Creating novel ornamentals via new strategies in the era of genome editing.

Ornamental breeding has traditionally focused on improving novelty, yield, quality, and resistance to biotic or abiotic stress. However, achieving these goals has often required laborious crossbreeding, while precise breeding techniques have been underutilized. Fortunately, recent advancements in plant genome sequencing and editing technology have opened up exciting new frontiers for revolutionizing ornamental breeding. In this review, we provide an overview of the current state of ornamental transgenic breeding and propose four promising breeding strategies that have already proven successful in crop breeding and could be adapted for ornamental breeding with the help of genome editing. These strategies include recombination manipulation, haploid inducer creation, clonal seed production, and reverse breeding. We also discuss in detail the research progress, application status, and feasibility of each of these tactics.

Heat stress promotes haploid formation during CENH3-mediated genome elimination in Arabidopsis.

Impaired activity of centromeric histone CENH3 causes inaccurate chromosome segregation and in crosses between the Arabidopsis recombinant CENH3 mutant GFP-tailswap and CENH3G83E with wild-type pollen it results in chromosome loss with the formation of haploids. This genome elimination in the zygote and embryo is not absolute as also aneuploid and diploid progeny is formed. Here, we report that a temporal and moderate heat stress during fertilization and early embryogenesis shifts the ratio in favour of haploid progeny in CENH3 mutant lines. Micronuclei formation, a proxy for genome elimination, was similar in control and heat-treated flowers, indicating that heat-induced seed abortion occurred at a late stage during the development of the seed. In the seeds derived from heat-treated crosses, the endosperm did not cellularize and many seeds aborted. Haploid seeds were formed, however, resulting in increased frequencies of haploids in CENH3-mediated genome elimination crosses performed under heat stress. Therefore, heat stress application is a selective force during genome elimination that promotes haploid formation and may be used to improve the development and efficacy of in vivo haploid induction systems.

The chromosome-level genome of Gypsophila paniculata reveals the molecular mechanism of floral development and ethylene insensitivity.

Gypsophila paniculata, belonging to the Caryophyllaceae of the Caryophyllales, is one of the most famous worldwide cut flowers. It is commonly used as dried flowers, whereas the underlying mechanism of flower senescence has not yet been addressed. Here, we present a chromosome-scale genome assembly for G. paniculata with a total size of 749.58 Mb. Whole-genome duplication signatures unveil two major duplication events in its evolutionary history: an ancient one occurring before the divergence of Caryophyllaceae and a more recent one shared with Dianthus caryophyllus. The integrative analyses combining genomic and transcriptomic data reveal the mechanisms regulating floral development and ethylene response of G. paniculata. The reduction of AGAMOUS expression probably caused by sequence polymorphism and the mutation in miR172 binding site of PETALOSA are associated with the double flower formation in G. paniculata. The low expression of ETHYLENE RESPONSE SENSOR (ERS) and the reduction of downstream ETHYLENE RESPONSE FACTOR (ERF) gene copy number collectively lead to the ethylene insensitivity of G. paniculata, affecting flower senescence and making it capable of making dried flowers. This study provides a cornerstone for understanding the underlying principles governing floral development and flower senescence, which could accelerate the molecular breeding of the Caryophyllaceae species.

A Hypomorphic Mutant of PHD Domain Protein Male Meiocytes Death 1.

Meiosis drives reciprocal genetic exchanges and produces gametes with halved chromosome number, which is important for the genetic diversity, plant viability, and ploidy consistency of flowering plants. Alterations in chromosome dynamics and/or cytokinesis during meiosis may lead to meiotic restitution and the formation of unreduced microspores. In this study, we isolated an Arabidopsis mutant male meiotic restitution 1 (mmr1), which produces a small subpopulation of diploid or polyploid pollen grains. Cytological analysis revealed that mmr1 produces dyads, triads, and monads indicative of male meiotic restitution. Both homologous chromosomes and sister chromatids in mmr1 are separated normally, but chromosome condensation at metaphase I is slightly affected. The mmr1 mutant displayed incomplete meiotic cytokinesis. Supportively, immunostaining of the microtubular cytoskeleton showed that the spindle organization at anaphase II and mini-phragmoplast formation at telophase II are aberrant. The causative mutation in mmr1 was mapped to chromosome 1 at the chromatin regulator Male Meiocyte Death 1 (MMD1/DUET) locus. mmr1 contains a C-to-T transition at the third exon of MMD1/DUET at the genomic position 2168 bp from the start codon, which causes an amino acid change G618D that locates in the conserved PHD-finger domain of histone binding proteins. The F1 progenies of mmr1 crossing with knockout mmd1/duet mutant exhibited same meiotic defects and similar meiotic restitution rate as mmr1. Taken together, we here report a hypomorphic mmd1/duet allele that typically shows defects in microtubule organization and cytokinesis.

Conservation of centromeric histone 3 interaction partners in plants.

The loading and maintenance of centromeric histone 3 (CENH3) at the centromere are critical processes ensuring appropriate kinetochore establishment and equivalent segregation of the homologous chromosomes during cell division. CENH3 loss of function is lethal, whereas mutations in the histone fold domain are tolerated and lead to chromosome instability and chromosome elimination in embryos derived from crosses with wild-type pollen. A wide range of proteins in yeast and animals have been reported to interact with CENH3. The histone fold domain-interacting proteins are potentially alternative targets for the engineering of haploid inducer lines, which may be important when CENH3 mutations are not well supported by a given crop. Here, we provide an overview of the corresponding plant orthologs or functional homologs of CENH3-interacting proteins. We also list putative CENH3 post-translational modifications that are also candidate targets for modulating chromosome stability and inheritance.

Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China.

OBJECTIVE: Hemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. A wide range of mutations, showing extensive molecular heterogeneity, have been described in hemophilia B patients. Our study was aimed at genetic analysis and prenatal diagnosis of hemophilia B in order to further elucidate the pathogenesis of the hemophilia B pedigree in China. MATERIALS AND METHODS: Polymerase chain reaction amplification and direct sequencing of all the coding regions was conducted in hemophilia B patients and carriers. Prenatal diagnosis of the proband was conducted at 20 weeks. RESULTS: We identified the novel point mutation 10.389 A>G, located upstream of the intron 3 acceptor site in hemophilia B patients. The fetus of the proband's cousin was identified as a carrier. CONCLUSION: Our identification of a novel mutation in the F9 gene associated with hemophilia B provides novel insight into the pathogenesis of this genetically inherited disorder and also represents the basis of prenatal diagnosis.

SOX9 overexpression plays a potential role in idiopathic congenital talipes equinovarus.

The collagen, type IX, alpha 1 (COL9A1) gene was previously identified as a candidate gene for idiopathic congenital talipes equinovarus (ICTEV), a congenital lower limb deformity in humans. In the present study, increased expression levels of COL9A1 were identified in the abductor hallucis muscle of ICTEV patients compared with those in control samples. The COL9A1 gene is regulated by SRY (sex­determining region Y)­box 9 (SOX9). Immunofluorescence analysis of SOX9 and COL9A1 proteins identified colocalization to the sarcolemma, endomysium and muscle membrane in muscle samples of ICTEV. No mutations in the exons and promoters of SOX9 were detected in blood samples of 84 ICTEV patients by denaturing gradient gel electrophoresis. mRNA and protein expression levels of SOX9 were detected by real­time polymerase chain reaction and western blot analysis, respectively and were found to be significantly higher in ICTEV muscle samples compared with those in control samples. Based on present observations, we hypothesize that overexpression of the SOX9 gene may play a role in the genetic etiology of ICTEV.

[Mechanism of GLI3 gene transcription regulation in idiopathic congenital talipes equinovarus].

OBJECTIVE: To investigate the mechanism of transcription regulation of GLI3 gene in idiopathic congenital talipes equinovarus. METHODS: pGL3-Gli3 luciferase report vectors were constructed, and the activity of Gli3 promoter was explored. A P-Match software was used to analyze the sequence upstream of the transcription start site of rat Gli3 gene, which was subsequently verified with chromatin immunoprecipitation assay (CHIP) and electrophoretic mobility shift assay (EMSA). Expression of the Gli3 gene was analyzed in L6 cells transfected with Hoxd13 small interference RNA(siRNA) and Hoxd13 expression vectors. RESULTS: The 5' region of rat Gli3 gene contains two potential binding sites for the Hoxd13 protein. CHIP and EMSA assays both confirmed that Hoxd13 can directly bind with site 2. As shown in L6 cells, expression of Gli3 may be enhanced with silencing of Hoxd13, whilst exogenous expression of Hoxd13 can down-regulate transcription of Gli3. CONCLUSION: Hoxd13 can directly regulate the expression of Gli3 gene through a Hoxd13 binding site in the limb of rat embryo.

[Possible role of GLI3 gene in the pathogenesis of idiopathic congenital talipes equinovarus].

OBJECTIVE: To investigate the relationship between GLI3 gene and pathogenesis of idiopathic congenital talipes equinovarus (ICTEV). METHOD: Potential mutations in the coding region of GLI3 were detected among 84 patients with ICTEV by denaturing gradient electrophoresis. Expression of GLI3 in the ICTEV patients' disease tissues was assessed by reverse transcription PCR. Following generation of rat model for ICTEV, mRNA and protein levels of GLI3 were evaluated by real-time PCR and immunohistochemistry and Western blotting. RESULTS: No mutation was found in exons 1 - 8 and 13 of GLI3 gene among the 84 ICTEV patients. No expression of GLI3 gene was detected in the flexor hallucis longus of ICTEV patients or normal controls. Expression of Gli3, in terms of both mRNA and protein, was stronger in the hindlimb of ICTEV rat embryos compared with normal controls. CONCLUSION: Mutation in the coding region of GLI3 may not be responsible for the occurrence of ICTEV. However, there may still be connection between abnormal expression of the gene and pathogenesis of ICTEV.

[Expression of COL9A1 gene and its polymorphism in children with idiopathic congenital talipes equinovarus].

OBJECTIVE: COL9A1 gene is located in the susceptibility region of idiopathic congenital talipes equinovarus (ICTEV) (6q12-13). This study aimed to investigate the expression of the COL9A1 gene and the distribution of single nucleotide polymorphism (SNP) of COL9A1 gene in patients with ICTEV and normal controls. METHODS: Immunohistochemistry was used to detect the expression of COL9A1 in 25 children with ICTEV and 5 normal controls. The frequencies of genotypes and allele of two SNPs in COL9A1 gene rs35470562 and rs1135056 were investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing in 118 patients with ICTEV and 100 normal controls. RESULTS: The COL9A1 protein expression was significantly higher in 22 (88%) out of 25 children with ICTEV than normal controls. There were significant differences in the frequencies of genotypes and allele of rs1135056 in COL9A1 gene between the ICTEV and the control groups: the G allele frequency was higher, the frequency of AA genotype was lower, and the frequencies of AG and GG genotypes were higher in ICTEV patients than those in healthy controls (P<0.05). CONCLUSIONS: COL9A1 protein is highly expressed in patients with ICTEV and rs1135056, which is located in the coding region of COL9A1 gene, may be associated with the pathogenesis of ICTEV.

[The regulation of UTROPHIN expression by EN1].

To investigate possible factors up-regulating the expression of UTROPHIN, potential regulatory elements in the promoter of the human UTROPHIN was predicted by P-match software and verified by EMSA and ChIP. The mechanism of EN1 regulation of the human UTROPHIN expression was evaluated by RNA interference and real-time PCR analyses. Two potential EN1 binding sites in UTROPHIN promoter region were predicted by P-Match software but only the second site was verified to interact directly with EN1 by EMSA and ChIP. The results from RNA interference and real-time PCR showed that the mRNA level of UTROPHIN increased in HeLa cells after EN1 was knockdowned by siRNA. It indicated that EN1 might be a negative regulatory factor for UTROPHIN. Our study suggested that UTROPHIN might be a new target for DMD therapy.

Duchenne or Becker muscular dystrophy: a clinical, genetic and immunohistochemical study in China.

BACKGROUND AND OBJECTIVE: Duchenne and Becker muscular dystrophies are X-linked diseases caused by mutations in the dystrophin gene, which affect approximately 1 in 3,500 and 1 in 18,000 boys, respectively. The aim of this work was to develop a method to assist the diagnosis and classification of the disease. MATERIALS AND METHODS: A large data set of dystrophin mutations was detected in 167 Chinese patients by multiplex ligation-dependent probe amplification and sequencing. Muscle biopsy, immunohistochemistry and STR analysis were also carried out in the patients and carriers. RESULTS: One hundred and three deletions, 23 duplications and two-point mutations. The deletion of one or more exons was detected in 103 (61.7%) patients. The region spanning exons 44-55 was the most frequent deletion. The duplication was identified in 23 (13.8%) patients, which was more common than previously reported. Most duplications were found in exons 2-18. Six out of the 45 muscle biopsies analyzed showed the presence of other muscle diseases. CONCLUSIONS: This study may be important to enable comparisons of mutation type and the most appropriate analytical approach for samples from different geographical areas and ethnicities.

[Molecular and prenatal diagnosis of a pedigree with spinocerebellar ataxia].

OBJECTIVE: To identify the type of a pedigree with spinocerebellar ataxia, and carry out asymptomatic carrier detection and prenatal diagnosis. METHODS: The blood samples of two patients in the spinocerebellar ataxia pedigree were collected. Based on the clinical characteristics of the pedigree and the disease incidence in China, the regions containing the CAG repeat of the SCA1, SCA2 and SCA3/MJD genes were amplified by polymerase chain reaction (PCR). The numbers of CAG repeats in the normal and abnormal allele fragments were identified by using agarose gel electrophoresis and DNA sequencing. We further carried out tests on the children of the patients and fetus to identify the presence of the abnormal allele. RESULTS: The numbers of CAG repeat in the SCA1 and SCA2 genes were in the normal range. The CAG repeat number in one allele of SCA3/MJD gene was in the normal range, while that in the other allele was in the abnormal range. One of the children of the patients and the fetus carried the abnormal allele. CONCLUSION: It was confirmed that the pedigree was SCA3/MJD by gene diagnosis. One of the children of the patients was asymptomatic carrier and the fetus also carried the abnormal allele.

[Teaching experience in integrated course of human development and genetics].

Establishment of integrated course system in human development and genetics is an important part of course reformation, and the improvement of this system is achieved by integrating the content of course, stabilizing teaching force, building teaching materials and applying problem-based learning. Integrity-PBL teaching model is founded and proved to be feasible and effective by teaching practice. Therefore, it maybe play an important role in improving teaching effect and cultivating ability of students to analyse and solve problems.

The expression of Gli3, regulated by HOXD13, may play a role in idiopathic congenital talipes equinovarus.

BACKGROUND: Idiopathic congenital talipes equinovarus (ICTEV) is a congenital limb deformity. Based on extended transmission disequilibrium testing, Gli-Kruppel family member 3 (Gli3) has been identified as a candidate gene for ICTEV. Here, we verify the role of Gli3 in ICTEV development. METHODS: Using the rat ICTEV model, we analyzed the differences in Gli3 expression levels between model rats and normal control rats. We used luciferase reporter gene assays and ChIP/EMSA assays to analyze the regulatory elements of Gli3. RESULTS: Gli3 showed higher expression levels in ICTEV model rats compared to controls (P < 0.05). We identified repressor and activator regions in the rat Gli3 promoter. The Gli3 promoter also contains two putative Hoxd13 binding sites. Using EMSA, the Hoxd13 binding site 2 was found to directly interact with Hoxd13 in vitro. ChIP assays of the Hoxd13-Gli3 promoter complex from a developing limb confirmed that endogenous Hoxd13 interacts with this region in vivo. CONCLUSION: Our findings suggest that HoxD13 directly interacts with the promoter of Gli3. The increase of Gli3 expression in ICTEV model animal might result from the low expression of HoxD13.

[Prenatal diagnosis of Duchenne and Becker muscular dystrophy by multiplex ligation-dependent probe amplification].

Duchenne/Becker muscular dystrophy (DMD/BMD) is an X-linked lethal recessive disease caused by mutation in the DMD gene. There is no efficient treatment for this serious and disabling disease. We established a combination method to detect carriers and performed prenatal diagnosis. Using multiplex ligation-dependent probe amplification (MLPA) and linkage analysis of short tandem repeats (STR) methods, 26 prenatal diagnosis were performed for pregnancies at risk of having a DMD/BMD baby through amniocentesis. Seven out of 26 male fetuses were affected and the pregnancies were terminated. Four out of 26 female fetuses were found to be carriers. MLPA can be the method of choice for initial screening of DMD/BMD patients for deletions and duplications mutations. When combined with STR-based analysis, it can improve the rate of DMD/BMD prenatal diagnosis.

[Gene diagnosis for spinal muscular atrophy and its application study].

OBJECTIVE: To establish an effective testing system for gene diagnosis, carrier detection and prenatal diagnosis for spinal muscular atrophy (SMA). METHODS: Twenty-six patients with SMA were directly tested with PCR-RFLP for exon 7 deletion in the SMN1 gene. Carrier detection was carried out with multi-PCR-DHPLC. Amniotic fluid was taken at the middle stage of gestation from pregnant women who had given birth to affected children. RESULTS: Twenty-five out of 26 patients were diagnosed as having SMN1 gene deletion. Fifty-two of their parents were found to be carriers of exon 7 deletion. Eight of 20 fetuses were diagnosed as having SMN1 gene deletion by PCR-RFLP. CONCLUSION: PCR-RFLP and multi-PCR-DHPLC techniques can provide rapid diagnosis for exon 7 deletion detection and carrier detection. PCR-RFLP may also be adapted for prenatal gene diagnosis of exon 7 deletion in SMN1 gene.

Characteristics of dystrophin gene mutations among Chinese patients as revealed by multiplex ligation-dependent probe amplification.

AIMS: To verify whether dystrophin gene mutations among Chinese patients feature different types and frequencies from other populations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) in combination with multiplex PCR (mPCR) and/or short tandem repeat (STR)-based linkage analysis were applied in a large series of Chinese families affected with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). There were a total of 19 cases seeking prenatal diagnosis during their second pregnancies. RESULTS: Of the 59 family trios (51 with DMD and 8 with BMD), 40 were found to have carried various mutations of the dystrophin gene. In addition to deletions and duplications within the mutational hotspots identified by both methods, 10 mutations missed by mPCR were detected by MLPA, among which at least 3 were of rare types. Combined MLPA and linkage analysis also achieved prenatal diagnoses in all of the 19 amniocentesis samples. CONCLUSIONS: Mutations of dystrophin gene among Chinese patients showed a diverse spectrum, with similarity to as well as discrepancies from other populations. For the comprehensive coverage of all exons of the dystrophin gene, MLPA should be the method of choice for initial screening of DMD/BMD patients. When combined with STR-based analysis, it can achieve diagnosis in as much as 70-80% of all referred cases.

[The functions of GLI3 and EN1 genes in idiopathic congenital talipes equinovarus].

To investigate the role of gene Gli3 in idiopathic congenital talipes equinovarus (ICTEV), we constructed the Gli3 luciferase reporter gene expression vectors to analyze the promoter activity of the rat gene Gli3. The regulatory element in the promoter region of the rat Gli3 was predicted using P-Match software and further verified by ChIP experiment. Meanwhile, the correlation between the rat En1 and ICTEV was evaluated by RT-PCR, immunohistochemistry, and Western blotting analyses. The result from P-Match software prediction showed that only one of the three possible En1 binding sites in Gli3 promoter region was interacted directly with En1 in vivo, which was confirmed by ChIP analysis. The results from RT-PCR, immunohistochemistry and Western blotting analyses suggested that En1 was down-regulated in ICTEV model rats compared to the controls. Our results indicated that En1 might be the negative regulatory element in the upstream of Gli3. The low expression level of EN1 in ICTEV could contribute to the up-regulation of GLI3, which led to the genesis of ICTEV.