCAs, a total of 14 QTLs were detected for the GCAs of the four traits. Especially, one pleiotropic QTL was identified on chromosome 1 between the SNP SYN16067 and the SNP PZE-101169244 which was simultaneously associated with the GCAs of the EH, the KR, and the YPP. These QTLs pave the way for further dissecting the genetic architecture underlying GCAs of the traits, and they may be used to enhance GCAs of inbred lines under the fixed heterotic pattern ReidxSipingtou in China through a marker-assisted selection approach.

Construction of high-density SNP genetic maps and QTL mapping for dwarf-related traits in Litchi chinensis Sonn

Litchi chinensis Sonn is widely cultivated in subtropical regions and has an important economic value.A high-density genetic map is a valuable tool for mapping quantitative trait loci(QTL)and marker-assisted breeding programs.In this study,a single nucleotide polymorphism(SNP)-based high-density linkage map was constructed by a genotyping-by-sequencing(GBS)protocol using an F1 population of 178 progenies between two commercial litchi cultivars,‘Ziniangxi’(dwarf)and‘Feizixiao’(vigorous).The genetic map consisted of 3027 SNP markers with a total length of 1711.97 cM in 15 linkage groups(LGs)and an average marker distance of 0.57 cM.Based on this high-density linkage map and three years of phenotyping,a total of 37 QTLs were detected for eight dwarf-related traits,including length of new branch(LNB),diameter of new branch(DNB),length of common petiole(LCP),diameter of common petiole(DCP),length of internode(LI),length of single leaf(LSL),width of single leaf(WSL),and plant height(PH).These QTLs could explain 8.0 to 14.7%(mean=9.7%)of the phenotypic variation.Among them,several QTL clusters were observed,particularly on LG04 and LG11,which might show enrichment for genes regulating the dwarf-related traits in litchi.There were 126 candidate genes identified within the QTL regions,55 of which are differentially expressed genes by RNA-seq analysis between‘Ziniangxi’and‘Feizixiao’.These DEGs were found to participate in the regulation of cell development,material transportation,signal transduction,and plant morphogenesis,so they might play important roles in regulating plant dwarf-related traits.The high-density genetic map and QTLs identification related to dwarf traits can provide a valuable genetic resource and a basis for marker-assisted selection and genomic studies of litchi.

QTL Mapping for Fiber Quality Traits Based on a Dense Genetic Linkage Map with SSR,TRAP,SRAP and AFLP Markers in Cultivated Tetraploid Cotton

Cotton is one of the most important economic crops in the world,and it provides natural fiber for the textile industry.With the advancement of the textile technology and increased consumption demands on cotton fiber,both cotton yield and quality should be enhanced.However,cotton yield

Genetic analysis and QTL mapping of growth period traits and plant height traits in soybean recombinant inbred lines from Dongnong 47 × PI 317334-B

High and stable yield is the main goal of soybean genetic improvement.In this study,association analysis was used to detect the quantitative trait loci(QTL)for the plant height,and soybean growth period using 182 SSR markers in the RIL population of 136 F_(4:8) lines,which developed from a cross between photoperiod-insensitive cultivar‘Dongnong 47’and photoperiod-sensitive variety PI317334–B.The results showed that 33 QTLs related to soybean growth period and plant height traits were detected by compound interval mapping,and were located on 12 linkage groups including N,C1,C2,J,D1a,B2,E,G,A2,O,L,I,with the contribution rate of 7.85–33.84%.These QTL loci and linkage markers related to soybean photoperiod sensitivity,would be helpful to identify key genes that control soybean photoperiod sensitivity,and provide an important basis for the breeding of new photoperiod-insensitive soybean varieties based on molecular design breeding.

Breeding design in wheat by combining the QTL information in a GWAS panel with a general genetic map and computer simulation

A large amount of genome-wide association study(GWAS)panels together with quantitative-trait locus(QTL)information associated with breeding-targeted traits have been described in wheat(Triticum aestivum L.).However,the application of mapping results from a GWAS panel to conventional wheat breeding remains a challenge.In this study,we first report a general genetic map which was constructed from 44 published linkage maps.It permits the estimation of genetic distances between any two genetic loci with physical map positions,thereby unifying the linkage relationships between QTL,genes,and genomic markers from multiple genetic populations.Second,we describe QTL mapping in a wheat GWAS panel of 688 accessions,identifying 77 QTL associated with 12 yield and grain-quality traits.Because these QTL have known physical map positions,they could be mapped onto the general map.Finally,we present a design approach to wheat breeding by using known QTL information and computer simulation.Potential crosses between parents in the GWAS panel may be evaluated by the relative frequency of the target genotype,trait correlations in simulated progeny populations,and genetic gain of selected progenies.It is possible to simultaneously improve yield and grain quality by suitable parental selection,progeny population size,and progeny selection scheme.Applying the design approach will allow identifying the most promising crosses and selection schemes in advance of the field experiment,increasing predictability and efficiency in wheat breeding.

Quantitative Trait Loci(QTL)Mapping and Marker Analysis of Fatty Acids in Peanut

Peanut,with high oil content,has been a major oil and food crop globally.The compositions of the fatty acids are the common factors in determining the oil quality.In the present study,an F2 segregated population with 140 individuals derived from the cross of Weihua8(a cultivar)and 12L49(a line with high oleic acid concentration)was used to construct a genetic map and conduct QTL mapping analysis.A total of 103 polymorphic SSR primers were utilized for genotyping the RILs and finally generating the SSR loci.Within the 103 SSR loci,a genetic linkage map,covering a total length of 3592.35 cM of the whole peanut genome,was constructed.Based on the genetic map,sixteen QTLs located on nine linkage groups related to peanut fatty acids were finally identified.Among them,four QTLs were detected associated with various traits simultaneously,which showed genetic stability in relation to fatty acids of peanut.Except for the QTLs for oleic acid,linoleic acid,and linolenic acid,three novel QTLs for arachidic acid and behenic acid were also detected.These QTLs might be helpful for further fine mapping analysis and marker-assisted selection of fatty acids in peanut.

Mapping of S-b Locus for F_1 Pollen Sterility in Cultivated Rice Using PCR Based Markers

In cultivated rice ( Oryza sativa L.), F-1 pollen sterility is controlled by at least 6 loci of the F, pollen sterility genes. To map S-b, one of loci, rice variety Taichung 65 (T65) carrying S-b(j)/S-b(j) and its near-isogenic line TIST2 carrying S-b(i)/S-b(i) were used to develop the mapping population. One hundred and fifty-eight microsatellite markers were selected to survey T65 and TISL2. RM13 on chromosome 5 was found to be polymorphic between them. Cosegregation indicated that RM13 was closely linked with locus S-b. Eleven RFLP markers were selected on the corresponding region from the genetic map of Rice Genome Research Program (RGP) of Japan to convert into sequence-tagged site (STS) markers. Amplicon length polymorphism (ALP) was carried out, but none of them was found to be polymorphic between T65 and TISL2. Then PCR-based RFLP (PBR) was done using six 4-nucleotide recognizing restriction endonucleases. Polymorphism was detected when PCR products of R830STS and R2213SSTS were digested with Taq I. Genetic analysis indicated that the distance between locus S-b and markers, R830STS, RM13 and R2213SSTS were 3.3 cM (centi-Morgan), 5.2 cM and 5.5 cM, respectively. These PCR-based markers could be directly used in marker-assisted selection. The technical system combining genetic mapping and PCR-based marker-assisted selection will facilitate the development of molecular breeding.

Construction of Molecular Genetic Linkage Map Based on an RIL Population of Rice and Detection of QTLs for Tiller Angle

In this study, an RIL （recombinant inbred line） population containing 240 lines was developed by single seed descent method （SSD） based on a parent com- bination of small-grain indica cultivar Kasalath and large-grain japanica cultivar TD70 with significant differences in plant type traits, to construct the molecular genetic linkage map. Totally 838 SSR （Simple Sequence Repeat） markers were used for polymorphism screening between parents, 302 SSR markers with polymorphism were detected, with a frequency of 36.04%; 141 SSR markers with clear amplified bands and uniform distribution in the genome were finally used for genotype analysis of the RIL population. According to the experimental results, the frequency of male and female genotype in this RIL population was respectively 53% and 47%, suggesting good balance in population structure. A molecular genetic linkage map of rice was constructed by 141 markers based on a RIL population of 240 lines, with a total genetic distance of about 1 832.47 cM covering all 12 chromosomes, an average genetic distance between markers of 12.70 cM and a range of genetic distance be- tween markers of 0.43-36.11 cM, which is consistent with basic requirements of quantitative trait locus （QTL） mapping. Except for few markers on chromosomes 1 and 8, the order and location of markers is similar to the published sequences of Nipponbare. QTL analysis for the tiller angle was conducted with this RIL population of 240 lines, and results showed that three QTLs controlling tiller angle were detected on chromosome 8, 9 and 11, which were named qTA8, qTA9 and qTA11, with a contribution rate of 4.10%, 26.08% and 4.35%, respectively. To be specific, qTA9 contained Tiller Angle Controlling （TAC1） gene. The construction of this molecular genetic linkage map laid the foundation for genetic analysis and QTL mapping of various traits in the progeny of indica and japonica.

Linkage map and QTL mapping of red flesh locus in apple using a R1R1×R6R6 population

The red flesh in apple fruit is a desired trait by consumers and it is associated to the anthocyanin content,which is mainly controlled by MdMYB10 with a R6 promoter.In this study,a high-density linkage group was constructed using the‘Fuji’x‘Red3’population which contained homozygous alleles R1R1 and R6R6,respectively.The linkage group consists of 7630 SNPs along 17 linkage groups,spanning 2270.21 cM,with an average density of 0.30 cM permarker.The cyanidin-3-galactoside concentration was used as the phenotypic data in QTL analysis.Moreover,one QTL peak which was flaked by two markers,marker2187260 to marker2173766,with LOD scores of 4.49 was detected.This QTL ranged from 0 to 40.79 cM on the top of linkage group(LG16).In addition one candidate molecular marker(marker2175442)in this QTL was identified,which was significant correlated with the flesh cyanidin-3-galactoside concentration.These genetic findings enrich the breeding basis of fruit flesh coloration in apple.

Status and Advances of Molecular Genetic Improvement of Poplar Species in China

Poplars are among the most important deciduous tree species in China. China is replete with natural resources of poplars. Poplars have a number of good characteristics, including fast growth rate, high yield, many uses, easiness of tissue culture and small gene group that make them well suited as a model system for the application of genetic engineering in forest trees. In the last decade, much progress has been made in genetic improvement of poplar species in China. Modern biotechnology is an important tool for genetic improvement in forest trees, and its applications to genetic improvement in poplars, which covers genetic transformation, gene expression, construction of genetic linkage map, QTLs (quantitative trait loci) identification and molecular assisted selection are reviewed in this paper. At the same time, the existing problems and outlook about the application of modern biotechnology to genetic improvement in forest trees are also discussed.

Mapping a leaf senescence gene els1 by BSR-Seq in common wheat

Leaf senescence is normally the last stage of plant development. Early senescence of functional leaves significantly reduces the photosynthetic time and efficiency, seriously affecting grain yield and quality in wheat. Discovering genes responsible for early leaf senescence(els) are necessary for developing novel germplasms and cultivars with delayed leaf-senescence through molecular manipulation and marker assisted selection. In this study, we identified an early leaf senescence line M114 in a derivative of a wheat breeding population. Genetic analysis indicated that early leaf senescence in M114 is controlled by a single recessive gene, provisionally designated els1. By applying bulked segregant analysis and RNA-Seq(BSR-Seq), seven polymorphic markers linked to els1 were developed and the gene was located on chromosome arm 2 BS in a 1.5 c M genetic interval between markers WGGB303 and WGGB305. A co-segregating marker, WGGB302, provide a starting point for fine mapping and map-based cloning of els1.