Cleaved Amplified Polymorphic Sequence Research Articles

Marker-Assisted Breeding Techniques for the Development of Gluten-Free Wheat Varieties: A Comprehensive Review

Ingestion of gluten proteins from wheat, barley and rye poses a significant health risk. The sole treatment for various disease is a lifelong adherence to a strict gluten-free diet. Conventional breeding methods have limitations in producing wheat varieties that maintain baking quality due to the complexity of the wheat genome and the multitude of gluten genes. This review explores the use of RNA interference (RNAi) silencing and CRISPR/Cas9 gene editing techniques. Efficient screening and selection processes for identifying lines with reduced celiac disease epitopes at both the DNA and protein levels, as well as maintaining baking quality, are discussed. The integration of gene editing for the production of wheat products holds significant potential in meeting the growing demand for safer dietary options while ensuring compliance with regulatory standards and addressing the complex challenges associated with disease management. Marker-assisted selection (MAS) offers several advantages over conventional selection methods in plant breeding, including timesaving, cost-effectiveness and goal-oriented outcomes. This review aims to delineate various molecular markers, encompassing sequence-tagged sites (STS), simple sequence repeats (SSR), genotyping by sequencing (GBS), single nucleotide polymorphism (SNP) arrays, exome capture, Competitive Allele Specific PCR (KASP), cleaved amplified polymorphic sequences (CAPS), semi-thermal asymmetric reverse PCR (STARP), and genotyping by target sequencing (GBTS). Additionally, we compile quantitative trait loci (QTL)/genes and their associated markers, which hold potential utility in MAS applications. The rapid advancement of wheat genomics is poised to expedite marker development, QTL mapping, gene cloning, and wheat breeding processes.

GmAP1d regulates flowering time under long-day photoperiods in soybean

Flowering time is important for adaptation of soybean (Glycine max) to different environments. Here, we conducted a genome-wide association study of flowering time using a panel of 1490 cultivated soybean accessions. We identified three strong signals at the qFT02-2 locus (Chr02: 12037319–12238569), which were associated with flowering time in three environments: Gongzhuling, Mengcheng, and Nanchang. By analyzing linkage disequilibrium, gene expression patterns, gene annotation, and the diversity of variants, we identified an AP1 homolog as the candidate gene for the qFT02-2 locus, which we named GmAP1d. Only one nonsynonymous polymorphism existed among 1490 soybean accessions at position Chr02:12087053. Accessions carrying the Chr02:12087053-T allele flowered significantly earlier than those carrying the Chr02:12087053-A allele. Thus, we developed a cleaved amplified polymorphic sequence (CAPS) marker for the SNP at Chr02:12087053, which is suitable for marker-assisted breeding of flowering time. Knockout of GmAP1d in the ‘Williams 82’ background by gene editing promoted flowering under long-day conditions, confirming that GmAP1d is the causal gene for qFT02-2. An analysis of the region surrounding GmAP1d revealed that GmAP1d was artificially selected during the genetic improvement of soybean. Through stepwise selection, the proportion of modern cultivars carrying the Chr02:12087053-T allele has increased, and this allele has become nearly fixed (95%) in northern China. These findings provide a theoretical basis for better understanding the molecular regulatory mechanism of flowering time in soybean and a target gene that can be used for breeding modern soybean cultivars adapted to different latitudes.

Identification of genetic loci and candidate genes underlying freezing tolerance in wheat seedlings.

Ten stable loci for freezing tolerance (FT) in wheat were detected by genome-wide association analysis. The putative candidate gene TaRPM1-7BL underlying the major locus QFT.ahau-7B.2 was identified and validated. Frost damage restricts wheat growth, development, and geographical distribution. However, the genetic mechanism of freezing tolerance (FT) remains unclear. Here, we evaluated FT phenotypes of 245 wheat varieties and lines, and genotyped them using a Wheat 90K array. The association analysis showed that ten stable loci were significantly associated with FT (P < 1 × 10-4), and explained 6.45-26.33% of the phenotypic variation. In particular, the major locus QFT.ahau-7B.2 was consistently related to all nine sets of FT phenotypic data. Based on five cleaved amplified polymorphic sequence (CAPS) markers closely linked to QFT.ahau-7B.2, we narrowed down the target region to the 570.67-571.16Mb interval (0.49Mb) on chromosome 7B, in which four candidate genes were annotated. Of these, only TaRPM1-7BL exhibited consistent differential expression after low temperature treatment between freezing-tolerant and freezing-sensitive varieties. The results of cloning and whole-exome capture sequencing indicated that there were two main haplotypes for TaRPM1-7BL, including freezing-tolerant Hap1 and freezing-sensitive Hap2. Based on the representative SNP (+1956, A/G), leading to an amino acid change in the NBS domain, a CAPS marker (CAPS-TaRPM1-7BL) was developed and validated in 431 wheat varieties (including the above 245 materials) and 318 F2 lines derived from the cross of 'Annong 9267' (freezing-tolerant) × 'Yumai 9' (freezing-sensitive). Subsequently, the TaRPM1-7BL gene was silenced in 'Yumai 9' by virus-induced gene silencing (VIGS), and these silenced wheat seedlings exhibited enhanced FT phenotypes, suggesting that TaRPM1-7BL negatively regulates FT. These findings are valuable for understanding the complex genetic basis of FT in wheat.

Genetic characterization of new source of resistance for tomato leaf curl New Delhi virus (ToLCNDV) from snapmelon (C. melo var. momordica)

AbstractThis study aimed to understand the genetics of tomato leaf curl New Delhi virus (ToLCNDV) in the identified novel source of resistance from Indian melon germplasm DSM-19 (Cucumis melo var. momordica) as viral diseases in muskmelon cause significant economic yield loss. To achieve this, a cross was made between the highly susceptible genotype Pusa Sarda (C. melo var. inodorus), known for its desirable fruit characters, and the resistant source DSM-19 to generate the suitable populations for inheritance study. These populations were screened under natural epiphytotic conditions and further validated through challenge inoculation with viruliferous whiteflies. The inheritance of ToLCNDV resistance in snapmelon germplasm DSM-19 was identified as monogenic recessive in both the screening methods. Moreover, a cleaved amplified polymorphic sequence (CAPS) marker named ‘CAPS 16 (2)’ was designed near to SNP marker D16 located on chromosome 11 of melon, and it was found to be linked to the ToLCNDV resistance gene in DSM-19. This is the first report on genetics of ToLCNDV resistance in snapmelon germplasm from India. Snapmelon line DSM-19 can be used as a source for fine mapping and introgression of the ToLCNDV resistance into susceptible muskmelon cultivars.

The Resistance of Soybean Variety Heinong 84 to Apple Latent Spherical Virus Is Controlled by Two Genetic Loci.

Apple latent spherical virus (ALSV) is widely used as a virus-induced gene silencing (VIGS) vector for function genome study. However, the application of ALSV to soybeans is limited by the resistance of many varieties. In this study, the genetic locus linked to the resistance of a resistant soybean variety Heinong 84 was mapped by high-throughput sequencing-based bulk segregation analysis (HTS-BSA) using a hybrid population crossed from Heinong 84 and a susceptible variety, Zhonghuang 13. The results showed that the resistance of Heinong 84 to ALSV is controlled by two genetic loci located on chromosomes 2 and 11, respectively. Cleaved amplified polymorphic sequence (CAPS) markers were developed for identification and genotyping. Inheritance and biochemical analyses suggest that the resistance locus on chromosome 2 plays a dominant dose-dependent role, while the other locus contributes a secondary role in resisting ALSV. The resistance locus on chromosome 2 might encode a protein that can directly inhibit viral proliferation, while the secondary resistance locus on chromosome 11 may encode a host factor required for viral proliferation. Together, these data reveal novel insights on the resistance mechanism of Heinong 84 to ALSV, which will benefit the application of ALSV as a VIGS vector.

MYB transcription factors encoded by diversified tandem gene clusters cause varied Morella rubra fruit color.

Chinese bayberry (Morella rubra) is a fruit tree with a remarkable variation in fruit color, ranging from white to dark red as determined by anthocyanin content. In dark red "Biqi" (BQ), red "Dongkui" (DK), pink "Fenhong" (FH), and white "Shuijing" (SJ), we identified an anthocyanin-related MYB transcription factor-encoding gene cluster of four members, i.e. MrMYB1.1, MrMYB1.2, MrMYB1.3, and MrMYB2. Collinear analysis revealed that the MYB tandem cluster may have occurred in a highly conserved region of many eudicot genomes. Two alleles of MrMYB1.1 were observed; MrMYB1.1-1 (MrMYB1.1n) was a full-length allele and homozygous in "BQ", MrMYB1.1-2 (MrMYB1.1d) was a nonfunctional allele with a single base deletion and homozygous in "SJ", and MrMYB1.1n/MrMYB1.1d were heterozygous in "DK" and "FH". In these four cultivars, expression of MrMYB1.1, MrMYB1.2, and MrMYB2 was enhanced during ripening. Both alleles were equally expressed in MrMYB1.1n/MrMYB1.1d heterozygous cultivars as revealed by a cleaved amplified polymorphic sequence marker. Expression of MrMYB1.3 was restricted to some dark red cultivars only. Functional characterization revealed that MrMYB1.1n and MrMYB1.3 can induce anthocyanin accumulation while MrMYB1.1d, MrMYB1.2, and MrMYB2 cannot. DNA-protein interaction assays indicated that MrMYB1.1n and MrMYB1.3 can directly bind to and activate the promoters of anthocyanin-related genes via interaction with a MYC-like basic helix-loop-helix protein MrbHLH1. We concluded that the specific genotype of MrMYB1.1 alleles, as well as the exclusive expression of MrMYB1.3 in some dark red cultivars, contributes to fruit color variation. The study provides insights into the mechanisms for regulation of plant anthocyanin accumulation by MYB tandem clusters.

A 1-bp deletion in the MC04g1399 is highly associated with failure to produce fruit wart in bitter gourd (Momordica charantia L.)

Fruit wart is an important appearance trait influencing consumer preferences of bitter gourd. 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 F2 population consisting of 2 360 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.

Molecular Mapping of Putative Genomic Regions Controlling Fruit and Seed Morphology of Watermelon.

The genetic regulatory basis of qualitative and quantitative phenotypes of watermelon is being investigated in different types of molecular and genetic breeding studies around the world. In this study, biparental F2 mapping populations were developed over two experimental years, and the collected datasets of fruit and seed traits exhibited highly significant correlations. Whole-genome resequencing of comparative parental lines was performed and detected single nucleotide polymorphism (SNP) loci were converted into cleaved amplified polymorphic sequence (CAPS) markers. The screened polymorphic markers were genotyped in segregating populations and two genetic linkage maps were constructed, which covered a total of 2834.28 and 2721.45 centimorgan (cM) genetic lengths, respectively. A total of 22 quantitative trait loci (QTLs) for seven phenotypic traits were mapped; among them, five stable and major-effect QTLs (PC-8-1, SL-9-1, SWi-9-1, SSi-9-1, and SW-6-1) and four minor-effect QTLs (PC-2-1 and PC-2-2; PT-2-1 and PT-2-2; SL-6-1 and SSi-6-2; and SWi-6-1 and SWi-6-2) were observed with 3.77-38.98% PVE. The adjacent QTL markers showed a good fit marker-trait association, and a significant allele-specific contribution was also noticed for genetic inheritance of traits. Further, a total of four candidate genes (Cla97C09G179150, Cla97C09G179350, Cla97C09G180040, and Cla97C09G180100) were spotted in the stable colocalized QTLs of seed size linked traits (SL-9-1 and SWi-9-1) that showed non-synonymous type mutations. The gene expression trends indicated that the seed morphology had been formed in the early developmental stage and showed the genetic regulation of seed shape formation. Hence, we think that our identified QTLs and genes would provide powerful genetic insights for marker-assisted breeding aimed at improving the quality traits of watermelon.

A Novel PCR-Based Functional Marker of Rice Blast Resistance Gene Pi25

Rice blast is arguably the most devastating fungal disease of rice. Utilization of resistance genes to breed resistant cultivars is one of the most economical and environmentally friendly approaches to combat the disease. Pi25, a major resistance gene conferring broad-spectrum resistance to both leaf and neck blast, is an ideal gene resource to improve the resistance of rice varieties to blast. Recently, several allele-specific markers were developed. However, they were deficiently efficient due to either an additional process of restriction enzyme digestion for cleaved amplified polymorphic sequence (CAPS) markers or the risk of false-positive error in identifying susceptible Tetep allele (Pi25TTP) for PCR-based markers. In this study, based on a conserved single nucleotide polymorphism (SNP) between resistant and susceptible alleles, a tetra-primer amplification refractory mutation system (ARMS)-PCR marker was developed. The new marker, namely Pi25-2687R3, could effectively distinguish the resistant Gumei 2 (GM2) allele (Pi25GM2) and the susceptible allele Pi25TTP. Moreover, a perfect consistency of genotyping was exhibited between Pi25-2687R3 and published CAPS marker CAP3/Hpy 99I. A more accurate genotyping was also displayed compared to the previous PCR-based SNP marker Pi25-2566. Our finding proved that Pi25-2687R3 could achieve the same result as CAP3/Hpy 99I with less workload and cost and could promote the accuracy in the identification of genotypes superior to Pi25-2566. This study provided a quick and reliable functional marker for discriminating Pi25 alleles, which would be a valuable tool for genotypic assay and rice molecular breeding of blast resistance.

Construction of a Tomato (Solanum lycopersicum L.) Introgression Line Population and Mapping of Major Agronomic Quantitative Trait Loci

Tomato as a fresh fruit has a large market share in China, but few new materials have been developed for such cultivar breeding in recent years. This study aims to create innovative breeding materials for fresh fruit tomatoes with consistent genetic backgrounds and take advantage of beneficial genes from wild germplasm resources. An introgression line (IL) population was constructed using freshly cultivated tomato S. lycopersicum 1052 and wild tomato S. pennellii LA0716 through hybridization and five consecutive backcrossings, with molecular marker-assisted selection techniques during seedling stages. A total of 447 cleaved amplified polymorphic sequences (CAPS) and 525 simple sequence repeat (SSR) markers were used to screen polymorphic markers among the two parental lines, resulting in 216 polymorphic CAPS and 236 polymorphic SSR markers, with 46.5% parental polymorphism. Then, 200 molecular markers uniformly distributed over the entire genome were further selected, and 107 ILs were finally obtained from 541 BC5 candidate plants. The physical distance between adjacent markers was 6.3~10.0 cm, with an average interval of 7.29 cm, and the IL population constructed covered the whole genome of S. pennillii LA0716, with an average introgression segment of 31.5 cm. Moreover, phenotype data of major agronomic traits in BC5 progeny after selfing two times, were analyzed for quantitative trait locus (QTL) mapping, and a total of 11 QTLs distributed on 6 chromosomes were identified, including 3 QTLs regulating plant height, 1 QTL regulating leaf size, 1 QTL regulating fruit color, 4 QTLs regulating fruit weight, and 2 QTLs regulating soluble solids content in ripening fruits. The IL population constructed in this study provided good materials for fresh fruit tomato breeding with improved yield and quality in the future.

Fine-Mapping of qECL7.1, a Quantitative Trait Locus Contributing to Epicotyl Length in Adzuki Bean (Vigna angularis)

Increasing the epicotyl length (ECL) of adzuki bean cultivars enhances the suitability for mechanical weeding during the vegetative stages and harvesting at pod maturity. To explore the genetic control of ECL, and to identify molecular markers that could facilitate breeding for increased ECL, recombinant inbred lines (RILs) were developed from a cross between Toiku161 (long epicotyls) and Chihayahime (ordinary length epicotyls). In this study, four quantitative trait loci (QTLs) were identified for ECL by QTL-seq analysis, one each on chromosomes 2, 7, 10 and 11. Insertion and deletion (InDel)-based mapping also detected QTLs on chromosomes 7, qECL7.1, and 10, qECL10.1. Substitution mapping using InDel, cleaved amplified polymorphic sequence (CAPS), derived cleaved amplified polymorphic sequence (dCAPS), and single nucleotide polymorphism (SNP) markers narrowed the chromosomal location of qECL7.1 to a 418 kb region flanked by DNA markers TC99_10,211,134 bp and TC102_10,628,880 bp. A total of 35 genes were predicted within the qECL7.1 region. The ECL QTLs and molecular markers identified here will contribute towards marker-assisted selection of desirable long ECL genotypes that allow for increased mechanization and more efficient adzuki bean production.

Construction of a high-density genetic linkage map and QTL mapping for bioenergy-related traits in sweet sorghum [Sorghum bicolor (L.) Moench

Sorghum is an important but arguably undervalued cereal crop, grown in large areas in Asia and Africa due to its natural resilience to drought and heat. There is growing demand for sweet sorghum as a source of bioethanol as well as food and feed. The improvement of bioenergy-related traits directly affects bioethanol production from sweet sorghum; therefore, understanding the genetic basis of these traits would enable new cultivars to be developed for bioenergy production. In order to reveal the genetic architecture behind bioenergy-related traits, we generated an F2 population from a cross between sweet sorghum cv. 'Erdurmus' and grain sorghum cv. 'Ogretmenoglu'. This was used to construct a genetic map from SNPs discovered by double-digest restriction-site associated DNA sequencing (ddRAD-seq). F3 lines derived from each F2 individual were phenotyped for bioenergy-related traits in two different locations and their genotypes were analyzed with the SNPs to identify QTL regions. On chromosomes 1, 7, and 9, three major plant height (PH) QTLs (qPH1.1, qPH7.1, and qPH9.1) were identified, with phenotypic variation explained (PVE) ranging from 10.8 to 34.8%. One major QTL (qPJ6.1) on chromosome 6 was associated with the plant juice trait (PJ) and explained 35.2% of its phenotypic variation. For fresh biomass weight (FBW), four major QTLs (qFBW1.1, qFBW6.1, qFBW7.1, and qFBW9.1) were determined on chromosomes 1, 6, 7, and 9, which explained 12.3, 14.5, 10.6, and 11.9% of the phenotypic variation, respectively. Moreover, two minor QTLs (qBX3.1 and qBX7.1) of Brix (BX) were mapped on chromosomes 3 and 7, explaining 8.6 and 9.7% of the phenotypic variation, respectively. The QTLs in two clusters (qPH7.1/qBX7.1 and qPH7.1/qFBW7.1) overlapped for PH, FBW and BX. The QTL, qFBW6.1, has not been previously reported. In addition, eight SNPs were converted into cleaved amplified polymorphic sequences (CAPS) markers, which can be easily detected by agarose gel electrophoresis. These QTLs and molecular markers can be used for pyramiding and marker-assisted selection studies in sorghum, to develop advanced lines that include desirable bioenergy-related traits.

Dissecting the genetic basis of grain color and pre-harvest sprouting resistance in common wheat by association analysis

Pre-harvest sprouting (PHS) adversely affects wheat quality and yield, and grain color (GC) is associated with PHS resistance. However, the genetic relationship between GC and PHS resistance remains unclear. In this study, 168 wheat varieties (lines) with significant differences in GC and PHS resistance were genotyped using an Illumina 90K iSelect SNP array. Genome-wide association study (GWAS) based on a mixed linear model showed that 67 marker-trait associations (MTAs) assigned to 29 loci, including 17 potentially novel loci, were significantly associated with GC, which explained 1.1–17.0% of the phenotypic variation. In addition, 100 MTAs belonging to 54 loci, including 31 novel loci, were significantly associated with PHS resistance, which accounted for 1.1–14.7% of the phenotypic variation. Subsequently, two cleaved amplified polymorphic sequences (CAPS) markers, 2B-448 on chromosome 2B and 5B-301 on chromosome 5B, were developed from the representative SNPs of the major common loci Qgc.ahau-2B.3/Qphs.ahau-2B.4 controlling GC/PHS resistance and PHS resistance locus Qphs.ahau-5B.4, respectively. Further validation in 171 Chinese mini-core collections confirmed significant correlations of the two CAPS markers with GC and PHS resistance phenotypes under different environments (P<0.05). Furthermore, the wheat public expression database, transcriptomic sequencing, and gene allelic variation analysis identified TraesCS5B02G545100, which encodes glutaredoxin, as a potential candidate gene linked to Qphs.ahau-5B.4. The new CAPS marker CAPS-356 was then developed based on the SNP (T/C) in the coding sequences (CDS) region of TraesCS5B02G545100. The high-density linkage map of the Jing 411/Hongmangchun 21 recombinant inbred lines (RILs) constructed based on specific locus amplified fragment sequencing markers showed that CAPS-356 collocated with a novel QTL for PHS resistance, supporting the role of TraesCS5B02G545100 as the potential candidate gene linked to Qphs.ahau-5B.4. These results provide valuable information for the map-based cloning of Qphs.ahau-5B.4 and breeding of PHS resistant white-grained varieties.

Interaction and association analysis of malting related traits in barley.

Barley is considered as a foundation of the brewing and malting industry. Varieties with superior malt quality traits are required for efficient brewing and distillation processes. Among these, the Diastatic Power (DP), wort-Viscosity (VIS), β-glucan content (BG), Malt Extract (ME) and Alpha-Amylase (AA) are controlled by several genes linked to numerous quantitative trait loci (QTL), identified for barley malting quality. One of the well-known QTL, QTL2, associated with barley malting trait present on chromosome 4H harbours a key gene, called as HvTLP8 that has been identified for influencing the barley malting quality through its interaction with β-glucan in a redox-dependent manner. In this study, we examined to develop a functional molecular marker for HvTLP8 in the selection of superior malting cultivars. We first examined the expression of HvTLP8 and HvTLP17 containing carbohydrate binding domains in barley malt and feed varieties. The higher expression of HvTLP8 prompted us to further investigate its role as a marker for malting trait. By exploring the 1000 bp downstream 3' UTR region of HvTLP8, we found single nucleotide polymorphism (SNP) in between Steptoe (feed variety) and Morex (malt variety), which was further validated by Cleaved Amplified Polymorphic Sequence (CAPS) marker assay. Analysis of 91 individuals from the Steptoe x Morex doubled haploid (DH) mapping population revealed CAPS polymorphism in HvTLP8. Highly significant (p<0.001) correlations among ME, AA and DP malting traits were observed. The correlation coefficient (r) between these traits ranged from 0.53 to 0.65. However, the polymorphism in HvTLP8 did not correlate effectively with ME, AA, and DP. Altogether, these findings will help us to further design the experiment regarding the HvTLP8 variation and its association with other desirable traits.

A rapid method for detection of the root-knot nematode resistance gene, Mi-1.2, in tomato cultivars.

Molecular markers have been widely used in plant breeding to improve the accuracy and efficiency of trait selection. In particular, molecular markers are powerful in facilitating the introgression of resistance genes by circumventing costly and time-consuming infection assays. To achieve their practical use, it is important to ensure the tight linkage between the markers and the traits. Here we report a new cleaved amplified polymorphic sequence (CAPS) marker, Mi1713, for the root-knot nematode resistance gene, Mi-1.2, in cultivated tomato. The Mi1713 marker is designed in the conserved region of Mi-1.2 and its homologs in tomato and other nightshade species. Combined with a single-step procedure for preparing PCR templates, the Mi1713 marker enables rapid and reliable screening for the presence of Mi-1.2. The approach described in this study is applicable in designing CAPS markers for various genes or alleles of interest in tomato and other crops.

A nonsynonymous mutation in an acetolactate synthase gene (Gh_D10G1253) is required for tolerance to imidazolinone herbicides in cotton

BackgroundHerbicide tolerance in crops enables them to survive when lethal doses of herbicides are applied to surrounding weeds. Herbicide-tolerant crops can be developed through transgenic approaches or traditional mutagenesis approaches. At present, no transgenic herbicide tolerant cotton have been commercialized in China due to the genetically-modified organism (GMO) regulation law. We aim to develop a non-transgenic herbicide-tolerant cotton through ethyl methanesulfonate (EMS) mutagenesis, offering an alternative choice for weed management.ResultsSeeds of an elite cotton cultivar Lumianyan 37 (Lu37) were treated with EMS, and a mutant Lu37-1 showed strong tolerance to imidazolinone (IMI) herbicides was identified. A novel nonsynonymous substitution mutation Ser642Asn at acetolactate synthase (ALS) (Gh_D10G1253) in Lu37-1 mutant line was found to be the potential cause to the IMI herbicides tolerance in cotton. The Ser642Asn mutation in ALS did not present among the genomes of natural Gossypium species. Cleaved amplified polymorphic sequence (CAPS) markers were developed to identify the ALS mutant allele. The Arabidopsis overexpressing the mutanted ALS also showed high tolerance to IMI herbicides.ConclusionThe nonsynonymous substitution mutation Ser642Asn of the ALS gene Gh_D10G1253 is a novel identified mutation in cotton. This substitution mutation has also been identified in the orthologous ALS genes in other crops. This mutant ALS allele can be used to develop IMI herbicide-tolerant crops via a non-transgenic or transgenic approach.Graphical abstract

Fine-Mapping Analysis of the Genes Associated with Pre-Harvest Sprouting Tolerance in Rice (Oryza sativa L.)

Pre-harvest sprouting (PHS) of rice (Oryza sativa L.) causes severe economic problems due to reduced grain quality and yield. Fine mapping was carried out to identify genes associated with PHS; the detected quantitative trait locus (QTL) was narrowed down to 50 Kbp using F3:4 populations, four polymorphic insertion and deletion (InDel) markers, and two cleaved amplified polymorphic sequence (CAPS) markers. In one region, five candidate genes were detected, and the SNP and InDel in each gene (Os01g0111400 and Os01g0111600) were confirmed to show the differences and resulting amino acid changes between parent plants. Based on haplotype, expression, and co-segregation analysis, the InDel in Os01g0111600 was confirmed to be associated with the PHS trait. The results of this study could be applied to improve the PHS tolerance of Japonica rice varieties, and they also improved our understanding of the genetic basis underlying PHS tolerance.

Molecular Markers and Their Applications in Marker-Assisted Selection (MAS) in Bread Wheat (Triticum aestivum L.)

As one of the essential cereal crops, wheat provides 20% of the calories and proteins consumed by humans. Due to population expansion, dietary shift and climate change, it is challenging for wheat breeders to develop new varieties for meeting wheat production requirements. Marker-assisted selection (MAS) has distinct advantages over conventional selection in plant breeding, such as being time-saving, cost-effective and goal-oriented. This review makes attempts to give a description of different molecular markers: sequence tagged site (STS), simple sequence repeat (SSR), genotyping by sequencing (GBS), single nucleotide polymorphism (SNP) arrays, exome capture, Kompetitive Allele Specific PCR (KASP), cleaved amplified polymorphic sequence (CAPS), semi-thermal asymmetric reverse PCR (STARP) and genotyping by target sequencing (GBTS). We also summarize some quantitative trait loci (QTL)/genes as well as their linked markers, which are potentially useful in MAS. This paper provides updated information on some markers linked to critical traits and their potential applications in wheat breeding programs.

A LuALS Mutation with High Sulfonylurea Herbicide Resistance in Linum usitatissimum L.

The cultivation of herbicide-resistant crops is an effective tool for weed management in agriculture. Weed control in flax (Linum usitatissimum L.) remains challenging due to the lack of available herbicide-resistant cultivars. In this study, a mutant resistant to acetolactate synthase (ALS)-inhibiting herbicides was obtained by ethyl methanesulphonate (EMS) mutagenesis using an elite cultivar, Longya10. Whole-plant dose-response assays revealed that, compared to Longya10, the mutant was 11.57-fold more resistant to tribenuron-methyl (TBM) and slightly resistant to imazethapyr (resistance index (mutant/Longya10) < 3). In vitro acetolactate synthase assays showed that the relative resistance of the mutant was 12.63 times more than that of Longya10. A biochemical analysis indicated that there was a Pro197Ser (relative to the Arabidopsis thaliana ALS sequence) substitution within the LuALS1, conferring high resistance to sulfonylurea herbicides in the mutant. Additionally, two cleaved amplified polymorphic sequence (CAPS) markers, BsaI-LuALS1 and EcoO109I-LuALS1, were developed based on the mutation site for marker assistant selection in breeding. Moreover, the mutant did not cause losses in natural field conditions. We find a mutant with ALS-inhibiting herbicide resistance chemically induced by EMS mutagenesis, providing a valuable germplasm for breeding herbicide-resistant flax varieties.

Primary mapping of quantitative trait loci regulating multivariate horticultural phenotypes of watermelon (Citrullus lanatus L.).

Watermelon fruits exhibit a remarkable diversity of important horticultural phenotypes. In this study, we initiated a primary quantitative trait loci (QTL) mapping to identify the candidate regions controlling the ovary, fruit, and seed phenotypes. Whole genome sequencing (WGS) was carried out for two differentiated watermelon lines, and 350 Mb (96%) and 354 Mb (97%) of re-sequenced reads covered the reference de novo genome assembly, individually. A total of 45.53% non-synonymous single nucleotide polymorphism (nsSNPs) and 54.47% synonymous SNPs (sSNPs) were spotted, which produced 210 sets of novel SNP-based cleaved amplified polymorphism sequence (CAPS) markers by depicting 46.25% co-dominant polymorphism among parent lines and offspring. A biparental F2:3 mapping population comprised of 100 families was used for trait phenotyping and CAPS genotyping, respectively. The constructed genetic map spanned a total of 2,398.40 centimorgans (cM) in length and averaged 11.42 cM, with 95.99% genome collinearity. A total of 33 QTLs were identified at different genetic positions across the eight chromosomes of watermelon (Chr-01, Chr-02, Chr-04, Chr-05, Chr-06, Chr-07, Chr-10, and Chr-11); among them, eight QTLs of the ovary, sixteen QTLs of the fruit, and nine QTLs of the seed related phenotypes were classified with 5.32-25.99% phenotypic variance explained (PVE). However, twenty-four QTLs were identified as major-effect and nine QTLs were mapped as minor-effect QTLs across the flanking regions of CAPS markers. Some QTLs were exhibited as tightly localized across the nearby genetic regions and explained the pleiotropic effects of multigenic nature. The flanking QTL markers also depicted significant allele specific contributions and accountable genes were predicted for respective traits. Gene Ontology (GO) functional enrichment was categorized in molecular function (MF), cellular components (CC), and biological process (BP); however, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were classified into three main classes of metabolism, genetic information processing, and brite hierarchies. The principal component analysis (PCA) of multivariate phenotypes widely demonstrated the major variability, consistent with the identified QTL regions. In short, we assumed that our identified QTL regions provide valuable genetic insights regarding the watermelon phenotypes and fine genetic mapping could be used to confirm them.