Identification of Large Deletion of Ccs Responsible for Non-Red Fruit Color in Pepper (Capsicum annuum) and Development of DNA Marker to Distinguish the Deletion

Chili pepper (Capsicum spp.) fruit color is an important agronomical trait. It has been known that a large deletion in the 5' upstream region of the Ccs gene generates non-red fruit color in pepper, but the accurate size and position of the deletion and whether all the non-red cultivars had the same large deletion or not were unclarified. In this study, to identify the Ccs upstream large deletion, we carried out diagnostic PCR using six forward primers at 300 - 900 bp intervals in the 5' untranslated region of Ccs with a fixed reverse primer for a yellow fruit pepper “Sonia Gold”. Then it was revealed that 4430 bp from -3234 bp position in upstream region to 1196 bp position in exon was deleted in Ccs of “Sonia Gold”. The allele having this deletion was named ccs-del. Probably this allele is substantially the same as ccs-p1 having 4879 bp deletion reported previously. Based on the sequence determined, we developed a PCR marker to distinguish ccs-del. Genotyping of 16 cultivars of C. annuum showed that 14 had ccs-del and the remaining two had another mutant allele ccs-3. This result indicates that ccs-del is the most common allele and widely shared in non-red fruit cultivars in C. annuum. Genotyping of 16 cultivars of C. chinense clarified that one cultivar each possessed ccs-del and ccs-3. These results indicate that major alleles responsible for non-red fruit color in C. annuum were shared across species throughout interspecific introgression.

A novel large deletion(exons 12,13)and a missense mutation(p.G46R)in the PAH in a Japanese patient with phenylketonuria

Background:Phenylketonuria(PKU)is caused by a defect in phenylalanine hydroxylase(PAH).More than 500 mutations have been reported for the gene encoding PAH.However,approximately l%-5%of these include large deletions and large duplications that cannot be detected by conventional methods.Methods:In this report we tried to fully characterize a PAH-deficient patient.The patient was a 2-year-old Japanese boy who was diagnosed with classical PKU at the time of neonatal screening,which was confirmed by the tetrahydrobiopterin-loading test.PCR-related direct sequencing and multiplex ligation-dependent probe amplification(MLPA)were used to analyze of the PAH of the patient.Results:Using PCR-related direct sequencing method,we could detect only a heterozygous novel missense mutation:p.136G>C(p.G46R).A second mutation was detected by MLPA.The patient was heterozygous for a novel large deletion of exons 12 and 13:c.1200-?_1359+?del(EX12_13del).For genetic counseling,an accurate genetic diagnosis is often necessary.Conclusions:Through a combination of MLPA and conventional methods,the success rate of PAH mutation identification can be close to 100%.

APC gene mutations in Chinese familial adenomatous polyposis patients

AIM:To study the characteristics of APC(adenomatous polyposis coli)gene germline mutation in Chinese patients with familial adenomatous polyposis(FAP).METHODS:APC gene from 14 FAP families was amplified by polymerase chain reaction(PCR)and underwent direct sequencing to determine the micromutation type.For the samples without micromutation,the large fragment deletion of APC gene was examined by multiplex ligation-dependent probe amplification(MLPA).RESULTS:There were gene micromutations in 9 families with a micromutation detection rate of 64.3%(9/14),including 6 frameshift mutations(66.7%),1 nonsense mutation(11.1%)and 2 splicing mutations(22.2%).Large fragment deletions were detected by MLPA in 2 families.The total mutation detection rate of micromutations and large fragment deletions was 78.6%(11/14).CONCLUSION:The detection rate of APC gene germline mutation can be improved by direct sequencing combined with MLPA large fragment deletion detection.

Genetic diagnosis strategy of hereditary non-polyposis colorectal cancer

AIM: To study the characteristics of mismatch repair gene mutation of Chinese hereditary non-polyposis colorectal cancer (HNPCC) and hMLH1 gene promoter methylation, and to improve the screening strategy and explore the pertinent test methods. METHODS: A systematic analysis of 30 probands from HNPCC families in the north of China was performed by immunohistochemistry, microsatellite instability (MSI), gene mutation and methylation detection. RESULTS: High frequency microsatellite instability occurred in 25 probands (83.3%) of HNPCC family. Loss of hMLH1 and hMSH2 protein expression accounted for 88% of all microsatellite instability. Pathogenic muta-tion occurred in 14 samples and 3 novel mutational sites were discovered. Deletion of exons 1-6, 1-7 and 8 of hMSH2 was detected in 3 samples and no large fragment deletion was found in hMLH1. Of the 30 probands, hMLH1 gene promoter methylation occurred in 3 probands. The rate of gene micromutation detection combined with large fragment deletion detection was 46.7%-56.7%. The rate of the two methods in combination with methylation detection was 63.3%. CONCLUSION: Scientific and rational detection strategy can improve the detection rate of HNPCC. Based on traditional molecular genetics and combined with epigenetics, multiple detection methods can accurately diagnose HNPCC.

A rice mutant displaying a heterochronically elongated internode carries a 100 kb deletion

We have isolated a recessive rice mutant, designated as indeterminate growth （ing）, which displays creeping and apparent heterochronic phenotypes in the vegetative period with lanky and winding culms. Rough mapping and subsequent molecular characterization revealed that the ing mutant carries a large deletion, which corresponds to a 103 kb region in the Nipponbare genome, containing nine annotated genes on chromosome 3. Of these annotated genes, the SLR1 gene encoding a DELLA protein is the only one that is well characterized in its function, and its null mutation, which is caused by a single base deletion in the middle of the intronless SLR1 gene, confers a slender phenotype that bears close resemblance to the ing mutant phenotype. The primary cause of the ing mutant phenotype is the deletion of the SLR1 gene, and the ing mutant appears to be the first characterized mutant having the entire SLR1 sequence deleted. Our results also suggest that the deleted region of 103 kb does not contain an indispensable gene, whose dysfunction must result in a lethal phenotype.

Gene diagnosis and targeted breeding for blast-resistant Kongyu 131without changing regional adaptability

The fungus Magnaporthe oryzae threatens the rice production of Kongyu 131 (KY131),a leading japonica variety in Northeast China.In this study,two rice lines,KP1 and KP2-Hd1,were obtained by introgressing the blast resistance genes Pi1 and Pi2 into KY131,respectively.However,both lines headed later than KY131.RICE60K SNP array analysis showed that Hd1 closely linked to Pi2 was introgressed into KP2-Hd1,and the linkage drag of Hd1 was broken by recombination.On the other hand,no known flowering genes were introgressed into KP1.Gene diagnosis by resequencing six flowering genes showed that KP1 carried functional Hd16 and Ghd8 alleles.Due to its suppression role in heading under long-day conditions,Ghd8 was chosen as the target for gene editing to disrupt its function.Four sgRNAs targeting different sites within Ghd8 were utilized to induce large-deletion mutations,which were easy to detect via agarose gel electrophoresis.All the ghd8-mutated KP1 lines were resistant to rice blast disease and headed earlier than the control KP1,even than KY131,under natural long-day conditions,which ensures its growth in Northeast China.This study confirmed that a combination of gene diagnosis and targeted gene editing is a highly efficient way to quickly eliminate undesired traits in a breeding line.

Analysis of EX5del4232ins268 and EX5del955 PAH gene mutations in Ukrainian patients with phenylketonuria

Phenylketonuria(PKU)is an autosomal recessive metabolic disorder caused by deficiency of phenylalanine hydroxylase(PAH).The major molecular defects causing PKU are missense mutations of PAH gene.Large deletions of exon 5(EX5del955 and EX5del4232ins)were first reported by the Czech study and were later found also in the Polish,Slovak,Slovenian and Italian PKU-patients.These observations demonstrate the existence of a common subset of this mutation predominantly among Central European populations of Slavic descent.That is why we suggest that EX5del1955 and EX5del4232ins268 mutations might be frequent causes of PKU in Ukrainian patients.EX5del955 and EX5del4232ins268 mutations were analyzed in 106 unrelated PKU patients negative for PAH gene mutations on one or both alleles from our previous analysis.The simultaneous detection of EX5del4232ins268 and EX5del955 mutations was performed by PCR amplification of mutant alleles.EX5del955 mutation was not detected in the Ukrainian patients.This relative alleles frequency of EX5del4232ins268 mutation in the Ukrainian PKU population was determined as 1,66%.Our findings can be the one more evidence of Central European Slavic origin of EX5del4232ins268 mutation,suggested previously.This finding is important for the improvement of DNA diagnosis necessary for the management of PKU patients from Ukraine.