Cloning Mapping Research Articles

Natural variations in the Cis-elements of GhRPRS1 contributing to petal colour diversity in cotton.

The cotton genus comprises both diploid and allotetraploid species, and the diversity in petal colour within this genus offers valuable targets for studying orthologous gene function differentiation and evolution. However, the genetic basis for this diversity in petal colour remains largely unknown. The red petal colour primarily comes from C, G, K, and D genome species, and it is likely that the common ancestor of cotton had red petals. Here, by employing a clone mapping strategy, we mapped the red petal trait to a specific region on chromosome A07 in upland cotton. Genomic comparisons and phylogenetic analyses revealed that the red petal phenotype introgressed from G. bickii. Transcriptome analysis indicated that GhRPRS1, which encodes a glutathione S-transferase, was the causative gene for the red petal colour. Knocking out GhRPRS1 resulted in white petals and the absence of red spots, while overexpression of both genotypes of GhRPRS1 led to red petals. Further analysis suggested that GhRPRS1 played a role in transporting pelargonidin-3-O-glucoside and cyanidin-3-O-glucoside. Promoter activity analysis indicated that variations in the promoter, but not in the gene body of GhRPRS1, have led to different petal colours within the genus. Our findings provide new insights into orthologous gene evolution as well as new strategies for modifying promoters in cotton breeding.

Genetic analysis of yield components in buckwheat using high-throughput sequencing analysis and wild resource populations.

Fagopyrum tataricum, an important medicinal and edible crop, possesses significant agricultural and economic value. However, the development of buckwheat varieties and yields has been hindered by the delayed breeding progress despite the abundant material resources in China. Current research indicates that quantitative trait loci (QTLs) play a crucial role in controlling plant seed type and yield. To address these limitations, this study constructed recombinant inbred lines (RILs) utilizing both cultivated species and wild buckwheat as raw materials. In total, 84,521 Single Nucleotide Polymorphism (SNP) markers were identified through Genotyping-by-Sequencing (GBS) technology, and high-resolution and high-density SNP genetic maps were developed, which had significant value for QTL mapping, gene cloning and comparative mapping of buckwheat. In this study, we successfully identified 5 QTLs related to thousand grain weight (TGW), 9 for grain length (GL), and 1 for grain width (GW) by combining seed type and TGW data from 202 RIL populations in four different environments, within which one co-located QTL for TGW were discovered on the first chromosome. Transcriptome analysis during different grain development stages revealed 59 significant expression differences between the two materials, which can serve as candidate genes for further investigation into the regulation of grain weight and yield enhancement. The mapped major loci controlling TGW, GL and GW will be valuable for gene cloning and reveal the mechanism underlying grain development and marker-assisted selection in Tartary buckwheat.

Spatial Mapping of Hematopoietic Clones in Human Bone Marrow.

CH occurs commonly in humans and can predispose to hematologic malignancy. Although well characterized in blood, it is poorly understood how clones are spatially distributed in the bone marrow. To answer this, we developed methods for spatially aware somatic mutation profiling to describe clonal heterogeneity in human bone marrow. See related commentary by Austin and Aifantis, p. 139.

Genome-wide association study reveals GmFulb as candidate gene for maturity time and reproductive length in soybeans (Glycine max).

The ability of soybean [Glycine max (L.) Merr.] to adapt to different latitudes is attributed to genetic variation in major E genes and quantitative trait loci (QTLs) determining flowering time (R1), maturity (R8), and reproductive length (RL). Fully revealing the genetic basis of R1, R8, and RL in soybeans is necessary to enhance genetic gains in soybean yield improvement. Here, we performed a genome-wide association analysis (GWA) with 31,689 single nucleotide polymorphisms (SNPs) to detect novel loci for R1, R8, and RL using a soybean panel of 329 accessions with the same genotype for three major E genes (e1-as/E2/E3). The studied accessions were grown in nine environments and observed for R1, R8 and RL in all environments. This study identified two stable peaks on Chr 4, simultaneously controlling R8 and RL. In addition, we identified a third peak on Chr 10 controlling R1. Association peaks overlap with previously reported QTLs for R1, R8, and RL. Considering the alternative alleles, significant SNPs caused RL to be two days shorter, R1 two days later and R8 two days earlier, respectively. We identified association peaks acting independently over R1 and R8, suggesting that trait-specific minor effect loci are also involved in controlling R1 and R8. From the 111 genes highly associated with the three peaks detected in this study, we selected six candidate genes as the most likely cause of R1, R8, and RL variation. High correspondence was observed between a modifying variant SNP at position 04:39294836 in GmFulb and an association peak on Chr 4. Further studies using map-based cloning and fine mapping are necessary to elucidate the role of the candidates we identified for soybean maturity and adaptation to different latitudes and to be effectively used in the marker-assisted breeding of cultivars with optimal yield-related traits.

Toward digital twin of the ocean: from digitalization to cloning

The forthcoming wave of progress in oceanographic technology is the digital twin of the ocean, a concept that integrates marine big data and artificial intelligence (AI). This development is a logical consequence of combining data science and marine science and is considered superior to previous models, such as the digital ocean, transparent ocean, and smart ocean. Amid the swift advancement of next-generation information technology, the conditions are favorable for developing a prototype digital twin of the ocean, which will integrate various functionalities—data fusion, situation presentation, phenomenon mining, autonomous learning, and intelligent prediction. The salient distinction between a digital twin of the ocean and traditional forms of virtual or augmented reality is because of the intelligence beyond digitalization exhibited by the former, primarily facilitated by AI-based cloning. Hence, herein, we initially propose a structured architecture for the generative digital twin ocean, encompassing elements from real-time data pools to key technologies and proof-of-concept applications. The core components of this prototype system include a data pool, an AI-based oceanographic model, and three-dimensional visualization interactions. Future research and objectives for the digital twin ocean will principally focus on the following: four-dimensional (comprising three-dimensional space along with time) digital cloning and real-time mapping of global ocean parameters, cooperative observation coupled with human–computer interactions, and intelligent prediction along with cutting-edge applications. Prospectively, this transformative technology holds the potential to considerably enhance our understanding of the ocean, yielding groundbreaking discoveries that will profoundly influence the marine economy and sustainable development.

A refinement of Reznick's Positivstellensatz with applications to quantum information theory

In his solution of Hilbert's 17th problem Artin showed that any positive definite polynomial in several variables can be written as the quotient of two sums of squares. Later Reznick showed that the denominator in Artin's result can always be chosen as an N-th power of the squared norm of the variables and gave explicit bounds on N. By using concepts from quantum information theory (such as partial traces, optimal cloning maps, and an identity due to Chiribella) we give simpler proofs and minor improvements of both real and complex versions of this result. Moreover, we discuss constructions of Hilbert identities using Gaussian integrals and we review an elementary method to construct complex spherical designs. Finally, we apply our results to give improved bounds for exponential quantum de Finetti theorems in the real and in the complex setting.

Identification of QTL underlying the main stem related traits in a doubled haploid barley population.

Lodging reduces grain yield in cereal crops. The height, diameter and strength of stem are crucial for lodging resistance, grain yield, and photosynthate transport in barley. Understanding the genetic basis of stem benefits barley breeding. Here, we evaluated 13 stem related traits after 28 days of heading in a barley DH population in two consecutive years. Significant phenotypic correlations between lodging index (LI) and other stem traits were observed. Three mapping methods using the experimental data and the BLUP data, detected 27 stable and major QTLs, and 22 QTL clustered regions. Many QTLs were consistent with previously reported traits for grain filling rate, internodes, panicle and lodging resistance. Further, candidate genes were predicted for stable and major QTLs and were associated with plant development and adverse stress in the transition from vegetative stage to reproductive stage. This study provided potential genetic basis and new information for exploring barley stem morphology, and laid a foundation for map-based cloning and further fine mapping of these QTLs.

Process-optimized phase-covariant quantum cloning

After the appearance of the no-cloning theorem, approximate quantum cloning machines (QCMs) has become a well-studied subject in quantum information theory. Among several measures to quantify the performance of a QCM, single-qudit fidelity and global fidelity have been most widely used. In this paper we compute the optimal global fidelity for phase-covariant cloning machines via semidefinite programming optimization, thereby completing a remaining gap in the previous results on QCMs. We also consider optimal simulations of the cloning and transpose cloning map, both by a direct optimization and by a composition of component-wise optimal QCMs. For the cloning map the composition method is suboptimal whereas for the transpose cloning map the method is asymptotically optimal.

A gene regulatory network for tiller development mediated by Tin8 in maize.

The complex gene regulatory network underlying tiller development in maize remains largely unknown. Here we identified two major quantitative trait loci for tiller number, Tin8 on chromosome 8 and the previously known Tb1 on chromosome 1, in a population derived from a teosinte-maize cross. Map-based cloning and association mapping revealed that Tin8, corresponding to Zcn8 encoding a phosphatidylethanolamine-binding-related kinase, is down-regulated in transcription, which results in decreased tiller number. A strong interaction between Tin8 and the key gen Tb1 was detected for tiller number. Further RNA-seq analysis showed that the expression of 13 genes related to tiller development was controlled by Tin8. Our results support the existence of a complex gene regulatory network for the outgrowth of the tiller bud in maize, in which Zcn8 controls 13 tiller-related genes, including four genes for hormonal responses. In particular, Zcn8 represses Gt1, D14, and Tru1 through the interaction with Tb1.

A growing genetic tree in the soil of prostate.

In this issue of Cell Stem Cell, Grossmann etal. (2021) reconstructed the developmental history of the prostate gland in a 59-year-old male by 3D tracking of somatic mutations across 319 micro-dissected specimens. It provides a genetic picture of how the human prostate gland is shaped during embryogenesis, puberty, and adult life.

A Large Transposon Insertion in the stiff1 Promoter Increases Stalk Strength in Maize.

Stalk lodging, which is generally determined by stalk strength, results in considerable yield loss and has become a primary threat to maize (Zea mays) yield under high-density planting. However, the molecular genetic basis of maize stalk strength remains unclear, and improvement methods remain inefficient. Here, we combined map-based cloning and association mapping and identified the gene stiff1 underlying a major quantitative trait locus for stalk strength in maize. A 27.2-kb transposable element insertion was present in the promoter of the stiff1 gene, which encodes an F-box domain protein. This transposable element insertion repressed the transcription of stiff1, leading to the increased cellulose and lignin contents in the cell wall and consequently greater stalk strength. Furthermore, a precisely edited allele of stiff1 generated through the CRISPR/Cas9 system resulted in plants with a stronger stalk than the unedited control. Nucleotide diversity analysis revealed that the promoter of stiff1 was under strong selection in the maize stiff-stalk group. Our cloning of stiff1 reveals a case in which a transposable element played an important role in maize improvement. The identification of stiff1 and our edited stiff1 allele pave the way for efficient improvement of maize stalk strength.

Krn1, a major quantitative trait locus for kernel row number in maize.

Kernel row number is a fundamental component of maize (Zea mays) yield and an important target for maize breeding. The revolutionary transition from the two-rowed teosinte to maize with increased kernel row numbers dramatically enhanced yields during domestication. Kernel row number is controlled by many quantitative trait loci (QTLs), however most genes responsible for these QTLs remain uncharacterised and the molecular genetic mechanisms are unknown. Here, we combined map-based cloning and association mapping to identify a major QTL for kernel row number, krn1, which is likely to correspond to an existing gene (ids1/Ts6) encoding an AP2 domain protein homologous to the product of the wheat key domestication gene Q. The increased expression of ids1/Ts6 in two maize mutants increased spikelet pair meristem numbers and then enhanced kernel row numbers. Nucleotide diversity analysis further revealed that ids1/Ts6 and Q were under strong parallel selection in maize and wheat that increased their yields during domestication or improvement. RNA-seq revealed that ids1/Ts6 is involved in multiple pathways regulating spikelet pair meristem development, involving several key genes such as fea3, fea4 and ra3. The cloning of the krn1 gene will pave a new way to efficiently improve maize yield in the near future.

Accessing a Russian Wheat Aphid Resistance Gene in Bread Wheat by Long-Read Technologies.

Russian wheat aphid (RWA) ( Kurdjumov) is a serious invasive pest of small-grain cereals and many grass species. An efficient strategy to defy aphid attacks is to identify sources of natural resistance and transfer resistance genes into susceptible crop cultivars. Revealing the genes helps understand plant defense mechanisms and engineer plants with durable resistance to the pest. To date, more than 15 RWA resistance genes have been identified in wheat ( L.) but none of them has been cloned. Previously, we genetically mapped the RWA resistance gene into an interval of 0.83 cM on the short arm of chromosome 7D and spanned it with five bacterial artificial chromosome (BAC) clones. Here, we used a targeted strategy combining traditional approaches toward gene cloning (genetic mapping and sequencing of BAC clones) with novel technologies, including optical mapping and long-read nanopore sequencing. The latter, with reads spanning the entire length of a BAC insert, enabled us to assemble the whole region, a task that was not achievable with short reads. Long-read optical mapping validated the DNA sequence in the interval and revealed a difference in the locus organization between resistant and susceptible genotypes. The complete and accurate sequence of the region facilitated the identification of new markers and precise annotation of the interval, revealing six high-confidence genes. Identification of as the most likely candidate opens an avenue for its validation through functional genomics approaches.

Molecular marker development and primary physical map construction for the tuber shape Ro gene locus in diploid potato (Solanum tuberosum L.)

Potato (Solanum tuberosum L.) is an important crop worldwide that is consumed fresh and processed in many different ways. The tuber shape is a critical trait in potato breeding, because it directly affects processing quality. Previous studies have demonstrated that the Ro locus on chromosome X is the major locus controlling tuber shape, and the round shape allele, Ro, is dominant over the long shape allele, ro. However, no reports on the cloning or physical mapping of this locus have been published. In this study, whole genome sequencing and bacterial artificial chromosome (BAC) library construction were applied to develop molecular markers and construct a physical map of Ro. Eight round-tuber genotypes were used to construct the BAC library, which contains about 40,000 clones with an average insert size of 85 kb and represents approximately four haploid potato genomes. A total of 66 individuals with extreme round or long tubers were selected to construct a round-tuber shape DNA bulk and a long-tuber shape DNA bulk for WGS. Single-nucleotide polymorphisms (SNPs) identified from WGS data were used to develop nine polymorphic markers linked to Ro. These markers were used to screen the BAC library, and eight positive BAC clones were ultimately identified and then analyzed by restriction enzyme digestion and BAC end sequencing and used to develop markers and identify new positive BAC clones. Finally, a primary physical map of Ro was constructed using a chromosome walking strategy. This map provides an important reference for cloning the potato tuber shape gene and studying tuber shape variation.

New Approaches for Genome Assembly and Scaffolding.

Affordable, high-throughput DNA sequencing has accelerated the pace of genome assembly over the past decade. Genome assemblies from high-throughput, short-read sequencing, however, are often not as contiguous as the first generation of genome assemblies. Whereas early genome assembly projects were often aided by clone maps or other mapping data, many current assembly projects forego these scaffolding data and only assemble genomes into smaller segments. Recently, new technologies have been invented that allow chromosome-scale assembly at a lower cost and faster speed than traditional methods. Here, we give an overview of the problem of chromosome-scale assembly and traditional methods for tackling this problem. We then review new technologies for chromosome-scale assembly and recent genome projects that used these technologies to create highly contiguous genome assemblies at low cost.

Erysimum cheiranthoides, an ecological research system with potential as a genetic and genomic model for studying cardiac glycoside biosynthesis

At least twelve plant families contain species that synthesize cardiac glycosides as defense against herbivory. These inhibitors of animal Na+, K+-ATPases also have medical uses in treating congestive heart failure and other diseases. However, despite extensive ecological research and centuries of use in both traditional and modern medicine, the complete cardiac glycoside biosynthesis pathway has yet to be elucidated in any plant species. To a large extent, this research deficit results from the fact that cardiac glycosides are produced exclusively by non-model plant species such as Digitalis that have not been amenable to the development of mutagenesis, cloning, and genetic mapping approaches. Recent advances in genome sequencing, transcript profiling, plant transformation, transient expression assays, and plant metabolite analysis have provided new opportunities for the investigation and elucidation of cardiac glycoside biosynthesis pathways. The genetic tools that have been developed for Brassicaceae, in particular Arabidopsis thaliana, may be directly applicable to Erysimum, a Brassicaceae genus that characteristically produces cardiac glycosides as defensive metabolites. We propose that Erysimum cheiranthoides (wormseed wallflower), a rapid-cycling, self-pollinating species with a relatively small, diploid genome, would be a suitable model system to advance research on the biosynthesis of cardiac glycosides in plants.

ZmCCT9 enhances maize adaptation to higher latitudes

From its tropical origin in southwestern Mexico, maize spread over a wide latitudinal cline in the Americas. This feat defies the rule that crops are inhibited from spreading easily across latitudes. How the widespread latitudinal adaptation of maize was accomplished is largely unknown. Through positional cloning and association mapping, we resolved a flowering-time quantitative trait locus to a Harbinger-like transposable element positioned 57 kb upstream of a CCT transcription factor (ZmCCT9). The Harbinger-like element acts in cis to repress ZmCCT9 expression to promote flowering under long days. Knockout of ZmCCT9 by CRISPR/Cas9 causes early flowering under long days. ZmCCT9 is diurnally regulated and negatively regulates the expression of the florigen ZCN8, thereby resulting in late flowering under long days. Population genetics analyses revealed that the Harbinger-like transposon insertion at ZmCCT9 and the CACTA-like transposon insertion at another CCT paralog, ZmCCT10, arose sequentially following domestication and were targeted by selection for maize adaptation to higher latitudes. Our findings help explain how the dynamic maize genome with abundant transposon activity enabled maize to adapt over 90° of latitude during the pre-Columbian era.

Universal asymmetric quantum cloning revisited

This paper revisits the universal asymmetric 1 → 2 quantum cloning problem. We identify the symmetry properties of this optimization problem, giving us access to the optimal quantum cloning map. Furthermore, we use the bipolar theorem, a famous method from convex analysis, to completely characterize the set of achievable single quantum clone qualities using the fidelity as our figure of merit; from this it is easier to give the optimal cloning map and to quantify the quality tradeoff in universal asymmetric quantum cloning. Additionally, it allows us to analytically specify the set of achievable single quantum clone qualities using a range of different figures of merit.

PBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping.

Applications that use Bacterial Artificial Chromosome (BAC) libraries often require paired-end sequences and knowledge of the physical location of each clone in plates. To facilitate obtaining this information in high-throughput, we generated pBACode vectors: a pool of BAC cloning vectors, each with a pair of random barcodes flanking its cloning site. In a pBACode BAC library, the BAC ends and their linked barcodes can be sequenced in bulk. Barcode pairs are determined by sequencing the empty pBACode vectors, which allows BAC ends to be paired according to their barcodes. For physical clone mapping, the barcodes are used as unique markers for their linked genomic sequence. After multi-dimensional pooling of BAC clones, the barcodes are sequenced and deconvoluted to locate each clone. We generated a pBACode library of 94,464 clones for the flounder Paralichthys olivaceus and obtained paired-end sequence from 95.4% of the clones. Incorporating BAC paired-ends into the genome preassembly improved its continuity by over 10-fold. Furthermore, we were able to use the barcodes to map the physical locations of each clone in just 50 pools, with up to 11 808 clones per pool. Our physical clone mapping located 90.2% of BAC clones, enabling targeted characterization of chromosomal rearrangements.

Development of a high-density genetic linkage map and identification of flowering time QTLs in adzuki bean (Vigna angularis).

A high-density linkage map is crucial for the identification of quantitative trait loci (QTLs), positional cloning, and physical map assembly. Here, we report the development of a high-density linkage map based on specific length amplified fragment sequencing (SLAF-seq) for adzuki bean and the identification of flowering time-related QTLs. Through SLAF library construction and Illumina sequencing of a recombinant inbred line (RIL) population, a total of 4425 SLAF markers were developed and assigned to 11 linkage groups (LGs). After binning the SLAF markers that represented the same genotype, the final linkage map of 1628.15 cM contained 2032 markers, with an average marker density of 0.80 cM. Comparative analysis showed high collinearity with two adzuki bean physical maps and a high degree of synteny with the reference genome of common bean (Phaseolus vulgaris). Using this map, one major QTL on LG03 and two minor QTLs on LG05 associated with first flowering time (FLD) were consistently identified in tests over a two-year period. These results provide a foundation that will be useful for future genomic research, such as identifying QTLs for other important traits, positional cloning, and comparative mapping in legumes.