Identification of Nucleus Sterility Candidate Genes using a Resequencing Technique in Sweet Pepper Sterile Line AB91

Background:Breeding hybrids with nuclear malesterile lines is an important method for the cross breeding of sweet peppers. To date, few reports have been published on the nuclear malesterility gene of sweet pepper.Yet, there are approximately 20 pepper nuclear malesterility lines in the world. Results: Using the self-developed testing material, sweet pepper nuclear malesterile dual-purpose line AB91, the genome-wide resequencing technique was applied to firstly find that the mutation site causing the abortion of sweet pepper nuclear malesterility AB91 is on chromosome #5. The mutation gene Capana05g000747 was filtered out and validated by the flight mass spectrometry genotyping and quantitative real-time PCR method and determined to be the gene causing the abortion of sweet pepper nuclear male sterility AB91.The gene Capana05g000747 mutation site is a non-synonymous mutation site located at the 6th exon,the base C mutated into A, and the amino acid changed from alanine to serine. The three-dimensional protein structure of fertile and sterile plant Capana05g000747 was predicted. The results showed that the three-dimensional structure of the two proteins was differed significantly. Sequence alignment analysis showed that the gene Capana05g000747 has a similar function to gene At2g02148. The gene At2g02148 contains a pentatricopeptide repeatprotein which has important physiological functions in the gene expression process of organelles and is closely related to the performance of malesterility genes. Conclusions: This study revealed that Capana05g000747 was selected as an important candidate gene for sweet pepper nuclear male sterile testingmaterial AB91.This gene provides a theoretical and technical foundation for further cloning as well as transformation and utilization of the gene.

Identification of Nucleus Sterility Candidate Genes using a Resequencing Technique in Sweet Pepper Sterile Line AB91

Background : Breeding hybrids with nuclear malesterile lines is an important method for the cross breeding of sweet peppers. To date, few reports have been published on the nuclear malesterility gene of sweet pepper.Yet, there are approximately 20 pepper nuclear malesterility lines in the world. Results: Using the self-developed testing material, sweet pepper nuclear malesterile dual-purpose line AB91, the genome-wide resequencing technique was applied to firstly find that the mutation site causing the abortion of sweet pepper nuclear malesterility AB91 is on chromosome #5. The mutation gene Capana05g000747 was filtered out and validated by the flight mass spectrometry genotyping and quantitative real-time PCR method and determined to be the gene causing the abortion of sweet pepper nuclear male sterility AB91.The gene Capana05g000747 mutation site is a non-synonymous mutation site located at the 6th exon,the base C mutated into A, and the amino acid changed from alanine to serine. The three-dimensional protein structure of fertile and sterile plant Capana05g000747 was predicted. The results showed that the three-dimensional structure of the two proteins was differed significantly. Sequence alignment analysis showed that the gene Capana05g000747 has a similar function to gene At2g02148. The gene At2g02148 contains a pentatricopeptide repeatprotein which has important physiological functions in the gene expression process of organelles and is closely related to the performance of malesterility genes. Conclusions : This study revealed that Capana05g000747 was selected as an important candidate gene for sweet pepper nuclear male sterile testingmaterial AB91.This gene provides a theoretical and technical foundation for further cloning as well as transformation and utilization of the gene.

Identification of Nucleus Sterility Candidate Genes using a Resequencing Technique in Sweet Pepper Sterile Line AB91

Breeding hybrids with nuclear male sterile lines is an important method for the cross breeding of sweet peppers. To date, few reports have been published on the nuclear male sterility gene of sweet pepper. Yet, there are approximately 20 pepper nuclear male sterility lines in the world. Using the self-developed testing material, sweet pepper nuclear male sterile dual-purpose line AB91, the genome-wide resequencing technique was applied to firstly find that the mutation site causing the abortion of sweet pepper nuclear male sterility AB91 is on chromosome #5. The mutation gene Capana05g000747 was filtered out and validated by the flight mass spectrometry genotyping method and determined to be the gene causing the abortion of sweet pepper nuclear male sterility AB91. The gene Capana05g000747 contains eight exons and seven introns, and its mutation site is a non-synonymous mutation site located at the 6th exon; the base C mutated into A, and the amino acid changed from alanine to serine. Sequence alignment analysis showed that the gene Capana05g000747 has a similar function to gene At2g02148. The gene At2g02148 contains a pentatricopeptide repeat protein which has important physiological functions in the gene expression process of organelles and is closely related to the performance of male sterility genes. Therefore, Capana05g000747 was selected as an important candidate gene for sweet pepper nuclear male sterile testing material AB91.

Identification of Nucleus Sterility Candidate Genes using a Resequencing Technique in Sweet Pepper Sterile Line AB91

Background: Breeding hybrids with nuclear male sterile lines is an important method for the cross breeding of sweet peppers. To date, few reports have been published on the nuclear male sterility gene of sweet pepper. Yet, there are approximately 20 pepper nuclear male sterility lines in the world. Results: Using the self-developed testing material, sweet pepper nuclear male sterile dual-purpose line AB91, the genome-wide resequencing technique was applied to firstly find that the mutation site causing the abortion of sweet pepper nuclear male sterility AB91 is on chromosome #5. The mutation gene Capana05g000747 was filtered out and validated by the flight mass spectrometry genotyping method and determined to be the gene causing the abortion of sweet pepper nuclear male sterility AB91. The gene Capana05g000747 contains eight exons and seven introns, and its mutation site is a non-synonymous mutation site located at the 6th exon; the base C mutated into A, and the amino acid changed from alanine to serine. Sequence alignment analysis showed that the gene Capana05g000747 has a similar function to gene At2g02148. Conclusion: The gene At2g02148 contains a pentatricopeptide repeat protein which has important physiological functions in the gene expression process of organelles and is closely related to the performance of male sterility genes. Therefore, Capana05g000747 was selected as an important candidate gene for sweet pepper nuclear male sterile testing material AB91.

The Application of Molecular Markers to Accelerate the Recovery of Cytoplasmic and Nuclear Male Sterility in South African Onion (Allium cepa L.) Hybrid Parental Lines

Male sterility is important to prevent self-pollination and loss of the onion hybrid genotype. Classic methods require 4-8 years of progeny testing before the cytoplasm type can be determined. An accurate and time-saving method was needed. Various types of markers were tested for application to South African onion parental lines of hybrid cultivars, and which could determine male sterility and maintainer genotypes accurately and easily with large numbers of samples. Five cytoplasmic (5’cob, orfA501, orf725, IGS and cob-type 2) and four nuclear markers (jnurf13, isotig34671_610, isotig30856_1351 and isotig29186_1830) were sourced. Genomic DNA was isolated from onion seedlings and young leaves growing from bulbs in the Agricultural Research Council (ARC) research field. PCR marker amplification products were separated by agarose or denaturing polyacrylamide gel electrophoresis (PAGE) gels. Real-time polymerase chain reaction (PCR) was performed with custom TaqMan® SNP genotyping assays containing primer/probe pairs designed to detect single nucleotide polymorphisms (SNPs) linked to the nuclear Ms locus. OrfA501 proved useful as a presence/absence marker for cytoplasmic male sterility, while TaqMan® SNP genotyping assays were superior to the jnurf13 nuclear marker in terms of rapid throughput. PCR molecular markers and custom TaqMan® SNP genotyping assays were efficient in screening the onion lines rapidly and accurately for their cytoplasmic and nuclear male sterility genotype. These methods reduced the time to identify the correct genotype of male sterile and maintainer lines, gave accurate genotypic information and proved to be useful on a larger scale. These molecular marker methods will facilitate the production of the correct seed for commercialization of onion lines worldwide.