Fine-mapping and transcriptome analysis of the photosensitive leaf-yellowing gene CaLY1 in pepper(Capsicum annuum L.)

Leaf color is directly related to altered photosynthesis.Hence,leaf yellowing mutants can be widely used for the researching plant physiology and functional genomes,for cultivating new varieties of popular horticultural plants,and for identifying hybrid purity(as markers).Here,we constructed a^(60)Co-γF_(2)population from the leaf-yellowing mutant R24 via radiation mutation with the inbred line WT21 of pepper.Genetic analysis showed that the leaf-yellowing of the mutant was controlled by a single recessive gene.By applying the Bulk Segregation Analysis and Kompetitive Allele Specific PCR markers,the leaf-yellowing gene CaLY1(Capsicum annuum Leaf yellow 1)was mapped on chromosome 9,SNP5791587-SNP6011215,with a size of 214.5 kb.One non-synonymous mutated gene Capana09g000166 was found in the interval.The gene encoded a Psb X,which is the core complex of PSⅡ.Transcriptome analysis further showed that 2301 differentially expressed genes were identified under shading treatment for 24 h in R24.The Gene Ontology enrichment pathways were related to photosynthesis light harvesting,cell wall,activity of quercetin 3-O-glucosyltransferase and flavonoid metabolic process,which likely regulate the response of pepper leaves to different light levels.Functional enrichment analysis indicated that the most abundant pathways were photosynthesis antenna proteins and metabolic.

Microarray-Assisted Fine-Mapping of Quantitative Trait Loci for Cold Tolerance in Rice

Many important agronomic traits, including cold stress resistance, are complex and controlled by quantitative trait loci （QTLs）. Isolation of these QTLs will greatly benefit the agricultural industry but it is a challenging task. This study explored an integrated strategy by combining microarray with QTL-mapping in order to identify cold-tolerant QTLs from a cold-tolerant variety ILl12 at early-seedling stage. All the early seedlings of IL112 survived normally for 9 d at 4-5℃, while Guichao2 （GC2）, an indica cultivar, died after 4 d under the same conditions. Using the F2-3 population derived from the progeny of GC2 and ILl12, we identified seven QTLs for cold tolerance. Furthermore, we performed Affymetrix rice whole-genome array hybridization and obtained the expression profiles of ILl12 and GC2 under both low-temperature and normal conditions. Four genes were selected as cold QTL-related candidates, based on microarray data mining and QTL-mapping. One candidate gene, LOC_Os07g22494, was shown to be highly associated with cold tolerance in a number of rice varieties and in the F2-3 population, and its overexpression transgenic rice plants displayed strong tolerance to low temperature at early-seedling stage. The results indicated that overexpression of this gene （LOC_Os07g22494） could increase cold tolerance in rice seedlings. Therefore, this study provides a promising strategy for identifying candidate genes in defined QTL regions.

Detection and fine-mapping of SC7 resistance genes via linkage and association analysis in soybean

Soybean mosaic virus（SMV） disease is one of the most serious and broadly distributed soybean（Glycine max（L.） Merr.） diseases. Here, we combine the advantages of association and linkage analysis to identify and fine-map the soybean genes associated with resistance to SMV strain SC7.A set of 191 soybean accessions from different geographic origins and 184 recombinant inbred lines（RILs） derived from Kefeng No.1（resistant） Nannong 1138-2（susceptible） were used in this study. The SC7 resistance genes were previously mapped to a 2.65 Mb region on chromosome 2 and a 380 kb region on chromosome 13. Among 19 single nucleotide polymorphisms（SNPs） detected via association analysis in the study, the SNP BARC-021625-04157 was located in the2.65 Mb region, and the SNP BARC-041671-08065 was located near the 380 kb region; three genes harboring the SNPs were probably related to SC7 resistance. The resistance gene associated with BARC-021625-04157 was then finemapped to a region of approximately 158 kb on chromosome2 using 184 RILs. Among the 15 genes within this region, one NBS-LRR type gene, one HSP40 gene and one serine carboxypeptidase-type gene might be candidate SC7 resistance genes. These results will be useful for map-based cloning and marker-assisted selection in soybean breeding programs.

A Sequential Quantitative Trait Locus Fine-Mapping Strategy Using Recombinant-Derived Progeny

A thorough understanding of the quantitative trait loci （QTLs） that underlie agronomically important traits in crops would greatly increase agricultural productivity. Although advances have been made in QTL cloning, the majority of QTLs remain unknown because of their low heritability and minor contributions to phenotypic performance. Here we summarize the key advantages and disad- vantages of current QTL fine-mapping methodologies, and then introduce a sequential QTL fine-mapping strategy based on both genotypes and phenotypes of progeny derived from recombinants. With this mapping strategy, experimental errors could be dramat- ically diminished so as to reveal the authentic genetic effect of target QTLs. The number of progeny required to detect QTLs atvarious R2 values was calculated, and the backcross generation suitable to start QTL fine-mapping was also estimated. This mapping strategy has proved to be very powerful in narrowing down QTL regions, particularly minor-effect QTLs, as revealed by fine-mapping of various resistance QTLs in maize. Application of this sequential QTL mapping strategy should accelerate cloning of agronomically important QTLs, which is currently a substantial challenge in crops.

Map-based cloning of q LP_(A01.1),a favorable allele from the Gossypium tomentosum chromosome segment line

Cotton is an important natural fiber crop worldwide which plays a vital role in our daily life.High yield is a constant goal of cotton breeding,and lint percentage(LP)is one of the important components of cotton fiber yield.A stable QTL controlling LP,qLP_(A01.1),was identified on chromosome A01 from Gossypium hirsutum introgressed lines with G.tomentosum chromosome segments in a previous study.To fine-map qLP_(A01.1),an F2 population with 986individuals was established by crossing G.hirsutum cultivar CCRI35 with the chromosome segment substitution line HT_390.A high-resolution genetic map including 47 loci and spanning 56.98 cM was constructed in the QTL region,and qLP_(A01.1)was ultimately mapped into an interval corresponding to an~80 kb genome region of chromosome A01in the reference genome,which contained six annotated genes.Transcriptome data and sequence analysis revealed that S-acyltransferase protein 24(GoPAT24)might be the target gene of qLP_(A01.1).This result provides the basis for cotton fiber yield improvement via marker-assisted selection(MAS)and further studies on the mechanism of cotton fiber development.

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.

QTG-Seq Accelerates QTL Fine Mapping through QTL Partitioning and Whole-Genome Sequencing of Bulked Segregant Samples

Deciphering the genetic mechanisms underlying agronomic traits is of great importance for crop improvement. Most of these traits are controlled by multiple quantitative trait loci (QTLs), and identifying the underlying genes by conventional QTL fine-mapping is time-consuming and labor-intensive. Here, we devised a new method, named quantitative trait gene sequencing (QTG-seq), to accelerate QTL fine-mapping. QTGseq combines QTL partitioning to convert a quantitative trait into a near-qualitative trait, sequencing of bulked segregant pools from a large segregating population, and the use of a robust new algorithm for identifying candidate genes. Using QTG-seq, we fine-mapped a plant-height QTL in maize (Zea mays L.), qPH7, to a 300-kb genomic interval and verified that a gene encoding an NF-YC transcription factor was the functional gene. Functional analysis suggested that qPH7-encoding protein might influence plant height by interacting with a CO-like protein and an AP2 domain-containing protein. Selection footprint ana卜 ysis indicated that qPH7 was subject to strong selection during maize improvement. In summary, QTG-seq provides an efficient method for QTL fine-mapping in the era of “big data".

Identification and fine mapping of quantitative trait loci for the number of vascular bundle in maize stem

Studies that investigated the genetic basis of source and sink related traits have been widely conducted.However, the vascular system that links source and sink received much less attention. When maize was domesticated from its wild ancestor, teosinte, the external morphology has changed dramatically; however, less is known for the internal anatomy changes. In this study, using a large maize-teosinte experimental population, we performed a high-resolution quantitative trait locus（QTL） mapping for the number of vascular bundle in the uppermost internode of maize stem.The results showed that vascular bundle number is dominated by a large number of small-effect QTLs, in which a total of 16 QTLs that jointly accounts for 52.2% of phenotypic variation were detected, with no single QTL explaining more than 6% of variation. Different from QTLs for typical domestication traits, QTLs for vascular bundle number might not be under directional selection following domestication.Using Near Isogenic Lines（NILs） developed from heterogeneous inbred family（HIF）, we further validated the effect of one QTL qVb9-2 on chromosome 9 and fine mapped the QTL to a 1.8-Mb physical region. This study provides important insights for the genetic architecture of vascular bundle number in maize stem and sets basis for cloning of qVb9-2.

Identification and Fine Mapping of rhm1 Locus for Resistance to Southern Corn Leaf Blight in Maize

rhml is a major recessive disease resistance locus for Southern corn leaf blight （SCLB）. To further narrow down its genetic position, F2 population and BCIFI population derived from the cross between resistant （H95rhm） and susceptible parents （H95） of maize （Zea mays） were constructed. Using newly developed markers, rhml was initially delimited within an interval of 2.5 Mb, and then finally mapped to a 8.56 kb interval between InDel marker IDP961-503 and simple sequence repeat （SSR） marker A194149--1. Three polymorphic markers IDP961-504, IDP B2-3 and A194149-2 were shown to be co-segregated with the rhml locus. Sequence analysis of the 8.56 kb DNA fragment revealed that it contained only one putative gene with a predicted amino acid sequence identical to lysine histidine transporter 1 （LHT1）. Comparative sequence analysis indicated that the LHT1 in H95rhrn harbors a 354 bp insertion in its third exon as compared with that of susceptible alleles in B73, H95 and Mo17. The 354 bp insertion resulted in a truncation of the predicted protein of candidate resistance allele （LHT1-H95rhm）. Our results strongly suggest LHTI as the candidate gene for rhml against SCLB. The tightly linked molecular markers developed in this study can be directly used for molecular breeding of resistance to Southern corn leaf blight in maize.

Genomic and functional evaluation of TNFSF14 in multiple sclerosis susceptibility

Among multiple sclerosis(MS)susceptibility genes,the strongest non-human leukocyte antigen(HLA)signal in the Italian population maps to the TNFSF14 gene encoding LIGHT,a glycoprotein involved in dendritic cell(DC)maturation.Through fine-mapping in a large Italian dataset(4,198 patients with MS and3,903 controls),we show that the TNFSF14 intronic SNP rs1077667 is the primarily MS-associated variant in the region.Expression quantitative trait locus(e QTL)analysis indicates that the MS risk allele is significantly associated with reduced TNFSF14 messenger RNA levels in blood cells,which is consistent with the allelic imbalance in RNA-Seq reads(P<0.0001).The MS risk allele is associated with reduced levels of TNFSF14 gene expression(P<0.01)in blood cells from 84 Italian patients with MS and 80 healthy controls(HCs).Interestingly,patients with MS are lower expressors of TNFSF14 compared to HC(P<0.007).Individuals homozygous for the MS risk allele display an increased percentage of LIGHT-positive peripheral blood myeloid DCs(CD11 c+,P=0.035)in 37 HCs,as well as in in vitro monocyte-derived DCs from 22 HCs(P=0.04).Our findings suggest that the intronic variant rs1077667 alters the expression of TNFSF14 in immune cells,which may play a role in MS pathogenesis.