초록

○ 연구결과
배 및 사과 품종의 화분관 검경에 의한 자가불화합성 정도 분석, 교배에 의한결실율 조사, S-RNase유전자의 PCR-RFLP분석, cloning 및 염기서열 분석에 의한 S-유전자형 결정 등을 수행하였다. S-genotype을 유묘기에 결정하는 방법(primer 및 제한효소 선발)을 개발하였으며, 배의 새로운 대립유전자를 발견하였다. 또한 S-호모접합체, 자가결실성 계통 및 속간잡종을 신속, 저비용으로 정확히 선별할 수 있는 S-RNase유전자의 PCR-RFLP 분석 체계를 개발하였다.

Abstract ▼

IV. Results of research & development and suggestions for utilization
This study performed analysis of self-incompatibility degree with pollen test, quality investigation by inbreeding, PCR-RFLP analysis of S-RNase gene, cloning and sequencing for determination of S-genotype in apple and pear cul

IV. Results of research & development and suggestions for utilization
This study performed analysis of self-incompatibility degree with pollen test, quality investigation by inbreeding, PCR-RFLP analysis of S-RNase gene, cloning and sequencing for determination of S-genotype in apple and pear cultivars. We were developing the method(primer design and restriction enzyme selection) for determination of S-genotype at young plant and found the new S-allele in pear. In addition, we develop the S-allele specific PCR-RFLP system, which is able to distinguish completely, and rapidity S-homozygotes, self-compatible strains and intergenus hybrid with low pay.
1. To elucidate the S-genotype of Korean-bred pear cultivars, whose parents are heterozygotes, the PCR amplification using S-RNase primers that are specific for each S-genotype was carried out in 15 Korean-bred pear cultivars and 5 Japanese-bred pear cultivars. The difference of the fragment length was shown in the following order: S₆ (355 bp) < S₇ (360bp) < S₁ (375 bp) < S₄ (376 bp) < S₃ and S₅ (384 bp) < S₈ (442 bp) < S₉ (1,323 bp) < S₂ (1,355 bp). The size of the introns was as follows: S₁ = 167 bp, S₂ = 1,153 bp, S₃ = 179 bp, S₄ = 168 bp, S₅ = 179 bp, S₆ = 147 bp, S₇ = 152 bp, S₈ = 234 bp, S₉ = 1,115 bp. There were five conservative and five hypervariable regions in the introns of S₁, S₃, S₄, S₅, S₆ and S₇-RNase.
A pairwise comparison of these introns of S-RNase revealed homologies as follows: 93.7% between S₁- and S₄-RNase, 93.3% between S₃- and S₅-RNase and 78.9% between S₆- and S₇-RNase.
2. PCR-RFLP and S-RNase sequencing determined the S-genotype of the pear cultivars. The S-genotype was S₄S₉ for Shinkou, S₃S₉ for Niitaka, S₃S₅ for Housui, S₁S₅ for Kimizukawase, S₁S₈ for Ichiharawase, S₃S₅ for Mansoo, S₃S₄ for Shinil, S₃S₄ for Whangkeumbae, S₃S₅ for Sunhwang, S₃S₅ for Whasan, S3S5 for Mihwang, S₅S for Chengsilri, S3S5 for Gamro, S3S4 for Yeongsanbae, S₃S₄ for Wonhwang, S₃S₅ for Gamcheonbae, S₃S₅ for Danbae, S₃S₄ for Manpoong, S₃S₄ for Soowhangbae and S4S6 for
Chuwhangbae. The information on the S-genotype of pear cultivars will be used for the pollinizer selection and breeding program.
3. S-genotype of the newly bred 22 Korean-bred pear strains, 2 Korean-bred cultivars and 3 Japanese cultivars were identified using PCR-CAPS analysis. S-RNase genes had an intron, which was hypervariable region, between exons whose homologies were fairly high among S1 to S7 RNase.
In addition, the specific restriction endonucleases for each S-allele were selected such as EcoRI(S₁- and S₄-RNase), NdeI(S₄-RNase), Eco0109I(S₃- and S₅-RNase), AlwNI(S₅-RNase), MluI(S₆- and S₇-RNase), and HinCII (S6-RNase).
4. A PCR amplification using newly designed S-RNase primers was carried out in 5 Korean-bred pear cultivars and 10 Japanese-bred pear cultivars. A new S-allele, designed as S₁₀, was discovered from Chengsilri, which intron S-RNase was constructed 1,513 bp nucleotide and exons were constructed 213 bp nucleotide sequence could be translated into 71 amino acid sequences. The S₁₀-RNase contained the conserved regions three cysteine residue characteristic of S-RNase and with one histidine residue essential for RNase activity in pear. The deduced nucleotide sequence of S1-RNase showed a high similarity to S₄-RNase (97.4%) and S₁₀-RNase shows 77.8 %(S5) to 84.4% (S₄) similarities with the other S-RNases.
S₁₀-RNase had specific restriction endonuclease site, 'HhaI', with digests 1,235 bp and 491 bp. The S-genotype of 'Chengsilri' was determined S₅S₁₀.
5. Breeding of the pear cultivars depends on artificial pollination that requires much labor, many worker and time period. The pear cultivar 'Osanijisseiki' (S₂S₄^sm; sm= stylar-part mutant) has been used as aa parent to breed a self-compatible cultivars with excellent fruit. We have developed a PCR-RFLP system that could screened out self-compatible strain from the offsprings between the self-incompatible cultivar and self-compatible cultivar. Recently, the nucleotide sequences of the S-RNase(S₁∼S₉) were determined from genomic DNA. Using the intron information, we could screened out the self-compatible strains with digestive only S₄-, S₅-, S₆- and S₉-RNase fragments and self-incompatible strain with digestive S₂- and S₉-RNase fragments. Also, we determined the S-genotype of eight self-incompatible strains and screened the eight self-compatible strains by S-allele based PCR-RFLP system.
6. To resolve the discrepancies in S-allele assignment, we have re-examined the identity of S-alleles known from apple cultivars. Based on nucleotide sequence of 16 cDNA, we designed allele-specific primer pairs to selectively amplify a single S-allele per reaction. In addition, S-RNase universal primer (ApFN and anti-ApRN) developed from conserved region of base sequence, which used to amplify S-gene in domestic bred apple cultivars and world apple cultivars. The amplification fragment was clone and analyzed nucleotide sequence. Therefore, we obtained information of 6 S-RNase sequences.
7. S₉-allele and new S-allele was identified from apple cultivar 'gamheong', 'Saenara' and 'Heongro' by PCR-RFLP and DNA sequencing
8. The pear and apple has gametophytic self-incompatibility that is not fertilization when used the pollen of same cultivar, the pollen tube extension is inhibit by the S-RNase in the stigma side. The self-fruiting rate of pear and apple is very low, so orchard must be mixed plant the pollinizer, which is high fruiting and compatible. Our research team performed investigation of quality and diallel cross for select pollinizer by using the information of S-genotype of pear and apple cultivar. The result of pollen test was show when self-incompatible gene is different; the pollen tube permeation almost became accomplished 100%. But one self-incompatible gene will be same it was positive for 50% to decrease and pollen and stigma has same S-RNase, pollen tube is can not invade wholly.
9. The non-affinity degree between the cultivars, which is publish to a pollen tube microscopic examination, and the result of S-genotype of apple and pear, which is decided by sequence and PCR-RFLP analysis, was visible agrees. With fruiting ratio and fertility ratio it received the environment effect at this year. However, investigation result of fruiting ratio and pollen tube test result agreed with each other.
10. We performed diallel cross and fruiting ratio investigation to investigate the quality of the fruit and investigated hormone content change and quality of the fruit which it interbreeds. When 'Yeongsanbae'(S₃S₅) with 'Chojuurou' (S₂S₃), 'Niitaka'(S₃S₉) with 'Nijisseiki'(S₂S₄), 'Whangkeumbae'(S₂S₄) with 'Nijisseiki'(S₂S₄) interbreeding in pear, it got the fruit which shows a most good quality. When 'Fuji'(S₁S₉) with 'Sekaiichi'(S₃S₉) interbreeding in apple, it got the fruit which shows a most good quality.
11. By bud pollination, 7 pear cultivars and 4 apple cultivars were performed selfing. It was success to raising the S-homozygotes. Moreover, we were screened four pear S-homozygotes and one apple S-homozygote by PCR-RFLP analysis.
12. To obtain the intergenus hybrids, pear and apple were crossed reciprocally.
We screened out five hybrids from offsprings between pear and apple.
Development of molecular methods is very useful for applying of breeding and determination of S-genotypes is able to apply in pollinizer selection.
Namely, we were able to obtain the information, which is when the artificial pollination, how much to use the amount of diluents is efficiently. In addition, developed skill will be able to apply to Prunus genus(plum, mume, almond, sweet cherry), which has self-incompatibility, with apple and pear.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 8
• CONTENTS ... 15
• 목차 ... 18
• 제1장 연구개발과제의 개요 ... 21
• 제1절 연구의 목적 및 중요성 ... 21
• 제2절 연구개발의 필요성 ... 22
• 제2장 국내.외의 연구동향 ... 25
• 제1절 국내.외의 관련연구의 기술현황 ... 25
• 제2절 연구의 전망 ... 27
• 제3장 연구개발수행 내용 및 결과 ... 29
• 제1절 S-RNase 염기서열과 PCR-RFLP 분석에 의한 배(Pyrus pyrifolia) 품종의 자가불화합성 유전자형 결정 ... 29
• 1. 서 론 ... 29
• 2. 연구재료 및 방법 ... 30
• 3. 연구결과 ... 31
• 가. 배 품종의 자가불화합성 유전자의 클로닝 및 염기서열 분석 ... 31
• 나. PCR-RFLP 분석에 의한 배 품종의 자가불화합성 유전자형 결정 ... 36
• 4. 고찰 ... 41
• 제2절 PCR-CAPS에 의한 국내 육성 배 육성계통의 자가불화합성 유전자형 결정 ... 43
• 1. 서론 ... 43
• 2. 연구재료 및 방법 ... 44
• 3. 연구결과 ... 45
• 가. 7개의 자가불화합성 유전자의 염기서열 결정 ... 45
• 나. 자가불화합성 유전자의 특이적인 제한효소 선발 ... 48
• 다. PCR-CAPS에 의한 배 계통의 자가불화합성 유전자형 결정 ... 49
• 4. 고찰 ... 52
• 제3절 배(Pyrus pyrifolia)의 새로운 S-RNase의 동정 및 PCR-RFLP에 의한 자가불화합성 유전자형의 결정 ... 53
• 1. 서론 ... 53
• 2. 연구재료 및 방법 ... 54
• 3. 연구결과 ... 55
• 가. PCR증폭에 의한 새로운 자가불화합성 유전자 동정 ... 55
• 나. 새로운 S-allele의 염기서열 분석 및 PCR-RFLP 분석 ... 56
• 4. 고찰 ... 61
• 제4절 PCR-RFLP법에 의한 자가화합성 계통 선발 ... 66
• 1. 서론 ... 66
• 2. 연구재료 및 방법 ... 67
• 3. 연구결과 ... 69
• 가. 자가화합계통 선발법 개발 ... 69
• 나. 자가불화합성 유전자의 염기서열 결정 및 제한효소 선발 ... 70
• 다. PCR-RFLP 체계에 의한 자가화합 계통선발 ... 72
• 4. 고찰 ... 77
• 제5절 S-allele 염기서열과 PCR-RFLP 분석에 의한 사과 품종의 자가불화합성 유전자형 결정 ... 79
• 1. 서론 ... 79
• 2. 연구재료 및 방법 ... 80
• 3. 연구결과 ... 84
• 가. RT-PCR에 의한 cDNA의 염기서열 결정 및 분석 ... 84
• 나. PCR-RFLP 분석에 의한 사과의 자가불화합성 유전자형 결정 ... 93
• 다. Genomic DNA를 이용한 자가불화합성 유전자의 염기서열분석 ... 96
• 라. 사과 품종의 자가불화합성 유전자형 정리 ... 101
• 4. 고찰 ... 102
• 제6절 화분관 검경 및 이면교배에 의한 교배친화성, 양.질적 형질분석 및 최적 수분수 선발 ... 104
• 1. 서론 ... 104
• 2. 연구재료 및 방법 ... 105
• 3. 연구결과 ... 106
• 가. 이면교배 후 교배친에 따른 화분관 행동조사 ... 106
• 나. 사과, 배 주요 육성품종별 교배조합에 따른 착과율 조사 ... 113
• 다. 수분수에 따른 품종의 형질조사 ... 120
• 4. 고찰 ... 123
• 제7절 자가불화합성을 이용한 S-호모접합체의 선발 ... 124
• 1. 서론 ... 124
• 2. 연구재료 및 방법 ... 124
• 3. 연구결과 ... 125
• 가. 배 품종의 S-호모접합체 육성 및 선발 ... 125
• 나. 사과 품종의 S-호모접합체 육성 및 선발 ... 127
• 4. 고찰 ... 128
• 제8절 자가불화합성 유전자의 PCR-RFLP법에 의한 배와 사과의 속간 교잡후대 선발 ... 129
• 1. 서론 ... 129
• 2. 연구재료 및 방법 ... 130
• 3. 결과 ... 132
• 가. 배와 사과간의 속간 교배 ... 132
• 나. RAPD에 의한 속간잡종 선별 ... 133
• 4. 고찰 ... 137
• 제4장 목표달성도 및 관련분야에의 기여도 ... 140
• 제1절 연구개발목표의 달성도 ... 140
• 제2절 관련분야의 기여도 ... 141
• 제5장 연구개발결과의 활용계획 ... 143
• 1. 추가연구의 필요성 ... 143
• 2. 타 연구에의 응용 ... 143
• 3. 기업화 추진방향 및 금후의 활용 ... 143
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 144
• 제1절 배 및 사과의 자가불화합성 관련 논문 정보(2001년 이후) ... 144
• 제2절 해외기관의 연구과제 관련 과학기술정보 ... 148
• 제7장 참고문헌 ... 151
• 끝페이지 ... 160