Chromosome Library Research Articles

Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli.

Chloroplast genomes present an alternative strategy for large-scale engineering of photosynthetic eukaryotes. Prior to our work, the chloroplast genomes of Chlamydomonas reinhardtii (204 kb) and Zea mays (140 kb) had been cloned using bacterial and yeast artificial chromosome (BAC/YAC) libraries, respectively. These methods lack design flexibility as they are reliant upon the random capture of genomic fragments during BAC/YAC library creation; additionally, both demonstrated a low efficiency (≤ 10%) for correct assembly of the genome in yeast. With this in mind, we sought to create a highly flexible and efficient approach for assembling the 117 kb chloroplast genome of Phaeodactylum tricornutum, a photosynthetic marine diatom. Our original article demonstrated a PCR-based approach for cloning the P. tricornutum chloroplast genome that had 90%-100% efficiency when screening as few as 10 yeast colonies following assembly. In this article, we will discuss this approach in greater depth as we believe this technique could be extrapolated to other species, particularly those with a similar chloroplast genome size and architecture. Key features • Large fragments of the chloroplast genome can be readily amplified through PCR from total algal DNA isolate. • Assembly protocol can be completed within a day, and yeast colonies harboring chloroplast genomes can be obtained in as few as 4-5 days. • Cloned genomes isolated from yeast transformants can be moved to Escherichia coli through electroporation.

Identifying candidate genes for sugar accumulation in sugarcane: an integrative approach

BackgroundElucidating the intricacies of the sugarcane genome is essential for breeding superior cultivars. This economically important crop originates from hybridizations of highly polyploid Saccharum species. However, the large size (10 Gb), high degree of polyploidy, and aneuploidy of the sugarcane genome pose significant challenges to complete genome sequencing, assembly, and annotation. One successful strategy for identifying candidate genes linked to agronomic traits, particularly those associated with sugar accumulation, leverages synteny and potential collinearity with related species.ResultsIn this study, we explored synteny between sorghum and sugarcane. Genes from a sorghum Brix QTL were used to screen bacterial artificial chromosome (BAC) libraries from two Brazilian sugarcane varieties (IACSP93-3046 and SP80-3280). The entire region was successfully recovered, confirming synteny and collinearity between the species. Manual annotation identified 51 genes in the hybrid varieties that were subsequently confirmed to be present in Saccharum spontaneum. This study employed a multifaceted approach to identify candidate genes for sugar accumulation, including retrieving the genomic region of interest, performing a gene-by-gene analysis, analyzing RNA-seq data for internodes from Saccharumofficinarum and S. spontaneum accessions, constructing a coexpression network to examine the expression patterns of genes within the studied region and their neighbors, and finally identifying differentially expressed genes (DEGs).ConclusionsThis comprehensive approach led to the discovery of three candidate genes potentially involved in sugar accumulation: an ethylene-responsive transcription factor (ERF), an ABA 8’-hydroxylase, and a prolyl oligopeptidase (POP). These findings could be valuable for identifying additional candidate genes for other important agricultural traits and directly targeting candidate genes for further work in molecular breeding.

Identification and functional characterization of the sugarcane (Saccharum spp.) AMT2-type ammonium transporter ScAMT3;3 revealed a presumed role in shoot ammonium remobilization

Sugarcane (Saccharum spp.) is an important crop for sugar and bioethanol production worldwide. To maintain and increase sugarcane yields in marginal areas, the use of nitrogen (N) fertilizers is essential, but N overuse may result in the leaching of reactive N to the natural environment. Despite the importance of N in sugarcane production, little is known about the molecular mechanisms involved in N homeostasis in this crop, particularly regarding ammonium (NH4+), the sugarcane’s preferred source of N. Here, using a sugarcane bacterial artificial chromosome (BAC) library and a series of in silico analyses, we identified an AMMONIUM TRANSPORTER (AMT) from the AMT2 subfamily, sugarcane AMMONIUM TRANSPORTER 3;3 (ScAMT3;3), which is constitutively and highly expressed in young and mature leaves. To characterize its biochemical function, we ectopically expressed ScAMT3;3 in heterologous systems (Saccharomyces cerevisiae and Arabidopsis thaliana). The complementation of triple mep mutant yeast demonstrated that ScAMT3;3 is functional for NH3/H+ cotransport at high availability of NH4+ and under physiological pH conditions. The ectopic expression of ScAMT3;3 in the Arabidopsis quadruple AMT knockout mutant restored the transport capacity of 15N–NH4+ in roots and plant growth under specific N availability conditions, confirming the role of ScAMT3;3 in NH4+ transport in planta. Our results indicate that ScAMT3;3 belongs to the low-affinity transport system (Km 270.9 µM; Vmax 209.3 µmol g−1 root DW h−1). We were able to infer that ScAMT3;3 plays a presumed role in NH4+ source–sink remobilization in the shoots via phloem loading. These findings help to shed light on the functionality of a novel AMT2-type protein and provide bases for future research focusing on the improvement of sugarcane yield and N use efficiency.

Abstract 5320: The RenMice™ HiTS (Hyperimmune target specific) Platform facilitates identification of novel therapeutic antibodies for challenging targets

Abstract In recent years, an increasing number of therapeutic antibodies have shown to be effective for the treatment of cancer and other diseases. However, limitations in the traditional discovery process, including immune tolerance of highly homologous genes, challenges with antibody sequence humanization, clone selection, and model selection for drug efficacy and safety evaluation, often hinder the process of identifying new therapeutic antibodies. The RenMice™ HiTS (Hyperimmune Target Specific) Platform is a library of chromosome engineered mice with fully human immunoglobulin variable domains replacing the mouse loci, each with a specific drug target gene knocked out. These mice are designed to establish robust immune responses and generate antibodies that bind to more epitopes of the target protein, including conserved domains. The platform is ideal for challenging targets, such as proteins with high homology across species, or multi-pass transmembrane proteins (e.g. GPCRs/ion channels). Here, we show that the platform can be used to generate antibodies that cross-react with multiple species, like human, monkey, dog, and mouse targets, by immunizing with both human and mouse or dog antigen. We provide examples for newer campaigns, including species cross-reactivity and internalization of novel antibodies targeting NECTIN-4, and high-throughput in vivo efficacy screening of novel anti-PD-1 antibodies in wild-type mice. In the future, we will evaluate the preliminary toxicity of these cross-reactive antibodies in preclinical animal models. Thus, selection of the best antibody candidate based on in vivo efficacy and safety allows for a streamlined and successful preclinical phase. In conclusion, the RenMice™ HiTS platform facilitates the generation of developable antibodies that recognize novel epitopes and challenging targets. Citation Format: Xiaoqian Zhang, Shufang Fu, Shujin Zhang, Xin Ji, Li Hui, James Jin, Jing Zhang. The RenMice™ HiTS (Hyperimmune target specific) Platform facilitates identification of novel therapeutic antibodies for challenging targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5320.

Major histocompatibility complex genomic investigation of endangered Chinese alligator provides insights into the evolution of tetrapod major histocompatibility complex and survival of critically bottlenecked species

BackgroundThe major histocompatibility complex (MHC) gene family, a vital immune gene family in vertebrates, helps animals defend against pathogens. The polymorphism of MHC genes is important for a species and is considered to be caused by the numerous alleles of MHC antigen-presenting genes. However, the mechanism of this process is unclear due to the lack of data on the MHC structure. The evolutionary trajectories of the tetrapod MHC are also unclear because of insufficient studies on the reptile MHC architecture. Here, we studied the Chinese alligator (Alligator sinensis), which experienced a population bottleneck, but the population increased rapidly in the past 30 years and is proposed to have a unique MHC system to face pathogenic challenges.ResultsWe successfully constructed a 2 Mb MHC region using bacterial artificial chromosome (BAC) library and genome data of the Chinese alligator and checked the antigen-presenting genes using transcriptome data and the rapid amplification of cDNA ends (RACE) technique. The MHC architecture reported here uncovers adjacent Class I and Class II subregions and a unique CD1 subregion. This newly added information suggested that the Class I-II structure pattern was more ancient in tetrapods and helped reconstruct the evolution of the MHC region architecture. We also found multiple groups of MHC class I (MHC-I) (12 duplicated loci, belonging to three groups, two of which were novel) and MHC class II (MHC-II) (11 duplicated loci, belonging to two groups) inside the 2 Mb MHC region, and there were three more duplicated MHC-I loci outside it. These highly duplicated antigen-presenting loci had differences in expression, amino acid length of antigen-presenting exons, and splice signal of exon and intron, which together promoted the polymorphism of duplicated genes. Although the MHC antigen-presenting genes were identified as monomorphic or oligomorphic in our previous population study, the loci with high copy numbers and many differences can make up for this loss, presenting another mechanism for polymorphism in antigen presentation. These MHC-I and MHC-IIB loci with low polymorphism for each locus, but high numbers in all, may also contribute to MHC antigen-presenting binding variability in a population.ConclusionTo summarize, the fine MHC region architecture of reptiles presented in this study completes the evolutionary trajectories of the MHC structure in tetrapods, and these distinctive MHC gene groups in the Chinese alligator may have helped this species to expand rapidly in the past recent years.

Identification of sex-specific markers and ZW-chromosome DNA clones from the genomic BAC library of the Chinese mitten crab Eriocheir sinensis

A ZW-ZZ sex determination system was previously proposed based on sex linkage analysis in the Chinese mitten crab, Eriocheir sinensis. In this study, we successfully identified a sex-specific marker from sex-linked markers in the genetic map and verified in different wild populations of E. sinensis. To further identify ZW chromosome DNA, we constructed a highly redundant genomic bacterial artificial chromosome (BAC) library for the Chinese mitten crab. The library was generated from high-molecular weight genomic DNA isolated from hemocytes and muscle in a female crab, which was comprising of 153,984 clones arrayed in 401,384-well microtiter plates. The average inserts size is 72 kb, representing 8.7-fold haploid genome coverage. By using sex specific markers, PCR-based screening was performed to identify ZW-derived BAC clones. Three W and Z-derived BAC clones were isolated, respectively. One of W-derived (P282E18) BAC clones was sequenced and assembled into a 43 kb contig without any gap. The W-derived BAC inserts can be mapped to an assembled contig of E. sinensis genome with 95% similarity in the aligned region, demonstrating that the BAC library is a valuable resource for discriminating Z and W chromosome contigs in the assembled haploid genome. The sex-specific marker provides a powerful tool not only for distinguishing the gender of larval or early juvenile E. sinensis without sexual dimorphism and sex-reversed individuals in monosex crab breeding, but also for identification of ZW-derived BAC clones used in the assembly of Z and W chromosomes in E. sinensis.

Expanding duplication of the testis PHD Finger Protein 7 (PHF7) gene in the chicken genome

Gene duplications increase genetic and phenotypic diversity and occur in complex genomic regions that are still difficult to sequence and assemble. PHD Finger Protein 7 (PHF7) acts during spermiogenesis for histone-to-histone protamine exchange and is a determinant of male fertility in Drosophila and the mouse. We aimed to explore and characterise in the chicken genome the expanding family of the numerous orthologues of the unique mouse Phf7 gene (highly expressed in the testis), observing the fact that this information is unclear and/or variable according to the versions of databases. We validated nine primer pairs by in silico PCR for their use in screening the chicken bacterial artificial chromosome (BAC) library to produce BAC-derived probes to detect and localise PHF7-like loci by fluorescence in situ hybridisation (FISH). We selected nine BAC that highlighted nine chromosomal regions for a total of 10 distinct PHF7-like loci on five Gallus gallus chromosomes: Chr1 (three loci), Chr2 (two loci), Chr12 (one locus), Chr19 (one locus) and ChrZ (three loci). We sequenced the corresponding BAC by using high-performance PacBio technology. After assembly, we performed annotation with the FGENESH program: there were a total of 116 peptides, including 39 PHF7-like proteins identified by BLASTP. These proteins share a common exon-intron core structure of 8–11 exons. Phylogeny revealed that the duplications occurred first between chromosomal regions and then inside each region. There are other duplicated genes in the identified BAC sequences, suggesting that these genomic regions exhibit a high rate of tandem duplication. We showed that the PHF7 gene, which is highly expressed in the rooster testis, is a highly duplicated gene family in the chicken genome, and this phenomenon probably concerns other bird species.

Streptomyces BAC Cloning of a Large-Sized Biosynthetic Gene Cluster of NPP B1, a Potential SARS-CoV-2 RdRp Inhibitor.

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

Activating cryptic biosynthetic gene cluster through a CRISPR-Cas12a-mediated direct cloning approach.

Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.

Exploring the Cunninghamia lanceolata(Lamb.) Hook Genome by BAC Sequencing.

Cunninghamia lanceolata (Lamb.)Hook is an important economic timber tree in China. However, its genome characteristics have not been extensively assessed. To better understand its genome information, the bacterial artificial chromosome (BAC) library of chinese fir was constructed. A total of 422 BAC clones were selected and divided into 10 pools and sequenced, and with an average insert size of 121 kb, ranging from 97 to 145 kb. A total of 61,902,523 bp of reference sequences were sequenced and assembled, and based on an estimated genome size of 11.6 Gb for Chinese fir, the BAC library was estimated to have a total coverage of 0.53% genome equivalents. Bioinformatics analyses were also performed for repeated sequences, tRNAs, coding gene prediction, and functional annotation. The results of this study provide insights into the brief structure of the Chinese fir genome and has generated gene data that will facilitate molecular investigations on the mechanisms underlying tree growth.

NLR1 is a strong candidate for the Rm3 dominant green peach aphid (Myzus persicae) resistance trait in peach.

The green peach aphid (GPA), Myzus persicae, is a polyphagous, sap-sucking aphid and a vector of many plant viruses. In peach, Prunus persica, three individual dominant GPA resistance loci have been genetically defined (Rm1-3), but knowledge of the underlying genes is limited. In this study, we focused on the Rm3 locus. Bulk segregant analysis (BSA) mapping in segregating progeny populations delimited Rm3 to an interval spanning 160kb containing 21 genes on chromosome 1. RNA-seq data provided no evidence of candidate genes, but chromosomal structural variations were predicted around a nucleotide-binding site-leucine-rich repeat (NLR) gene (ppa000596m) within the Rm3 fine-mapping interval. Following bacterial artificial chromosome (BAC) library construction for a GPA-resistant peach cultivar and the sequencing of three target BAC clones, a chromosomal structural variation encompassing two novel TIR-NLR-class disease resistance (R) protein-coding genes was identified, and the expressed NLR gene (NLR1) was identified as a candidate for M. persicae resistance. Consistent with its proposed role in controlling GPA resistance, NLR1 was only expressed in the leaves of resistant peach phenotypes. A molecular marker that was designed based on the NLR1 sequence co-segregated with the GPA-resistant phenotype in four segregating populations, 162 peach cultivars, and 14 wild relatives, demonstrating the dominant inheritance of the Rm3 locus. Our findings can be exploited to facilitate future breeding for GPA-resistance in peach.

Identification of Streptococcus thermophilus Genes Specifically Expressed under Simulated Human Digestive Conditions Using R-IVET Technology.

Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy industry, requires further documentation of its physiological status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library were designed to positively select activated genes. Second, recombinant clones were introduced into complementary models mimicking the human gut, the Netherlands Organization for Applied Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine, the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human feces as a colon model. All inserts of activated clones displayed a promoter activity that differed from one digestive condition to another. Our results also showed that S. thermophilus adapted its metabolism to stressful conditions found in the gastric and colonic competitive environment and modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses, a first stage in the identification of gut resistance markers and a key step in probiotic selection.

Genomic Characterization of the Fruity Aroma Gene, FaFAD1, Reveals a Gene Dosage Effect on γ-Decalactone Production in Strawberry (Fragaria × ananassa).

Strawberries produce numerous volatile compounds that contribute to the unique flavors of fruits. Among the many volatiles, γ-decalactone (γ-D) has the greatest contribution to the characteristic fruity aroma in strawberry fruit. The presence or absence of γ-D is controlled by a single locus, FaFAD1. However, this locus has not yet been systematically characterized in the octoploid strawberry genome. It has also been reported that the volatile content greatly varies among the strawberry varieties possessing FaFAD1, suggesting that another genetic factor could be responsible for the different levels of γ-D in fruit. In this study, we explored the genomic structure of FaFAD1 and determined the allele dosage of FaFAD1 that regulates variations of γ-D production in cultivated octoploid strawberry. The genome-wide association studies confirmed the major locus FaFAD1 that regulates the γ-D production in cultivated strawberry. With the hybrid capture-based next-generation sequencing analysis, a major presence–absence variation of FaFAD1 was discovered among γ-D producers and non-producers. To explore the genomic structure of FaFAD1 in the octoploid strawberry, three bacterial artificial chromosome (BAC) libraries were developed. A deletion of 8,262 bp was consistently found in the FaFAD1 region of γ-D non-producing varieties. With the newly developed InDel-based codominant marker genotyping, along with γ-D metabolite profiling data, we revealed the impact of gene dosage effect for the production of γ-D in the octoploid strawberry varieties. Altogether, this study provides systematic information of the prominent role of FaFAD1 presence and absence polymorphism in producing γ-D and proposes that both alleles of FaFAD1 are required to produce the highest content of fruity aroma in strawberry fruit.

Identification of satellited markers by microdissection and fluorescence in situ hybridization: a clinical case of isodicentric chromosome 22

BackgroundThe presence of small supernumerary marker chromosomes (sSMCs) in a karyotype leads to diagnostic questions because the resulting extra material may cause abnormal development depending on the origin of the duplication/triplication. Because SMCs are so small, their origin cannot be determined by conventional cytogenetic techniques, and new molecular cytogenetic methods are necessary. Here, we applied a target approach to chromosome rearrangement analysis by isolating a chromosome of interest via microdissection and using it in fluorescence in situ hybridization (FISH) as a probe in combination with whole-chromosome painting probes. This approach allows to identify origins of both the euchromatin and repeat-rich regions of a marker.Case presentationWe report a case of an adult male with congenital atresia of the rectum and anus, anotia, and atresia of the external auditory canal along with hearing loss. Karyotyping and FISH analysis with whole-chromosome painting probes of acrocentric chromosomes and the constructed microdissection library of the marker chromosome reliably identified an additional chromosome in some metaphases: mos 47,XY,+idic(22)(q11.2)[14]/46,XY [23].ConclusionWe propose to use whole-chromosome libraries and microdissected chromosomes in FISH to identify SMCs enriched with repeated sequences. We show that the methodology is successful in identifying the composition of a satellited marker chromosome.

The Japanese Wild-Derived Inbred Mouse Strain, MSM/Ms in Cancer Research.

Simple SummarySince human studies on tumor susceptibility alleles require vast numbers of DNA samples from both cancer patients and well-matched controls, their investigation and identification in humans have been complemented using mouse models. However, a number of confounding factors are associated with this type of research, including heterogeneity, weak genetic interactions, and lifestyle habits. Inbred strains relatively recently established from wild mice are often more resistant to carcinogenic stimulation and various pathogens than standard inbred mouse strains. A Japanese wild-derived inbred mouse strain, MSM/Ms has been used to map tumor resistance loci as well as other Quantitative Trait Loci (QTL) in Japan. Furthermore, genetic tools have been developed with MSM/Ms. MSM/Ms genomic sequences are currently available, which have greatly promoted the identification of tumor resistance loci as well as genes controlling quantitative variations and provide a more detailed understanding of gene function. MSM/Ms is a unique inbred mouse strain derived from the Japanese wild mouse, Mus musculus molossinus, which has been approximately 1 million years genetically distant from standard inbred mouse strains mainly derived from M. m. domesticus. Due to its genetic divergence, MSM/Ms has been broadly used in linkage studies. A bacterial artificial chromosome (BAC) library was constructed for the MSM/Ms genome, and sequence analysis of the MSM/Ms genome showed approximately 1% of nucleotides differed from those in the commonly used inbred mouse strain, C57BL/6J. Therefore, MSM/Ms mice are thought to be useful for functional genome studies. MSM/Ms mice show unique characteristics of phenotypes, including its smaller body size, resistance to high-fat-diet-induced diabetes, high locomotive activity, and resistance to age-onset hearing loss, inflammation, and tumorigenesis, which are distinct from those of common inbred mouse strains. Furthermore, ES (Embryonic Stem) cell lines established from MSM/Ms allow the MSM/Ms genome to be genetically manipulated. Therefore, genomic and phenotypic analyses of MSM/Ms reveal novel insights into gene functions that were previously not obtained from research on common laboratory strains. Tumorigenesis-related MSM/Ms-specific genetic traits have been intensively investigated in Japan. Furthermore, radiation-induced thymic lymphomas and chemically-induced skin tumors have been extensively examined using MSM/Ms.

Sequence Composition of Bacterial Chromosome Clones in a Transgressive Root-Knot Nematode Resistance Chromosome Region in Tetraploid Cotton.

Plants evolve innate immunity including resistance genes to defend against pest and pathogen attack. Our previous studies in cotton (Gossypium spp.) revealed that one telomeric segment on chromosome (Chr) 11 in G. hirsutum cv. Acala NemX (rkn1 locus) contributed to transgressive resistance to the plant parasitic nematode Meloidogyne incognita, but the highly homologous segment on homoeologous Chr 21 had no resistance contribution. To better understand the resistance mechanism, a bacterial chromosome (BAC) library of Acala N901 (Acala NemX resistance source) was used to select, sequence, and analyze BAC clones associated with SSR markers in the complex rkn1 resistance region. Sequence alignment with the susceptible G. hirsutum cv. TM-1 genome indicated that 23 BACs mapped to TM-1-Chr11 and 18 BACs mapped to TM-1-Chr 21. Genetic and physical mapping confirmed less BAC sequence (53–84%) mapped with the TM-1 genome in the rkn1 region on Chr 11 than to the homologous region (>89%) on Chr 21. A 3.1-cM genetic distance between the rkn1 flanking markers CIR316 and CIR069 was mapped in a Pima S-7 × Acala NemX RIL population with a physical distance ∼1 Mbp in TM-1. NCBI Blast and Gene annotation indicated that both Chr 11 and Chr 21 harbor resistance gene-rich cluster regions, but more multiple homologous copies of Resistance (R) proteins and of adjacent transposable elements (TE) are present within Chr 11 than within Chr 21. (CC)-NB-LRR type R proteins were found in the rkn1 region close to CIR316, and (TIR)-NB-LRR type R proteins were identified in another resistance rich region 10 cM from CIR 316 (∼3.1 Mbp in the TM-1 genome). The identified unique insertion/deletion in NB-ARC domain, different copies of LRR domain, multiple copies or duplication of R proteins, adjacent protein kinases, or TE in the rkn1 region on Chr 11 might be major factors contributing to complex recombination and transgressive resistance.

In Memoriam: Mortimer L. Mendelsohn: (1 December, 1925-8 January, 2020).

In Memoriam: Mortimer L. Mendelsohn: (1 December, 1925-8 January, 2020).

Evolution of multiple sex-chromosomes associated with dynamic genome reshuffling in Leptidea wood-white butterflies

Sex-chromosome systems tend to be highly conserved and knowledge about their evolution typically comes from macroevolutionary inference. Rapidly evolving complex sex-chromosome systems represent a rare opportunity to study the mechanisms of sex-chromosome evolution at unprecedented resolution. Three cryptic species of wood-white butterflies—Leptidea juvernica, L. sinapis and L. reali—have each a unique set of multiple sex-chromosomes with 3–4 W and 3–4 Z chromosomes. Using a transcriptome-based microarray for comparative genomic hybridisation (CGH) and a library of bacterial artificial chromosome (BAC) clones, both developed in L. juvernica, we identified Z-linked Leptidea orthologs of Bombyx mori genes and mapped them by fluorescence in situ hybridisation (FISH) with BAC probes on multiple Z chromosomes. In all three species, we determined synteny blocks of autosomal origin and reconstructed the evolution of multiple sex-chromosomes. In addition, we identified W homologues of Z-linked orthologs and characterised their molecular differentiation. Our results suggest that the multiple sex-chromosome system evolved in a common ancestor as a result of dynamic genome reshuffling through repeated rearrangements between the sex chromosomes and autosomes, including translocations, fusions and fissions. Thus, the initial formation of neo-sex chromosomes could not have played a role in reproductive isolation between these Leptidea species. However, the subsequent species-specific fissions of several neo-sex chromosomes could have contributed to their reproductive isolation. Then, significantly increased numbers of Z-linked genes and independent neo-W chromosome degeneration could accelerate the accumulation of genetic incompatibilities between populations and promote their divergence resulting in speciation.

Improved protocols for BAC insert DNA isolation, BAC end sequencing and FISH for construction of BAC based physical map of genes on the chromosomes.

Bacterial artificial chromosome (BAC) library is an important genomic resource useful in targeted marker development, positional cloning, physical mapping and a substrate for genome sequencing for better understanding the genome organization of a species. The present manuscript elucidates the improvement in protocols for economical and efficient BAC insert DNA isolation, BAC end sequencing and FISH for physical localization on the metaphase chromosome complements. BAC clones of Clarias magur, maintained in 384-well plate format in our laboratory, were used in this study. The protocols gave consistent and efficient results. We use routinely these protocols for BAC insert DNA extraction, generating end sequence data of the clone and constructing DNA probes to hybridize on the metaphase spreads of C. magur using FISH for physical their localization.

Construction and characterization of a BAC library for flue-cured tobacco line with high resistance to blank shank

<p indent="0mm">Tobacco (<italic>N. tabacum</italic>) is an important model crop in molecular biology research. In this study, a bacterial artificial chromosome (BAC) library of a flue-cured tobacco line 14–60 with high blank shank resistance and good quality was constructed. High molecular weight DNA was isolated using intact nuclei from tobacco, partially cleaved with <italic>Hin</italic>d III and cloned into the pIndigoBAC536-S vector. The BAC library consisted of 414,720 clones arrayed in one thousand and eighty 384-microtite plates, with an average insert size of 123 kb ranging from 97.0–145.5 kb. No empty insert clone was found. Based on an estimated genome size of 4500 Mb for common tobacco, the BAC library was estimated to cover 11 times of genome equivalents. The utility of the library was further confirmed by screening the library with the primers of tobacco <italic>hem</italic>A, <italic>NtFT</italic>, and<italic> eIF4E-</italic>1 genes. The high capacity library will serve as a giant genomic resource for map-based cloning of quantitative trait loci or genes associated with important agronomic and smoking quality traits or resistance to blank shank, physical mapping and comparative genome analysis.