Fine-mapping and primary analysis of candidate genes associated with seed coat color in mung bean(Vigna radiata L.)

Seed coat color affects the appearance and commodity quality of mung beans(Vigna radiata L.).The substances that affect mung bean seed coat color are mainly flavonoids,which have important medicinal value.Mapping the seed coat color gene in mung beans would facilitate the development of new varieties and improve their value.In this study,an F2 mapping population consisting of 546 plants was constructed using Jilv9(black seed coat)and BIS9805(green seed coat).Using bulk segregated analysis(BSA)sequencing and kompetitive allele-specific PCR(KASP)markers,the candidate region related to seed coat color was finally narrowed to 0.66 Mb on chromosome(Chr.)4 and included eight candidate genes.Combined transcriptome and metabolome analyses showed that three of the eight candidate genes(LOC106758748,LOC106758747,and LOC106759075)were differentially expressed,which may have caused the differences in flavonoid metabolite content between Jilv9 and BIS9805.These findings can provide a research basis for cloning the genes related to seed coat color and accelerate molecular markerassisted selection breeding in mung beans.

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.

Evaluation and Bulked Segregant Analysis of Major Yield QTL qtl12.1 Introgressed into Indigenous Elite Line for Low Water Availability under Water Stress

Near isogenic lines carrying large-effect QTL （qtl2.1）, which has a consistent influence on grain yield under upland drought stress conditions in a wide range of environments, were evaluated under water stress in the fields. The line which gave higher yield under drought was crossed with a local elite line, PMK3, and forwarded to F2：3 generation. Significant variation was found among the F2：3 lines for agronomic traits under water stress in the fields. Low to high broad sense heritability （H） for investigated traits was also found. Water stress indicators such as leaf rolling and leaf drying were negatively correlated with plant height, biomass and grain yield under stress. Bulked segregant analysis （BSA） was performed with the markers in the vicinity of qUl2.1, and RM27933 was found to be segregated perfectly well in individual components of drought resistant and drought susceptible bulks which were bulked based on yield under water stress among F2：3 lines. Hence, this simple and breeder friendly marker, RM27933, may be useful as a potentially valuable candidate marker for the transfer of the QTL qtl12.1 in the regional breeding program. Bioinformatic analysis of the DNA sequence of the qtl12.1 region was also done to identify and analyze positional candidate genes associated with this QTL and to ascertain the putative molecular basis of qUl2.1.

Tagging of Brown Planthopper Resistance Genes in F_2s of IR50 × Ptb33 of Rice by Using Bulked Segregant Analysis

Brown planthopper （Nilaparvata lugens Stal） is one of the most damaging pests causing hopper burn in rice, and thereby reducing the productivity and also the quality of the product. The effective management strategy to control this pest is the identification and transfer of desirable genes to local rice cultivars. The most important approach for developing resistant cultivars is the identification of markers, which can help in marker-assisted selection of more durable resistant genotype. The susceptible parent IR50 and the resistant parent Ptb33, and their F2 populations were used in bulked segregant analysis for identification of resistant genes with random amplified polymorphic DNA marker （RAPD） primers. The primers OPC7 and OPAG14 showed both dominant and susceptible specific banding pattern so called co-dominant markers. Moreover, OPC7697 and OPAG14680 showed resistant specific bands and thus being in coupling phase, whereas OPC7846 and OPAG14650 showed susceptible specific genotypic bands in bulked segregant analysis. Therefore, the coupling phase markers, OPC7697 and OPAG14680, are considered to be more useful in marker-assisted selection of rice genotypes in crop improvement.

Digging out molecular markers associated with low salinity tolerance of Nannochloropsis oceanica through bulked mutant analysis

Nannochloropsis oceanica is a marine microalgal species with both economic value and biological importance.It grows fast,contains rich oils,reproduces asexually,holds a small and haploidy genome,and is easy to be modified genetically.However,the genetic study of N.oceanica is scarce.Very less genetic bases of its traits have been deciphered,and no gene has been isolated from it with the function verified simultaneously via either genetic or reverse genetic approaches or both(de novo cloned).Changing medium salinity may aid to control harmful organisms met during large scale cultivation.As a stress,it may also facilitate the accumulation of desirable chemicals including fatty acids.In order to decipher the genetic basis of the low salinity tolerance of N.oceanica,we mutated N.oceanica with Zeocin.In total,five mutant bulks were constructed at equal number of cells,100 mutants each,which were tolerant to a discontinuous serial of salinities from that of 100%of f/2 to that of a mixture of 4%of f/2 and 94%of BG11.The bulks were genotyped through whole genome re-sequencing and analyzed with bulked mutant analysis(BMA)newly modified from bulked segregant analysis(BSA).In total,47 SNPs and 112 InDels were found to associate with the low salinity tolerance,and around them a set of low salinity tolerance associating genes were identified.A set of annotatable genes commonly found between control and different salinities indicated that the genes functioning in gene expression,energy metabolism and cellular structure may be involved in the low salinity tolerance.These associating molecular markers and genes around them were not enough for outlining the physiological mechanism underlining the tolerance;however they should aid to improve N.oceanica genetically.

Discovering Candidate Chromosomal Regions Linked to Kernel Size-Related Traits via QTL Mapping and Bulked Sample Analysis in Maize

Kernel size-related traits,including kernel length,kernel width,and kernel thickness,are critical components in determining yield and kernel quality in maize(Zea mays L.).Dissecting the phenotypic characteristics of these traits,and discovering the candidate chromosomal regions for these traits,are of potential importance for maize yield and quality improvement.In this study,a total of 139 F2:3 family lines derived from EHel and B73,a distinct line with extremely low ear height(EHel),was used for phenotyping and QTL mapping of three kernel sizerelated traits,including 10-kernel length(KL),10-kernel width(KWid),and 10-kernel thickness(KT).The results showed that only one QTL for KWid,i.e.,qKWid9 on Chr9,with a phenotypic variation explained(PVE)of 13.4%was detected between SNPs of AX-86298371 and AX-86298372,while no QTLs were detected for KL and KT across all 10 chromosomes.Four bulked groups of family lines,i.e.,Groups I to IV,were constructed with F2:3 family lines according to the phenotypic comparisons of KWid between EHel and B73.Among these four groups,Group I possessed a significantly lower KWid than EHel(P=0.0455),Group II was similar to EHel(P=0.34),while both Group III and Group IV were statistically higher than EHel(P<0.05).Besides,except Group IV exhibited a similar KWid to B73(P=0.11),KWid of Groups I to III were statistically lower than B73(P<0.00).By comparing the bulked genotypes of the four groups to EHel and B73,a stable chromosomal region on Chr9 between SNPs of AX-86298372 to AX-86263154,entirely covered by qKWid9,was identified to link KWid with the positive allele of increasing phenotypic effect to KWid from B73,similar to that of qKWid9.A large amount of enzyme activity and macromolecule binding-related genes were annotated within this chromosomal region,suggesting qKWid9 as a potential QTL for KWid in maize.

Integrated genomic and transcriptomic analysis reveals genes associated with plant height of foxtail millet

Foxtail millet(Setaria italica)is an important C4 model crop;however,due to its high-density planting and high stature,lodging at the filling stage resulted in a serious reduction in yield and quality.Therefore,it is imperative to identify and deploy the genes controlling foxtail millet plant height.In this study,we used a semi-dwarf line 263A and an elite high-stalk breeding variety,Chuang 29 to construct an F2 population to identify dwarf genes.We performed transcriptome analysis(RNA-seq)using internode tissues sampled at three jointing stages of 263A and Chuang 29,as well as bulk segregant analysis(BSA)on their F2 population.A total of 8918 differentially expressed genes(DEGs)were obtained from RNA-seq analysis,and GO analysis showed that DEGs were enriched in functions such as‘‘gibberellin metabolic process”and‘‘oxidoreductase activity”,which have previously been shown to be associated with plant height.A total 593 mutated genes were screened by BSA-seq method.One hundred and seventy-six out of the 593 mutated genes showed differential expression levels between the two parental lines,and seven genes not only showed differential expression in two or three internode tissues but also showed high genomic variation in coding regions,which indicated they play a crucial role in plant height determination.Among them,we found a gibberellin biosynthesis related GA20 oxidase gene(Seita.5G404900),which had a single-base at the third exon,leading to the frameshift mutation at 263A.Cleaved amplified polymorphic sequence assay and association analysis proved the single-base in Seita.5G404900 co-segregated with dwarf phenotype in two independent F2 populations planted in entirely different environments.Taken together,the candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height,and the molecular marker will be useful for marker-assisted dwarf breeding.

Fine-mapping of a candidate gene for web blotch resistance in Arachis hypogaea L.

Peanut(Arachis hypogaea L.)is a globally important oil crop.Web blotch is one of the most important foliar diseases affecting peanut,which results in serious yield losses worldwide.Breeding web blotch-resistant peanut varieties is the most effective and economically viable method for minimizing yield losses due to web blotch.In the current study,a bulked segregant analysis with next-generation sequencing was used to analyze an F2:3 segregating population and identify candidate loci related to web blotch resistance.Based on the fine-mapping of the candidate genomic interval using kompetitive allele-specific PCR(KASP)markers,we identified a novel web blotch resistance-related locus spanning approximately 169 kb on chromosome 16.This region included four annotated genes,of which only Arahy.35VVQ3 had a non-synonymous single nucleotide polymorphism in the coding region between the two parents.Two markers(Chr.16.12872635 and Chr.16.12966357)linked to this gene were shown to be co-segregated with the resistance of peanut web blotch by 72 randomly selected recombinant inbred lines(RIL),which could be used in marker-assisted breeding of resistant peanut varieties.

A 1-bp deletion in the MC04g1399 is highly associated with failure to produce fruit wart in bitter gourd

Fruit wart is an important appearance trait influencing consumer preferences of bitter gourd(Momordica charantia L.).The molecular genetic mechanisms underlying fruit wart formation in bitter gourd are largely unknown.In this study,genetic analysis based on four generations showed that fruit wart formation in bitter gourd was controlled by a single dominant locus named as Fwa.The Fwa locus was initially mapped into a 4.82 Mb region on pseudochromosome 4 by BSA-seq analysis and subsequently narrowed down to a 286.30 kb region by linkage analysis.A large F2population consisting of 2360 individuals was used to screen recombinants,and the Fwa locus was finally fine mapped into a 22.70 kb region harboring four protein-coding genes through recombination analysis.MC04g1399,encoding an epidermal patterning factor 2-like protein,was proposed as the best candidate gene for Fwa via sequence variation and expression analysis.In addition,a 1-bp insertion and deletion(InDel)variation within MC04g1399 was converted to a cleaved amplified polymorphic sequence(CAPS)marker that could precisely distinguish between the warty and non-warty types with an accuracy rate of 100%among a wide panel of 126 bitter gourd germplasm resources.Our results not only provide a scientific basis for deciphering the molecular mechanisms underlying fruit wart formation but also provide a powerful tool for efficient genetic improvement of fruit wart via marker-assisted selection.

Iodine Sorption Value and Surface Chemical Analysis of Regenerated Cellulosic Fibres

The surface chemical analysis and bulk analysis were conducted for fibres regenerated from waste garments and treated with iodine solution. The aim was to assess the fibre accessibility by the iodine solution and ascertain the location of the reagent within the fibres. X-ray Photoelectron Spectroscopy (XPS) analysis indicated that the fibres regenerated from indigo dyed waste denim garments (ReCell-Denim) had a relatively high accessibility by the iodine solution compared to the standard lyocell, ReCell-1 and ReCell-2 fibres. With the exception of ReCell-Denim, the standard lyocell, ReCell-1 and ReCell-2 fibre’s iodine sorption values correlated well with the percentage crystallinity. The high accessibility of the ReCell-Denim fibres was attributed to the presence of the positively charged nitrogen from the indigo dyes that improved the substantivity of the fibres to the iodine solution. The iodine sorption of the fibres is relatively higher in the bulk compared to the fibre surface.

Mapping of wheat stripe rust resistance gene Yr041133 by BSR-Seq analysis

Puccinia striiformis Westend. f. sp. tritici(Pst) pathotype CYR34 is widely virulent and prevalent in China.Here, we report identification of a strpie rust resistance(Yr) gene, designated Yr041133, in winter wheat line 041133. This line produced a hypersensitive reaction to CYR34 and conferred resistance to 13 other pathotypes. Resistance to CYR34 in line 041133 was controlled by a single dominant gene. Bulked segregant RNA sequencing(BSR-Seq) was performed on a pair of RNA bulks generated by pooling resistant and susceptible recombinant inbred lines. Yr041133 was mapped to a 1.7 c M genetic interval on the chromosome arm 7 BL that corresponded to a 0.8 Mb physical interval(608.9–609.7 Mb) in the Chinese Spring reference genome. Based on its unique physical location Yr041133 differred from the other Yr genes on this chromosome arm.

团状模塑料抗收缩机理研究

通过动态热机械分析仪(DMA)的静态热机械(TMA)分析法和示差扫描量热仪(DSC),测试得多种低收缩添加剂(LPA)的线性膨胀系数和玻璃化转变温度(Tg),加入不同LPA的团状模塑料(BMC)复合材料的线性收缩率,研究了BMC中LPA的抗收缩机理。结果表明:BMC复合材料的抗收缩效果不仅与LPA的线膨胀系数有关,还与LPA的玻璃化转变温度有关;同时还通过扫描电镜(SEM)观察不同种类的LPA在BMC复合材料中的分散及形成的空穴的大小佐证了以上的联系。

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.

Development of Sequence Characterized Amplified Region (SCAR) Primers for the Detection of Resistance to Sporisorium reiliana in Maize

Head smut of maize （Zea mays L.）, which was caused by Sporisorium reiliana, occurred in most of the maize growing areas of the world. The purpose of this study was to develop SCAR markers for map-based cloning of resistance genes and MAS. Two sets of BC3 progenies, one （BC3Q） derived from the cross Qi319 （resistance）×Huangzao 4 （susceptible）, the other （BC3M） from Mol7 （resistance）× Huangzao 4 （susceptible）, were generated. Huangzao 4 was the recurrent parent in both progenies. A combination of BSA （bulked segregant analysis） with AFLP （amplified fragment length polymorphism） method was applied to map the genes involving the resistance to S. reiliana, and corresponding resistant and susceptible bulks and their parental lines were used for screening polymorphic AFLP primer pairs. One fragment of PI3M61-152 was converted into SCAR （sequence charactered amplified fragment） marker S130. The marker was mapped at chromosome bin 2.09, the interval of a major QTL region previously reported to contribute to S. reiliana resistance. Furthermore, S130 was highly and facilitate map-based cloni associated with resistance to S. reiliana, and could be useful for marker-assisted selection ng of resistance genes.

AFLP and SCAR Markers Associated with Peel Color in Eggplant (Solanum melongena)

Peel color is an important breeding objective for eggplant. Dark purple and purplish red are the most common colors in commercial eggplant cultivars. A co-dominant amplified fragment length polymorphism （AFLP） marker which was associated with the peel color （each in coupling phase to dark purple and purplish red） was found in studying the genetic diversity in 58 eggplant accessions （contained cultivars and wild relatives）. The maker bands were sequenced and converted to SCAR marker, this polymorphism in sequence was from an inserted/deleted （indels） mutation. And DNA from 136 eggplant materials （inbred lines, F1, and wild relatives） were amplified with the designed SCAR primers as template, high correlation between the SCAR marker and peel color （dark purple and purplish red） was found. Then, bulked line analysis （BLA） combined with AFLP was further used to identify polymorphic fragments, and another six AFLP markers were tested and verified to be associated with peel color, which demonstrated that BLA was an useful method for identifying molecular markers of interested traits. In conclusion, these markers will facilitate the MAS （maker-assisted selection） of eggplant breeding for peel color.

DeepBSA: A deep-learning algorithm improves bulked segregant analysis for dissecting complex traits

Bulked segregant analysis(BSA)is a rapid,cost-effective method for mapping mutations and quantitative trait loci(QTLs)in animals and plants based on high-throughput sequencing.However,the algorithms currently used for BSA have not been systematically evaluated and are complex and fallible to operate.We developed a BSA method driven by deep learning,DeepBSA,for QTL mapping and functional gene cloning.DeepBSA is compatible with a variable number of bulked pools and performed well with various simulated and real datasets in both animals and plants.DeepBSA outperformed all other algorithms when comparing absolute bias and signal-to-noise ratio.Moreover,we applied DeepBSA to an F2 segregating maize population of 7160 individuals and uncovered five candidate QTLs,including three well-known plant-height genes.Finally,we developed a user-friendly graphical user interface for DeepBSA,by integrating five widely used BSA algorithms and our two newly developed algorithms,that is easy to operate and can quickly map QTLs and functional genes.The DeepBSA software is freely available to noncommercial users at http://zeasystemsbio.hzau.edu.cn/tools.html and https://github.com/lizhao007/DeepBSA.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

Identification RAPD Markers Linked to Texture Quality of Chinese Cabbage

[ Objectives] This study was conducted to identify the random amplification of polymorphic DNA （RAPD） markers linked to chewy texture-controlling gene of Chinese cabbage. [ Methods] The RAPD markers associated with chewy texture of Chinese cabbage were identified via bulked segregant analysis （BSA） in an F2 population derived from the cross between Hua 273 （female parent） and 114 Fushan （male parent）. [ Results] OPA06-1400 was identified to he linked to the chewy texture-controlling gene of Chinese cabbage. The genetic distance between the target gene and the RAPD marker was 24.8 cM. [ Conclusions] The resuits provide experimental evidence for breeding of Chinese cabbage.

AN ALGORITHM PROPOSED FOR BUSY-PERIOD SUBCOMPONENT ANALYSIS OF BULK QUEUES

This paper presents an algorthmic procedure for a busy-period subcomponent analysis of bulkqueues. A component of interest for many server queues is the period tto reduce congestion froma level k to level k-1. For an M(x)/M/c system with the possibility of total or partial rejection ofbatches, it is demonstrated that the expected length of busy periods, the proportion of delayed batchand the steady state queue length probabilities can be easily obtained. The procedure is based on thenested partial sums and monotonic properties of expected lengths of the busy periods.

Identifying a melanogenesis-related candidate gene by a high-quality genome assembly and population diversity analysis in Hypsizygus marmoreus

Hypsizygus marmoreus is one of the most important edible fungi in Basidiomycete division and includes white and gray strains.However,very limited knowledge is known about the genomic structures and the genetic basis for the white/gray diversity of this mushroom.Here,we report the near-complete high-quality H.marmoreus genome at the chromosomal level.Comparative genomics analysis indicates that chromosome structures were relatively conserved,and variations in collinearity and chromosome number were mainly attributed by chromosome split/fusion events in Aragicales,whereas the fungi genome experienced many genomic chromosome fracture,fusion,and genomic replication events after the split of Aragicales from Basidiomycetes.Resequencing of 57 strains allows us to classify the population into four major groups and associate genetic variations with morphological features,indicating that white strains were not originated independently.We further generated genetic populations and identified a cytochrome P450 as the candidate causal gene for the melanogenesis in H.marmoreus based on bulked segregant analysis (BSA)and comparative transcriptome analysis.The high-quality H.marmoreus genome and diversity data compiled in this study provide new knowledge and resources for the molecular breeding of H.marmoreus as well as the evolution of Basidiomycete.