Genetic And QTLs Analysis Of The Fertility Restorer Of Cytoplamic Male Sterility In Pepper (Capsicum Annuum L.) And Breeding Of Fertility Restorer Lines

Pepper (Capsicum annuum L.) is a very important vegetable crop in the world. In China the planting area has been kept around 1.3 million ha per year since 2000. Because of its obvious heterosis, pepper hybrid has been widely utilized to increase yields and improve the quality of peppers. At present the hybrid seed possesses 90% of the commercial seeds total. However, the hybrid seed production in China now is labour-intensive and relies on manual removal of male flowers and manual pollination, which has the risk of self-crossing and consequently the seed purity cannot be assured. Hybrid seed production by using Cytoplasmic Male Sterility (CMS) can lower labour costs, increase the genetic purity of the F₁ seeds, and additionally protect the intelligent property of new varieties. But development of the fertility restorater lines has been a problem in pepper breeding and 'One gene control of the restoration of the fertility' can not explain all the questions in breeding. So a deeper understanding of the fertility restoration mechanisms of the CMS is necessary. In this dissertation, the genetic properties, gene localization, homologous cloning and lines development of the restoration of CMS were studied.1. The restoration of Peterson's CMS in pepper was genetically analysed. The genetic mechanisms of fertility restoration was studied by using test cross between '77013A', a strict cytoplasmic-genic male sterile line, and a doubled haploid population of 114 pepper lines obtained from anther culture of the F1 hybrid ('Yolo Wonder' (sterility maintainer line) between 'Perennial' (fertility restorer line) ) and its parental lines. The fertility of the test-crossed lines was assessed under greenhouse and open field conditions with three criteria: Pollen Index (PI, visual estimation of pollen amount per flower), Pollen Number (PN, pollen counting under microscope), and Seed Number (SN, the number of seeds per fruit in open pollination). The correlation between every two criteria and the two cultivation methods was significant, and the correlation coefficient ranged from 0.55 to 0.84. Analysis of variance showed that the genotype (DH line) and environment were the significant sources of the variation in fertility. Narrow sense heritability of fertility restoration ranged from 0.38 to 0.92, depending on the criteria and environment. The distribution of the phenotype was continuous in the test-crossed lines indicating the quantitative inheritance of fertility restoration. The number of segregating genes regulating the restoration of CMS was estimated according the method of Snape et al. Results showed that three to four genetic factors were involved in pollen traits (PI and PN) and five to eight genetic factors in seed production (SN). The heredity analysis of the CMS will be helpful for understanding of the genetic mechanisms of the fertility restoration and the exploitation of the CMS in hybrid seed production.2. Fertility restoration of the Peterson's CMS in pepper is quantitative and environment-dependant. QTL (Quantitative Traits Locus) analysis of fertility restoration was performed based on the test-cross progeny between 77013A and a doubled haploid population of 114 lines obtained from the F₁ hybrid between Yolo wonder and Perennial. The fertility of the test-crossed lines was assessed under greenhouse and open field conditions using three criteria related to pollen or seed production. One major QTL for fertility restoration was mapped on the chromosome P6. It was significant in all the environments and for all the traits, accounting for 20% to 69% of the phenotypic variation, depending on the trait. Four additional minor QTLs were also detected on chromosomes P5, P2, and linkage groups PY3 and PY1, accounting for 7% to 17% of phenotypic variation. Most of the alleles increasing the fertility originated from the restorer parent of Perennial except for two alleles at minor QTLs. Phenotypic analysis and genetic dissection indicate that breeding pepper for complete sterility of female lines and high fertility hybrid requires complex combinations of alleles from both parents and a strict environment control.3. The transfer for restoration of CMS in sweet pepper was conducted with molecular marker assistance. A hot pepper fertility restorer line 'Xiangtanwan' and a sweet pepper CMS maintainer line '83-60' were used as materials, and a RAPD (Random amplified polymorphic DNA) marker, OPW19-800, as assisted selection. The restoration was assessed by using test-cross, and the node position and fruit size were investigated during 4 generations backcross and 2 generations self-cross. Using 'Zhongjiao No. 5' as control, the fruits size, fruit weight and yield were investigated in test-cross lines after four generations of back-cross followed by two generations of self-cross. Four lines, BC4(-12-2-l-3)I1, BC4(-12-2-l-4)I1, BC4(-16-2-6-7)I1 and BC4(-16-8-2-2)I1, were selected, whose fruit size of test-cross lines were 10.9 cm×7.0cm, 11.3 cm×7.7cm, 9.3 cm×6.9cm and 10.5 cm×6.5cm ('Zhongjiao No.5' 11.1cm×7.0cm), respectively, the average weight of fruits were 134.0g, 166.0g, 114.0g and 110.0g('Zhongjiao No.5' 151g), the average yield were 85.5%, 79.0%, 76.6% and 104.8% of 'ZhongjiaoNo. 5', and the fertility restoration reached 91.3%, 84%, 71%, and 62% of the normal lines.4. Genes related to fertility restoration of CMS in pepper were homologous cloned by using the information on Rf genes and PPR (Pentatrico Peptide Repeat). 'Perennial' and 'Yolo Wonder' were used as materials. Primers were designed based on the published conserved sequence in 5' end of the restoration gene and the conserved sequence in PPR gene (3' end primer) to amply the genomic DNA. PCR products were sequenced and BLAST was done in GenBank (www.ncbi.nih.gov). Comparison was done within the protein bank and the nucleic acid bank by using two commands of 'Blastx' and 'Blastn'. Three PCR products of 'Petuniua hybrida Vilm', six products of 'Perennial' and six products of 'Yolo Wonder' were obtained and then 11 products of them were sequenced. 'Y-1500', a l,200bp PCR product from 'Yolo Wonder', had 43.5% identity with an unknown functional gene in PPR in rice (Oryza sativa L.), which assumed that 'Y-1500' might be related to the fertility restoration of CMS in pepper.