초록 ▼

○ 연구결과
무의 자가불화합성 유전자의 분자적 특성을 분석하기 위하여 자가불화합성 유전자인 SLG, SRK 그리고 SP11를 포함한 거대단편을 해석하였다. 또한, 국내 무의 F₁ 품종 생산에 이용되고 있는 inbred line을 이용하여 PCR-RFLP에 의한 S haplotype의 동정 기술을 개발하였으며, S haplotype의 출현빈도를 분석하였다. SLG 특이적 primer 및 F₁ 순도 검정용 primer와 class I과 class II를 분류할 수 있는 primer를 SRK의

○ 연구결과
무의 자가불화합성 유전자의 분자적 특성을 분석하기 위하여 자가불화합성 유전자인 SLG, SRK 그리고 SP11를 포함한 거대단편을 해석하였다. 또한, 국내 무의 F₁ 품종 생산에 이용되고 있는 inbred line을 이용하여 PCR-RFLP에 의한 S haplotype의 동정 기술을 개발하였으며, S haplotype의 출현빈도를 분석하였다. SLG 특이적 primer 및 F₁ 순도 검정용 primer와 class I과 class II를 분류할 수 있는 primer를 SRK의 kinase domain에서 합성하여 적용하였다. 육성계통에 있어서 동정된 S haplotype의 종류와 출현빈도를 분석하였으며, S haplotype과 자가불화합성의 정도를 비교 분석하였다.

Abstract ▼

IV. Results of research & development and suggestions for utilization
Through analysis of genomic organization of the SLG/SRK/SP11 gene of the S locus in radish, we wish to secure molecular information. And, this research performed the PCR-RFLP system(primer design and restriction nzyme selectio

IV. Results of research & development and suggestions for utilization
Through analysis of genomic organization of the SLG/SRK/SP11 gene of the S locus in radish, we wish to secure molecular information. And, this research performed the PCR-RFLP system(primer design and restriction nzyme selection) development for determination of S haplotype in inbred lines.
1. The 72 kb fragment(obtained from Mlu I digested genomic DNA) that includes the SLG, SRK and SP11 genes of S⁹¹⁸ had cloned into Bacterial Artificial Chromosome(BAC) vector, and determined the nucleotide sequence of self-incompatibility genes. To confirm positive clone containing both SLG and SRK, we performed southern blot analysis using SLG⁴⁸ and SRK⁴⁸ probe of B. rapa class I S haplotype. These two probes detected the same bands in the positive clone, suggesting that the positive clone are includes the S-locus. One positive clone was digested Spe I or BamH I and subcloned into pBluescript II and pBIN19 plasmid vector. We determined the nucleotide sequence, and characterized the genomic organization of fragment by database searches and compares with S⁹ previously characterized S-locus of class I S haplotype in B. rapa. In this 72 kb genomic sequence, 12 putative genes, including SP11, SRK and SLG
were identified. The nucleotide and amino acid sequence of SLG-S⁹¹⁸ showed the highest homology to those of the SLG³³(B. oleracea, AB054730) 과 SLG^13-b(B. oleracea, AB024418). The predicted mature protein of SP11 gene contained 84 amino acids and eight cysteine residues, characteristic of members of the pollen coat protein(PCP) family of Brassica. SP11 was also showed the highest homology to those of the B. rapa SP11-60(AB067446) and B. oleracea SP11-15(AB089511) more than 95%.
2. This study investigated the PCR-RFLP system for the identification of S haplotypes in 192 inbred lines that are used as the parental lines of commercial radish F₁ hybrid cultivars in domestic 3 seed companies. PCR using the SLG-universal primer pair (USLf+USLr) amplified DNA fragments of about 1,200 bp from tested whole inbred lines. Digestion of the PCR products with restriction endonucleases (such as Sty I, Alu I, Msp I, Hinf I, Afa I) and subsequent Metaphor agarose gel electrophresis revealed polymorphism of the amplified DNA fragment. From several restriction endonucleases tested, the PCR products from the amplified with the SLG-universal primer pair showed the polymorphism of the electrophoretic profiles after digestion with Hinf I, Msp I and Afa I. When digested with Hinf I, the highest polymorphism as seventeen types based on their band patterns which were designated as S¹∼S¹7.The numbers of S haplotypes detected in the inbred lines were 17 and the highly frequent haplotypes were S¹(53%), S²(6.7%) and S³(15%). In Brassica vegetables, the most commom S haplotypes belong to class II; they exhibit a weaker self-incompatibility phenotype and are pollen-recessive to class I. This intense selection for uniformity could lead to the loss of S haplotypes and the accumulation of recessive S haplotypes in breeding.
3. The present investigation was performed with 88 inbred lines of radish belonging to used in F₁ hybrid production. DNA fragments of SLG alleles were amplified from all inbred lines by PCR using SLG universal primer pair USf and USr. Digestion of the PCR products with restriction endonucleases and subsequent Mataphor agarose gel electrophoresis revealed polymorphism of the amplified DNA fragment. Electrophoretic profiles of the PCR products after digestion with Hinf I and Msp I were classified into 11 band patterns(S¹∼S¹1) in all inbred lines. The numbers of S haplotypes detected in the inbred lines were 11 and the highly frequent S haplotypes were S⁷ (30.6%), S³ (23.8%), S¹1 (11.3%). Our presentinvestigation shows that the PCR-RFLP analysis using specific primer pairs of SLG is useful for identifying the S haplotypes in radish. To estimate the level of self-incompatibility of identified the S haplotypes by seeds set analysis, we calculated t e seeds set ratio, which corresponds to the ratio between number of seeds obtained and number of capsules after self-pollination. The seeds set ratio varied from 0% to 4.3% in all inbred lines. The average of seeds set ratio showed any significant statistical differences among different lines. The level of self-incompatibility for S-haplotype appeared to differ according to the genetic background of inbred lines. Consequently, it was expected that existence of other leading reason concerned in the degree of self-incompatibility.
4. One hundred seventy-eight inbred lines in R. sativus S homozygotes were used as plant materials. DNA fragments of the class I and class II S haplotypes were amplified by PCR using specific two primer pairs. Class I and class II primer pairs amplified a single DNA fragment of 900bp in 15 and of 1,050bp in 163 out of 178 inbred lines, respectively. These DNA fragments showed high polymorphism in electrophoresis after digestion with restriction endonucleases: 15 inbred lines amplified with class I primer pair were classified into 5 haplotypes and 163 inbred lines amplified with class II primer pair were classified into the 7 S haplotypes by EcoR II + Hinf I, respectively. The nucleotide sequences of the kinase domain of total 12 different S haplotypes were determined. Degrees of similarity of the nucleotide sequences to SRK12 (GenBank Acc. no: Z18921) of B. oleracea ranged from about 58% to 93%. Also, similarity of nucleotide sequences between SRKs in class I group was more than 88%, and in class II group was more than 90%. Nucleotide sequencing of the DNA fragments amplified from different types shows that the exons of SRK are highly conserved, and that there are high variations of the introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. In this study, the results of PCR-RFLP and sequencing were accurately the same. Therefore, the used class I and class II specific primer pairs, and endonucleases in PCR-RFLP analysis can be identify the characteristics of S-haplotypes which is used in breeding more precisely and quickly.
5. By analysis of 72 kb Mlu I genomic fragment containing both SLG and SRK genes, identified the SP11-S⁹¹⁸ of male determinant SI gene. For isolation of the other S haplotypes, We synthesized that could be amplified by PCR using the primer designed based on nucleotide sequence of SP11-S⁹¹⁸. PCR using the SP11-specific primer pair (SPC2f +SPC2r) amplified DNA fragments of about 400 bp from tested 5 radish inbred lines. We determined the nucleotide sequences of SP11 of total 5 different S haplotypes. The nucleotide sequence revealed a striking similarity in the region encoding the signal peptide such as Brassica. The high degree of sequence conservation between S haplotypes observed about 77∼96%. Also, the sequence similarity with class II S haplotype of B. rapa SP11-60 (AB067446) and B. oleracea SP11-15 (AB089511) exhibited 77∼93%, but with class I S haplotype of B. rapa SP11 S⁹ (BAA85458) lower degree of sequence conservation with approximately 20% sequence identity. The SP11 genes that isolated from inbred lines of radish predicted by class II S haplotype from the high degree of allelic sequence similarity with class II S haplotype of Brassica, originated from the same ancestral S haplotypes before speciation of these genera. Also, in the analysis of the 5'-flanking regions of SP11 genes by comparing the genomic sequences of the genera Brassica and Raphanus, alignment of these sequences are highly conserved (53∼96% identity) in the 5'-flanking region of SP11 genes. Similarities between the nucleotide sequences from the B. rapa and B. oleracea lines ranged from 76% to 96%, not significantly higher than the similarities of R. sativus lines (from 60% to 91%). In addition, the position and sequence of the putative TATA box were conserved in B. rapa, B. oleracea and R. sativus lines. Two direct repeats (TTTTAGATATAAA) and a putative pollen-specific element (CTTAAATTAGA) were identified from PCP-A class gene. From database searches in the 5'-flanking region, these repeats and a putative pollen-specific sequences were not also found in the 5'-flanking region of B. oleracea and R. sativus lines. Moreover other common repeat or palindromic sequence was not found in these sequences. Therefor, these sequences strongly support that the sprorophtic expression patterns in Brassica and Raphanus are controlled by different cis-regulatory elements in this region.
6. We developed methods for identification of S haplotypes by PCR-CAPS in inbred lines and primer pairs for evaluation of F₁ seed purity by using PCR in F₁ hybrid varieties. DNA fragments of SRK kinase domain were amplified from all inbred lines by PCR using primer pair SRK3f and SRK7r. Digestion of the PCR products with restriction endonucleases and subsequent revealed polymorphism of the amplified DNA fragmentand and determined the nucleotide sequences. Electrophoretic profiles of the PCR products after digestion with Hinf I and EcoR I were classified into 9 band patterns(R1∼R9) in tested 67 inbred lines. After ascertaining S haplotype by PCR-CAPS analysis, we synthesized the primer pairs for F₁ seeds purity evaluation for 5 S haplotypes of high frequence used in F₁ seed production. These primer pairs were classifiable the parental S haplotype of F₁ seeds with amplified size and specific amplification. We have evaluated F₁ seeds that is produ cted by crossing of inbred lines with R1/R2 and R5/R8. The parents of homozygous for the S-alleles was amplified by specific one fragment. The pure F₁ seeds of heterozygous for the S-alleles was amplified by specific two fragments with different size. But, seeds that is amplified one fragment of maternal line were found in commercial F₁ hybrid cultivars, and these seeds could presume to be pollution by crossing of S haplotype except maternal line or selfing.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 10
• CONTENTS ... 18
• 목차 ... 21
• 제1장 연구개발과제의 개요 ... 24
• 제1절 연구의 목적 및 중요성 ... 24
• 제2절 연구개발의 필요성 ... 24
• 제2장 국내·외의 연구동향 ... 28
• 제1절 국내·외의 관련연구의 기술현황 ... 28
• 제2절 연구의 전망 ... 29
• 제3장 연구개발수행 내용 및 결과 ... 30
• 제1절 무의 자가불화합성 유전자 좌(5-locus)의 genomic 구조 분석: S918 haplotype의 자가불화합성 유전자의 특성 및 동정 ... 30
• 1. 서론 ... 30
• 2. 연구재료 및 방법 ... 31
• 3. 연구결과 ... 36
• 가. BAC vector 구축 및 Southern blot 분석에 의한 5-locus의 확인 ... 36
• 나. 5-locus의 subcloning에 의한 주두측 자가불화합성 유전자 동정 ... 38
• 다. 화분측 자가불화합성 유전자의 단리 및 특성 ... 43
• 4. 고찰 ... 46
• 제2절 주두측 자가불화합성 유전자(SLG)의 PCR-RFLP에 의한 육성계통의 S haplotype 결정 ... 49
• 1. 서론 ... 49
• 2. 재료 및 방법 ... 50
• 3. 연구결과 ... 51
• 가. 주두측 자가불화합성 유전자(SLG)의 PCR-HPLP 분석 ... 51
• 나. 동정된 S haplotype의 출현빈도 분석 ... 54
• 4. 고찰 ... 55
• 제3절 무 육성계통의 자가불화합성 반수형 동정 및 자가불화합성 정도와의 관계 ... 57
• 1. 서론 ... 57
• 2. 연구재료 및 방법 ... 58
• 3. 연구결과 ... 60
• 가. 자가불화합성의 유전자형 동정 ... 60
• 나. 자가불화합성의 점도 분석 ... 63
• 4. 고찰 ... 66
• 제4절 SRK의 PCR-RFLP 및 DNA 염기서열 결정에 의한 무 육성계통의 S haplotype 동정 ... 67
• 1. 서론 ... 67
• 2. 연구재료 및 방법 ... 68
• 3. 연구결과 ... 70
• 가. SRK kinase domain의 PCR-RFLP 분석 ... 70
• 나. 염기서열 결정에 의한 S haplotype 분석 ... 74
• 4. 고찰 ... 78
• 제5절 화분측 자가불화합성 S 유전자(SP11/SCR) 및 화분 특이적 promoter의 분석 ... 80
• 1. 서론 ... 80
• 2. 연구재료 및 방법 ... 81
• 3. 연구결과 ... 82
• 가. 화분측 자가불화합성의 SP11 유전자의 단리 및 특성 분석 ... 82
• 나. 화분 특이적 Promoter 영역의 분석 ... 84
• 4. 고찰 ... 87
• 제6절 SRK의 kinase domain을 이용한 PCH-CAPS에 의한 S-alleles 동정 및 F1 종자 순도검정 ... 90
• 1. 서론 ... 90
• 2. 연구재료 및 방법 ... 91
• 3. 연구결과 ... 93
• 가. SRK kinase domain의 PCR-CAPS메 의한 S haplotype 동정 ... 93
• 나. F1 순도 검정용 pimer 합성 및 검정 ... 99
• 4. 고찰 ... 101
• 제4장 목표달성도 및 관련분야에의 기여도 ... 103
• 제1절 연구개발목표의 달성도 ... 103
• 제2절 관련분야의 기여도 ... 104
• 제5장 연구개발결과의 활용계획 ... 105
• 1. 추가연구의 필요성 ... 105
• 2. 타 연구에의 응용 ... 105
• 3. 기업화 추진방향 및 금후의 활용 ... 105
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 106
• 제1절 배추과 식물의 자가불화합성 연구 현황 및 금후 연구 방향 ... 106
• 제7장 참고문헌 ... 110
• 끝페이지 ... 117