Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth,Plutella xylostella

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing.In the current study,we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella,in addition to its known functions in the ovarian development in female adults and egg hatching.Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein(GFP)gene into autosomal Pxfl(2)d for labelling mutants could be achieved.However,this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity.We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9.The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences.We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein(CFP)expression cassettes on a Z-linked region in P.xylostella,which can also be used for early sex detection.By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain,the gene editing efficiency reached 29.73%,confirming the success of expressing a functional Cas9 gene.Taken together,we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P.xylostella,while the establishment of a heritable strain required the positioning of appropriate sites.This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

Loss of heterozygosity for chromosomes 16q in Wilms tumors predicts outcomes:A meta-analysis

BACKGROUND The research findings suggest that the prognosis of children with Wilms tumor(WT)is affected by various factors.Some scholars have indicated that loss of heterozygosity(LOH)on chromosome 16q is associated with a poor prognosis in patients with WT.AIM To further elucidate this relationship,we conducted a meta-analysis.METHODS This meta-analysis was registered in INPLASY(INPLASY2023100060).We systematically searched databases including Embase,PubMed,Web of Science,Cochrane,and Google Scholar up to May 31,2020,for randomized trials reporting any intrapartum fetal surveillance approach.The meta-analysis was performed within a frequentist framework,and the quality and network inconsistency of trials were assessed.Odds ratios and 95%CIs were calculated to report the relationship between event-free survival and 16q LOH in patients with WT.RESULTS Eleven cohort studies were included in this meta-analysis to estimate the relationship between event-free survival and 16q LOH in patients with WT(I^(2)=25%,P<0.001).As expected,16q LOH can serve as an effective predictor of eventfree survival in patients with WT(risk ratio=1.95,95%CI:1.52–2.49,P<0.001).CONCLUSION In pediatric patients with WT,there exists a partial correlation between 16q LOH and an unfavorable treatment prognosis.Clinical detection of 16q chromosome LOH warrants increased attention to the patient’s prognosis.

Characterization of a 4.1 Mb inversion harboring the stripe rust resistance gene YR86 on wheat chromosome 2AL

Wheat cultivar Zhongmai 895 was earlier found to carry YR86 in an 11.6 Mb recombination-suppressed region on chromosome 2AL when crossed with Yangmai 16.To fine-map the YR86 locus,we developed two large F2 populations from crosses Emai 580/Zhongmai 895 and Avocet S/Zhongmai 895.Remarkably,both populations exhibited suppressed recombination in the same 2AL region.Collinearity analysis across Chinese Spring,Aikang 58,and 10+wheat genomes revealed a 4.1 Mb chromosomal inversion spanning 708.5-712.6 Mb in the Chinese Spring reference genome.Molecular markers were developed in the breakpoint and were used to assess a wheat cultivar panel,revealing that Chinese Spring,Zhongmai 895,and Jimai 22 shared a common sequence named InvCS,whereas Aikang 58,Yangmai 16,Emai 580,and Avocet S shared the sequence named InvAK58.The inverted configuration explained the suppressed recombination observed in all three bi-parental populations.Normal recombination was observed in a Jimai 22/Zhongmai 895 F2 population,facilitating mapping of YR86 to a genetic interval of 0.15 cM corresponding to 710.27-712.56 Mb falling within the inverted region.Thirty-three high-confidence genes were annotated in the interval using the Chinese Spring reference genome,with six identified as potential candidates for YR86 based on genome and transcriptome analyses.These results will accelerate map-based cloning of YR86 and its deployment in wheat breeding.

Characterization of meiotic chromosome behavior in the autopolyploid Saccharum spontaneum reveals preferential chromosome pairing without distinct DNA sequence variation

Autopolyploidy and allopolyploidy may represent an evolutionary advantage and are more common in plants than assumed. However, less attention has been paid to autopolyploidy than to allopolyploidy,and its evolutionary consequences are largely unclear, especially for plants with high ploidy levels. In this study, we developed oligonucleotide(oligo)-based chromosome painting probes to identify individual chromosomes in S. spontaneum. Using fluorescence in situ hybridization(FISH), we investigated chromosome behavior during pachytene, metaphase, anaphase, and telophase of meiosis I(MI) in autotetraploid,autooctoploid, and autodecaploid S. spontaneum clones. All autopolyploid clones showed stable diploidized chromosome behavior;so that homologous chromosomes formed almost exclusively bivalents during MI. Two copies of homologous chromosome 8 with similar sizes in the autotetraploid clone showed preferential pairing with each other with respect to the other copies. However, sequence variation analysis showed no apparent differences among homologs of chromosome 8 and all other chromosomes. We suggest that either the stable diploidized pairing or the preferential pairing between homologous copies of chromosome 8 in the studied autopolyploid sugarcane are accounted for by unknown mechanisms other than DNA sequence similarity. Our results reveal evolutionary consequences of stable meiotic behavior in autopolyploid plants.

Chromosome 5P of Agropyron cristatum induces chromosomal translocation by disturbing homologous chromosome pairing in a common wheat background

Wide hybridization is a strategy for broadening the genetic basis of wheat. Because an efficient method for inducing wheat–alien chromosome translocations will allow producing useful germplasm, it is desirable to discover new genes that induce chromosomal variation. In this study, chromosome 5P from A.cristatum was shown to induce many types of chromosomal structural variation in a common wheat background, including nonhomoeologous chromosome translocations, as revealed by genomic in situ hybridization, fluorescence in situ hybridization, and DNA marker analysis. Aberrant meiosis was associated with chromosomal structural variation, and aberrant meiotic behavior was observed in wheat–A.cristatum 5P monosomic and disomic addition lines, suggesting that the effect of chromosome 5P was independent of the number of chromosome 5P copies. Chromosome 5P disturbed homologous chromosome pairing at pachytene stage in a common wheat background, resulting in a high frequency of univalent formation and reduced crossing over. Thirteen genes involved in DNA repair or chromatin remodeling, including RAD52-like and MSH6 genes, were differentially expressed(upregulated) in wheat–A. cristatum 5P addition lines according to transcriptome analysis, implicating chromosome 5P in the process of meiotic double-strand break repair. These findings provide a new, efficient tool for inducing wheat–alien chromosome translocations and producing new germplasm.

Chromosome-level genome assembly of Cylas formicarius provides insights into its adaptation and invasion mechanisms

Cylasformicarius is one of the most important pests of sweet potato worldwide, causing considerable ecological and economic damage.This study improved the effect of comprehensive management and understanding of genetic mechanisms by examining the functional genomics of C. formicarius.Using Illumina and PacBio sequencing, this study obtained a chromosome-level genome assembly of adult weevils from lines inbred for 15 generations.The high-quality assembly obtained was 338.84 Mb, with contig and scaffold N50 values of 14.97 and 34.23 Mb, respectively.In total, 157.51 Mb of repeat sequences and 11 907 protein-coding genes were predicted.A total of 337.06 Mb of genomic sequences was located on the 11 chromosomes, accounting for 99.03%of the total length of the associated chromosome.Comparative genomic analysis showed that C. formicarius was sister to Dendroctonus ponderosae, and C. formicarius diverged from D. ponderosae approximately 138.89 million years ago (Mya).Many important gene families expanded in the C. formicarius genome were involved in the detoxification of pesticides, tolerance to cold stress and chemosensory system.To further study the role of odorant-binding proteins (OBPs) in olfactory recognition of C. formicarius, the binding assay results indicated that Cfor OBP4–6 had strong binding affinities for sex pheromones and other ligands.The high-quality C. formicarius genome provides a valuable resource to reveal the molecular ecological basis, genetic mechanism, and evolutionary process of major agricultural pests;it also offers new ideas and new technologies for ecologically sustainable pest control.

Nannochloropsis artificial chromosomes(Nanno ACs)loom on the horizon

Species in genus Nannochloropsis,especially N.oceanica and N.gaditana,have been evolving as the model microalgae for both application and theory studies.The position effect of genome integration,the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments.The molecular tools including genetic transformation,homologous recombination,gene edition,gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species.The construction of animal and plant artificial chromosomes with“top down”strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes(NannoACs).It seems that the methods and materials to set the foundation for constructing NannoACs are at hands.In this review,we outlined the current status of transgenes in Nannochloropsis species,summarized the limitations of both integrative and non-integrative vectors,and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first,and then truncating the natural chromosomes.NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds,thus contributing towards its genetic improvement and synthetic biological studies.

Genetic effects of Agropyron cristatum 2P chromosome translocation fragments in a wheat background

Agropyron cristatum(2n=4x=28,PPPP)is a wild relative of common wheat which contains a large number of desirable genes that can be exploited for wheat improvement.Wheat–A.cristatum 2P alien translocation lines exhibit many desirable traits,such as small flag leaves,a high spikelet number and density,and a compact plant type.An agronomic trait evaluation and a genetic analysis were carried out on translocation lines and backcross populations of these lines carrying different translocation fragments.The results showed that a translocation fragment from 2PT-3(2PL)reduced the length of the flag leaves,while translocation fragments from 2PT-3(2PL)and 2PT-5(2PL(0.60–1.00))reduced the width of the flag leaves.A translocation fragment from 2PT-13(2PS(0.18–0.36))increased the length and area of the flag leaves.Translocation fragments from 2PT-3(2PL)and 2PT-8(2PL(0.86–1.00))increased the density of spikelets.Translocation fragments from 2PT-7(2PL(0.00–0.09)),2PT-8(2PL(0.86–1.00)),2PT-10(2PS),and 2PT-13(2PS(0.18–0.36))reduced plant height.This study provides a scientific basis for the effective utilization of wheat–A.cristatum translocation lines.

Rapid identification of Psathyrostachys huashanica Keng chromosomes in wheat background based on ND-FISH and SNP array methods

Psathyrostachys huashanica Keng(2n=2x=14,NsNs)is regarded as a valuable wild relative species for common wheat cultivar improvement because of its abundant beneficial agronomic traits.However,although the development of many wheat–P.huashanica-derived lines provides a germplasm base for the transfer of excellent traits,the lag in the identification of P.huashanica chromosomes in the wheat background has limited the study of these lines.In this study,three novel nondenaturing fluorescence in situ hybridization(ND-FISH)-positive oligo probes were developed.Among them,HS-TZ3 and HS-TZ4 could specifically hybridize with P.huashanica chromosomes,mainly in the telomere area,and HS-CHTZ5 could hybridize with the chromosomal centromere area.We sequentially constructed a P.huashanica FISH karyotype and idiogram that helped identify the homologous groups of introduced P.huashanica chromosomes.In detail,1Ns and 2Ns had opposite signals on the short and long arms,3Ns,4Ns,and 7Ns had superposed two-color signals,5Ns and 6Ns had fluorescent signals only on their short arms,and 7Ns had signals on the intercalary of the long arm.In addition,we evaluated different ways to identify alien introgression lines by using low-density single nucleotide polymorphism(SNP)arrays and recommended the SNP homozygosity rate in each chromosome as a statistical pattern.The 15K SNP array is widely applicable for addition,substitution,and translocation lines,and the 40K SNP array is the most accurate for recognizing transposed intervals between wheat and alien chromosomes.Our research provided convenient methods to distinguish the homologous group of P.huashanica chromosomes in a common wheat background based on ND-FISH and SNP arrays,which is of great significance for efficiently identifying wheat–P.huashanica-derived lines and the further application of Ns chromosomes.

Characterization of Two Human Lung Adenocarcinoma Cell Lines by Reciprocal Chromosome Painting

Lung cancer is a leading cause of cancer death worldwide. Some lung cancer patients correlate with a gas of radon besides smoking. To search for common chromosomal aberrations in lung cancer cell lines established from patients induced by different factors, a combined approach of chromosome sorting, forward and reverse chromosome painting was used to characterize karyotypes of two lung adenocarcinoma cell lines： A549 and GLC-82 with the latter line derived from a patient who has suffered long-term exposure to environmental radon gas pollution. The chromosome painting results revealed that complex chromosomal rearrangements occurred in these two lung adenocarcinoma cell lines. Thirteen and twenty-four abnormal chromosomes were identified An A549 and GLC-82 cell lines, respectively. Almost half of abnormal chromosomes in these two cell lines were formed by non-reciprocal translocations, the others were derived from deletions and duplication/or amplification in some chromosomal regions. Furthermore, two apparently common breakpoints, HSA8q24 and 12q14 were found in these two lung cancer cell lines.

Chromosome Location of the Male-sterility and Yellow Seedling Gene in Line 1066A of Foxtail Millet

Using foxtail millet (Setaria italica (L.) Beauv.) male-sterile line 1066A as female parent and Yugu 1 primary trisomic series (1 - 7) and tetrasomics 8, 9 as male parents, chromosome location of gene for male-sterility and yellow seedling in line 1066A was studied by primary trisomic analysis. The plants of F-1 generation of trisomics 2 - 9 were obtained by crossing with a great many plants of 1066A. F-1 generation of trisomics was similar to their male parent in morphologic characters, the color of their seedling was green, and pollen was partially fertile. The segregation ratio of fertility to sterility is 3:1 in F-2 generation of trisomics 2, 3, 4, 5, 7, 8 and 9; and 14:1 only in F-2 generation of trisomic 6 (chi(0.05)(2) = 0.012). The segregation ratio of green seedling to yellow seedling is 12:1 only in F-2 generation of trisomic 7 (chi(0.05)(2) = 0.31), but in other cases, this ratio is 3:1. The results indicated that the male-sterility gene was located on chromosome 6, and the gene for yellow seedling was monogenic recessive and located on chromosome 7. The rate of trisomics transmission by pollen was tested, trisomics 8 and 9 were the highest in rates of trisomics transmission and followed by trisomics 6 and 4.

Karyotypic evolution in family Hipposideridae (Chiroptera, Mammalia) revealed by comparative chromosome painting, G- and C-banding

Comparing to its sister-family （Rhinolophidae）, Hipposideridae was less studied by cytogenetic approaches. Only a few high-resolution G-banded karyotypes have been reported so far, and most of the conclusions on the karyotypic evolution in Hipposideridae were based on conventional Giemsa-staining. In this study, we applied comparative chromosome painting, a method of choice for genome-wide comparison at the molecular level, and G- and C-banding to establish comparative map between five hipposiderid species from China, using a whole set of chromosome-specific painting probes from one of them （Aselliscus stoliczkanus）. G-band and C-band comparisons between homologous segments defined by chromosome painting revealed that Robertsonian translocations, paracentric inversions and heterochromatin addition could be the main mechanism of chromosome evolution in Hipposideridae. Comparative analysis of the conserved chromosomal segments among five hipposiderid species and outgroup species suggests that bi-armed chromosomes should be included into the ancestral karyotype of Hipposideridae, which was previously believed to be exclusively composed of acrocentric chromosomes.

Chromosome Preparation and Preliminary Observation of Two Amphioxus Species in Xiamen

Amphioxus has an important evolutionary position as a result of their phylogenetic position relative to vertebrates. Understanding their chromosomes would provide key points in the study of evolutionary biology and comparative genomics. The difficulty in preparing amphioxus chromosomes currently provides a significant hurdle in this research. In the current study, we describe an improved method for metaphase preparation from amphioxus embryos and methodology for preparing metaphase spreads from regenerative somatic cells. Chromosomes of two amphioxus species from Xiamen waters in China are also observed. The diploid chromosome number was found to be 40 in Branchiostoma belcheri, while B. japonicum has 36, confirming the two are distinct species from cytotaxonomic viewpoint.

Identification of Quantitative Trait Loci (QTLs) Affecting Yield and Fiber Properties in Chromosome 16 in Cotton Using Substitution Line

Gossypium hirsutum L. and G. barbadense L. are the two cultivated tetraploid species of cotton. The first is characterized by a high yield and wide adaptation, and the second by its super fiber property. Substitution line in which a pair of intact chromosomes of TM_1 ( G. hirsutum ) were replaced by a pair of homozygous chromosomes of 3_79 ( G. barbadense ) is an excellent material for genetic research and molecular tagging. In this study, substitution line 16 (Sub 16) was used to evaluate the performance of the 16th chromosome in G. barbadense in TM_1 background. The genetic analysis using the major gene plus polygene mixed inheritance model in F 2∶3 family revealed that there might exist 2 QTLs respectively for boll size, lint percentage, lint index, fiber length and the first fruit branch node, 1 QTL for fiber elongation and flowering date, and no QTL for seed index, fiber strength and Micronaire in chromosome 16. However, 9 QTLs (LOD (logarithm of odds)≥3.0) controlling 6 quantitative traits were significantly identified in linkage group of chromosome 16 constructed in (TM_1×3_79) F 2by interval mapping. Among them, 1 QTL for boll size, fiber length, flowering date and fiber elongation could explain 15.2%, 19.7%, 12.1%, and 11.7% phenotypic variance respectively, 2 QTLs for lint index could explain 11.6% and 41.9%, and 3 QTLs for lint percentage could explain 8.7%, 9.6% and 29.2% phenotypic variance respectively. One unlinked SSR marker was associated with one QTL respectively for boll size and flowering date and they could explain 1.60% and 4.63% phenotypic variance. The traits associated significantly with chromosome 16 from Sub 16 were boll weight, lint percentage, lint index, fiber length, fiber elongation and flowering days.

Using Subtracted AFLP to Efficiently Mark an Alien Chromosome Fragment in Wheat Background

为解决AFLP中样品间共同片段对多态性片段识别的干扰 ,作者建立了一种“减法AFLP”标记技术 ,并运用这种技术成功对小麦中的外源染色体片段进行了标记。运用该技术 ,样品间的共同扩增片段显著减少 ,在非变性聚丙稀酰胺凝胶上带型差异十分明显 ,多态性片段很容易分辨。其真实性通过将其中两条成功转换成外源染色体片段特异的SCAR标记得到了验证。减法AFLP标记技术的建立为标记作物中的外源染色体片段提供了一种有效手段。

Genetic Analysis on Chromosome of Hybrid F_1 between Elymus canadensis and Elymus sibiricus

Morphologic and cytological characteristics of hybrid F1 between E. canadensis and E. sibidcus were studied. The results showed that hybrid F1 were obviously exceeded parents in plant growth vigor, and spike type of F1 was between parents. The karyotype formula of hybrid F1 which was true hybrid was 2n =4x=16M ＋ 10m ＋2st. At meiotic anaphase, chromosome lagging behaviors were frequently observed. The frequency of lagging chromosomes of F1 was 87.37%. At meiotic metaphase, there were many univalents and multivalents, and the rod bivalent formed greatly.

Isolation and Chromosomal Mapping of a Corn B Chromosome Specific RAPDs

B染色体存在于多种动植物中 ,具有很多独特的性状。B染色体与正常染色体在DNA组成方面十分相似 ,寻找B染色体特异序列一直是B染色体研究的难点和热点。通过对含有和不含有B染色体的两种玉米 (ZeamaysL .)基因组进行了RAPD分析 ,筛选到一个B染色体特异性分子标记B480。该标记与玉米的自主复制起始序列ARS1和ARS2同源 ,特别是该序列中的 2 5bp出现在多种模式生物基因组中。FISH的结果显示 。

Development and Utilization of 1S^1 Chromosomearm-specific Molecular Markers of Aegilops longissima

Introducing the 1S^1 chromosome of Aegilops longissima into wheat genome can significantly improve wheat grain quality and contents of iron and zinc. Therefore, the development of molecular markers specific to 1S^1 chromosome of A. longissima is of important significance for breeding high-quality wheat with high contents of iron and zinc in grains. In this study, nine molecular markers specific to 1S^1 chromosome of A. longissima were developed, including two 1S^1S specific markers,six 1S^1L specific markers and one 1S^1 specific marker which was located on both short and long arms. The practicability of these molecular markers were verified using hybrid population as materials. The results showed that hybrid population could be effectively screened and identified, which indicated that the developed 1S^1 chromosome-specific molecular markers could be used for screening and identification of hybrid population and could be used in marker-assisted breeding of high-quality wheat with high contents of Fe and Zn in grains.

Meiotic Behavior of 1BL/1RS Translocation Chromosome and Alien Chromosome in Two Tri-genera Hybrids

The behavior of wheat-rye translocation chromosome and alien chromosome including Thinopyrum and Haynaldia chromosome at meiosis was investigated in two hybrids by fluorescence in situ hybridization (FISH). Misdivision of translocation chromosome at anaphase I and rye chromatin micronucleus at tetrad stage were observed, A plant with one normal 1BL/1RS translocation chromosome and one 1BL/1RS translocation chromosome deleted about 1/3 of rye chromosome arm in length was identified. One plant with wheat-Thinopyrum non-Robertson translocation chromosome was also detected in the F-2 population of Yi4212 x Yi4095. That could be the results of unequal misdivision of wheat-rye 1BL/1RS translocation chromosome and Thinopyrum chromosome during meiosis. No interaction between translocation chromosome and alien chromosome at meiosis was supported by the data of the distribution frequencies of translocation chromosome and Thinopyrum or Haynaldia chromosome in the progeny of two hybrids. The results may be useful to cultivate new germplasms with different length of rye 1R short arm and wheat-alien non-Robertson translocation tines under wheat background.

Repeat Sequences and Base Correlations in Human Y Chromosome Palindromes

On the basis of information theory and statistical methods, we use mutual information, n- tuple entropy and conditional entropy, combined with biological characteristics, to analyze the long range correlation and short range correlation in human Y chromosome palindromes. The magnitude distribution of the long range correlation which can be reflected by the mutual information is PS〉PSa〉PSb （P5a and P5b are the sequences that replace solely Alu repeats and all interspersed repeats with random uneorrelated sequences in human Y chromosome palindrome 5, respectively）; and the magnitude distribution of the short range correlation which can be reflected by the n-tuple entropy and the conditional entropy is PS〉P5a〉PSb〉random uncorrelated sequence. In other words, when the Alu repeats and all interspersed repeats replace with random uneorrelated sequence, the long range and short range correlation decrease gradually. However, the random nncorrelated sequence has no correlation. This research indicates that more repeat sequences result in stronger correlation between bases in human Y chromosome. The analyses may be helpful to understand the special structures of human Y chromosome palindromes profoundly.