Molecular characterization of a novel vernalization allele <Emphasis Type="Italic">Vrn-B1d</Emphasis> and its effect on heading time in Chinese wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) landrace Hongchunmai

Flowering time of wheat cultivars contributes greatly to the adaptability to environmental conditions and it is largely controlled by vernalization genes. In this study, 262 Chinese mini-core wheat cultivars were used to identify the allelic variation at VRN-B1 locus. A novel dominant allele Vrn-B1d was found in Chinese spring wheat landrace cultivar Hongchunmai. This allele contained several genetic divergence within the first intron comparing to the recessive allele vrn-B1, including one large 6850-bp deletion (670–7519 bp), one small 187-bp deletion (7851–8037 bp), one unique SNP (T to C, 7845 bp), and one 4-bp mutation (TTTT to ACAA, 7847–7850 bp). Meanwhile, it was also different from the three known dominant alleles at VRN-B1 locus. Two pairs of primers were designed to identify the novel allele Vrn-B1d and other four known alleles of VRN-B1. A multiplex PCR was established to discriminate all five alleles simultaneously. The greenhouse experiment with high temperature (non-vernalizing condition) and long light showed that F₂ plants containing Vrn-B1d allele headed significantly earlier than those with recessive vrn-B1 allele, suggesting that Vrn-B1d is a dominant allele conferring the spring growth habit. This study provides a useful germplasm and molecular markers for wheat breeding.     

Role of common sarcomeric gene polymorphisms in genetic susceptibility to left ventricular dysfunction

Mutations in sarcomeric genes are common genetic cause of cardiomyopathies. An intronic 25-bp deletion in cardiac myosin binding protein C (MYBPC3) at 3 ^′ region is associated with dilated and hypertrophic cardiomyopathies in Southeast Asia. However, the frequency of sarcomeric gene polymorphisms and associated clinical presentation have not been established with left ventricular dysfunction (LVD). Therefore, the aim of the present study was to explore the association of MYBPC3 25-bp deletion, titin (TTN) 18 bp I/D , troponin T type 2 (TNNT2) 5 bp I/D and myospryn K2906N polymorphisms with LVD. This study includes 988 consecutive patients with angiographically confirmed coronary artery disease (CAD) and 300 healthy controls. Among the 988 CAD patients, 253 with reduced left ventricle ejection fraction (LVEF ≤ 45%) were categorized as LVD. MYBPC3 25-bp deletion, TTN 18 bp I/D and TNNT2 5 bp I/D polymorphisms were determined by direct polymerase chain reaction method, while myospryn K2906N polymorphism by TaqMan assay. Our results showed that MYBPC3 25-bp deletion polymorphism was significantly associated with elevated risk of LVD (LVEF <45) (healthy controls versus LVD: OR = 3.85, P< 0.001; and nonLVD versus LVD: OR = 1.65, P = 0.035), while TTN 18 bp I/D , TNNT2 5 bp I/D and myospryn K2906N polymorphisms did not show any significant association with LVD. The results also showed that MYBPC3 25-bp deletion polymorphism was significantly associated with other parameters of LV remodelling, i.e. LV dimensions (LV end diastole dimension, LVEDD: P = 0.037 and LV end systolic dimension, LVESD: P = 0.032). Our data suggests that MYBPC3 25-bp deletion may play significant role in conferring LVD as well as CAD risk in north Indian population.     

Comparison <Emphasis Type="Italic">CCR5del32</Emphasis> Mutation in the <Emphasis Type="Italic">CCR5</Emphasis> Gene Frequencies in Russians,Tuvinians, and in Groups of HIV-Infected Individuals

The 32-bp deletion (CCR5del32 mutation) in the CCR5 (chemokine (C-C motif) receptor 5) gene, encoding CCR5 chemokine receptor, is one of the factors determining natural resistance to human immunodeficiency virus (HIV-1) infection. In the present study, the samples of Russians (n = 102), Tuvinians (n = 50), and HIV-infected individuals (n = 107) were examined for the presence of CCR5del32 mutation in the CCR5 gene. The CCR5del32 allele frequency in Russians and Tuvinians constituted 7.84 and 2%, respectively. Among HIV-1 infected individuals, two groups, of macrophage-tropic HIV-1 strain- and T-cell-tropic HIV-1 strain-infected were distinguished. The CCR5del32 allele frequency in the first group (6.45%) was lower than in the second one (8.73%). Statistical treatment of the HIV-1 infected individuals typing data showed that the difference in the CCR5del32 allele frequencies between the groups of sexually (macrophage-tropic) and parenterally (T-cell-tropic) infected individuals observed was within the limit of random deviation.     

Impact of the 7-bp deletion in <Emphasis Type="Italic">HvGA20ox2</Emphasis> gene on agronomic important traits in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background

Alike to Reduced height-1 (Rht-1) genes in wheat and the semi dwarfing (sd-1) gene in rice, the sdw1/denso locus involved in the metabolism of the GA, was designated as the ‘Green Revolution’ gene in barley. The recent molecular characterization of the candidate gene HvGA20ox2 for sdw1/denso locus allows to estimate the impact of the functional polymorphism of this gene on the variation of agronomically important traits in barley.

Results

We investigated the effect of the 7-bp deletion in exon 1 of HvGA20ox2 gene (sdw1.d mutation) on the variation of yield-related and malting quality traits in the population of DHLs derived from cross of medium tall barley Morex and semi-dwarf barley Barke. Segregation of plant height, flowering time, thousand grain weight, grain protein content and grain starch was evaluated in two diverse environments separated from one another by 15° of latitude. The 7-bp deletion in HvGA20ox2 gene reduced plant height by approximately 13 cm and delayed flowering time by 3–5 days in the barley segregating DHLs population independently on environmental cue. On other hand, the sdw1.d mutation did not affect significantly either grain quality traits (protein and starch content) or thousand grain weight.

Conclusions

The beneficial effect of the sdw1.d allele could be associated in barley with lodging resistance and extended period of vegetative growth allowing to accumulate additional biomass that supports higher yield in certain environments. However, no direct effect of the sdw1.d mutation on thousand grain weight or grain quality traits in barley was detected.

     

Molecular characteristics of two new <Emphasis Type="Italic">waxy</Emphasis> mutations in China waxy maize

The waxy gene mutation causes waxy maize grain to have a sticky quality. China has numerous waxy maize landraces and is thought to be the place of origin of waxy maize. The most abundant waxy maize resources in China are located in the Yunnan province and its surrounding areas. We collected 57 waxy maize landraces from Yunnan province and cloned and sequenced the waxy gene from its fourth to eighth exon. Two new waxy gene mutations, named wx-Cin4 and wx-124, were identified. The wx-Cin4 mutation is a 466-bp retrotransposon inserted into exon six. The wx-124 mutation is a 116-bp miniature inverted-repeat transposable element inserted into exon seven. This is the first time a 124-type mutation has been found in a maize waxy gene. The discovery of the two specific waxy mutations from landraces collected in Yunnan province provides new evidence supporting the hypothesis that China is the origin area for waxy maize.     

Development of an HRM-based,safe and high-throughput genotyping system for two <Emphasis Type="Italic">low phytic acid</Emphasis> mutations in soybean

Two low phytic acid (lpa) mutants, Gm-lpa-ZC-2 (ZC-lpa) and Gm-lpa-TW-1 (TW-lpa), resulting from a G → A mutation in GmIPK1 and a 2-bp deletion in GmMIPS1, respectively, were previously developed to increase the nutritional value and environmental friendliness of soybean meal. Two functional CAPS markers were subsequently developed for genotyping plants carrying the two mutant genes; however, both are costly and time consuming and hence unsuitable for large-scale breeding use. In the present work, by integrating a quick DNA extraction protocol with an optimized high-resolution melting curve (HRM) analysis, we developed a fast and high-throughput genotyping system for the two mutations. In this system, (1) DNAs are extracted within half an hour using a protocol that only requires freezing and heating of leaf disks in two non-toxic solutions and can be directly used for PCR; (2) for genotyping, asymmetric PCRs with competitive primers are performed, and the samples are then discriminated and grouped through HRM analysis; and (3) all steps are performed in a 96-well plate, and hence adaptable to high-throughput genotyping. Although the system was developed for two lpa mutations, the general principle should be applicable to any other genes in soybean.     

Novel deletion of <Emphasis Type="Italic">SLC34A2</Emphasis> in Chinese patients of PAM shares mutation hot spot with fusion gene <Emphasis Type="Italic">SLC34A2</Emphasis>–<Emphasis Type="Italic">ROS1</Emphasis> in lung cancer

Pulmonary alveolar microlithiasis (PAM) is an autosomal recessive disorder with distinctive deposition of calcium phosphate microliths in the lungs. Mutation of the SLC34A2 gene was proved to be responsible for PAM. Here, we report the study of a family affected by PAM in China. Two daughters of an inbred family whose parents are cousins and are affected by PAM. Mutation analysis of the SLC34A2 gene by polymerase chain reaction (PCR) amplification and direct sequencing in both patients revealed that exon 5 was deleted on both alleles. Both parents of the patients are proved to be carriers of the mutated allele. Gap-PCR was performed to determine the breakpoints and a homologous deletion of 1152 bp encompassing exon 5 of the SLC34A2 gene (c.IVS4+1452_IVS5+660del) was confirmed. A 4-bp fragment of TGGG was located on the edge of both upstream and downstream breakpoints. The upstream breakpoint lies in the same region as the breakpoint of a fused gene SLC34A2–ROS1, which encodes a constitutive kinase in the lung cancer cell line HCC78 and nonsmall-cell lung cancer (NSCLC), suggesting that the deletion in this family is a hot spot for recombination, not only in cancer samples with somatic mutation, but also in PAM patients with germline genetic defects of SLC34A2.     

CRISPR/Cas9-mediated efficient editing in <Emphasis Type="Italic">phytoene desaturase</Emphasis> (<Emphasis Type="Italic">PDS</Emphasis>) demonstrates precise manipulation in banana cv. Rasthali genome

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species. The tissue-specific expression profile of RAS-PDS1 and RAS-PDS2 in Rasthali suggested differential regulation of the genes. A single 19-bp guide RNA (gRNA) was designed to target the conserved region of these two RAS-PDS and transformed with Cas9 in embryogenic cell suspension (ECS) cultures of cv. Rasthali. Complete albino and variegated phenotype were observed among regenerated plantlets. DNA sequencing of 13 plants confirmed the indels with 59% mutation frequency in RAS-PDS, suggesting activation of the non-homologous end-joining (NHEJ) pathway. The majority of mutations were either insertion (1–5) or deletion (1–4) of nucleotides near to protospacer adjacent motif (PAM). These mutations have created stop codons in RAS-PDS sequences which suggest premature termination of RAS-PDS protein synthesis. The decreased chlorophyll and total carotenoid contents were detected in mutant lines that revealed the functional disruption of both RAS-PDS genes. Our results demonstrate that genome editing through CRISPR/Cas9 can be applied as an efficient tool for banana genome modification.     

ACOS5 is required for primexine formation and exine pattern formation during microsporogenesis in Arabidopsis

Pollen exine, mainly composed of sporopollenin, plays important roles during microspore development. It has been reported that Acyl-CoA Synthetase5 (ACOS5) is required for sporopollenin biosynthesis in Arabidopsis. Here we show that ACOS5 is essential for primexine formation during Arabidopsis microspore development. Through genetic screen, we identified a point mutation of ACOS5 allele, acos5-2, showing abnormal microspore development. Its microspores were degenerated and aborted after released from the tetrads. Transmission electron microscopy showed that primexine formation was reduced in acos5-2 mutant as compared to that of the wild-type. Consequently, sporopollenin was aggregated and randomly deposited on the microspores. In situ hybridization indicated that the key regulators of tapetum development, DYT1 and TDF1, are required for the expression of ACOS5 in tapetum. Furthermore, the GUS reporter showed that the 593-bp promoter sequence was sufficient for the expression of ACOS5 in the anther. Our data provide evidence that ACOS5 is required for primexine formation and sporopollenin deposition during microspore development.     

Three Novel <Emphasis Type="Italic">NF1</Emphasis> Gene Mutations in a Cohort of Bulgarian Neurofibromatoses Patients

Neurofibromatosis (NF) is a clinically heterogeneous autosomal dominant disorder. Three distinct forms have been identified: neurofibromatosis type 1 (NF1), type 2 (NF2) and schwannomatosis. In the present study, we report clinical and genetic findings in the NF1 and NF2 genes in a cohort of 27 Bulgarian patients, with 18 cases (67%) genetically verified. Both NF1 and NF2 genes were screened by Sanger sequencing on DNA samples. The Sanger negative samples were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) for deletions and duplications. The results from genetic testing revealed three novel mutations and fifteen previously reported ones (13 in the NF1 gene and 2 in the NF2 gene). The novel variants in the NF1 gene are a splice site mutation c.4725-1G>A, a small deletion of five bases c.823delATCTT, p.Leu275ValfsTer14, and a single base duplication c.6547dupC, p.Arg2183ProfsTer11. The novel splice site mutation is manifested by multiple “café au lait” macules and neurofibromas. Both novel out of frame mutations were found in patients with multiple “café au lait” spots and focal epilepsy. A segmental neurofibromatosis (SNF1) is restricted to one or more body segments. Here we present a case with SNF1 caused by a somatic deletion of exons 1 to 12 of the NF1 gene which is manifested by multiple neurofibromas in the right hand. Two nonsense mutations are found in the NF2 gene. Our study adds three novel mutations to the NF1 mutation spectra and contributes to the clinical-genetic NF1-characterization. Here we report strikingly different phenotypic spectra caused by the same mutation in a single family. Our findings contribute to the genotype- phenotype correlations which are difficult to establish, due to the extremely complex NF phenotype being a combination of clinical features.     

Identification of a novel 15.5 kb <Emphasis Type="Italic">SHOX</Emphasis> deletion associated with marked intrafamilial phenotypic variability and analysis of its molecular origin

Haploinsufficiency of the short stature homeobox contaning SHOX gene has been shown to result in a spectrum of phenotypes ranging from Leri–Weill dyschondrosteosis (LWD) at the more severe end to SHOX-related short stature at the milder end of the spectrum. Most alterations are whole gene deletions, point mutations within the coding region, or microdeletions in its flanking sequences. Here, we present the clinical and molecular data as well as the potential molecular mechanism underlying a novel microdeletion, causing a variable SHOX-related haploinsufficiency disorder in a three-generation family. The phenotype resembles that of LWD in females, in males, however, the phenotypic expression is milder. The 15523-bp SHOX intragenic deletion, encompassing exons 3–6, was initially detected by array-CGH, followed by MLPA analysis. Sequencing of the breakpoints indicated an Alu recombination-mediated deletion (ARMD) as the potential causative mechanism.     

Detection of Insertions/Deletions Within <Emphasis Type="Italic">SIRT1</Emphasis>, <Emphasis Type="Italic">SIRT2</Emphasis> and <Emphasis Type="Italic">SIRT3</Emphasis> Genes and Their Associations with Body Measurement Traits in Cattle

Growth traits are complex quantitative traits controlled by numerous candidate genes, and they can be well-evaluated using body measurement traits. As the members of the nicotinamide adenine dinucleotide-dependent family of histone deacetylases, class I sirtuin genes (including SIRT1, SIRT2 and SIRT3) play crucial roles in regulating lipid metabolism, cellular growth and metabolism, suggesting that they are potential candidate genes affecting body measurement traits in animals. Hence, the objective of this work aimed to detect novel insertions/deletions (indels) of SIRT1, SIRT2 and SIRT3 genes in 955 cattle belonging to five breeds, as well as to evaluate their effects on body measurement traits. Herein, the novel 12-bp indel of SIRT1 gene, the 7-bp indel of SIRT2 gene and the 26-bp indel of SIRT3 gene were firstly reported, respectively. The association analysis indicated that the indels within SIRT1 and SIRT2 genes were significantly associated with body measurement traits such as body weight, chest circumference, height at hip cross, hip width, body height, etc. (P?<?0.05 or P?<?0.01). Therefore, based on these findings, the two novel indel variants within bovine SIRT1 and SIRT2 genes could be considered as potential molecular markers for growth traits in cattle selection practices and breeding.