【摘要】：梨屬于薔薇科蘋果亞科梨屬植物,是典型的配子體自交不親和性植物(gametophytic self-incompatibility,GSI),其自交不親和性由單一基因位點(diǎn)的S-RNase復(fù)等位基因(S基因)所控制。在梨栽培中必須配以不同的S基因型授粉品種才能保證授粉座果的順利進(jìn)行。梨品種S-RNase基因的鑒定可以為梨栽培過程中授粉品種的準(zhǔn)確配置和育種過程中親本的合理選擇提供理論依據(jù)。我國梨品種S基因型復(fù)雜,不管是地方栽培品種還是野生型個(gè)體都蘊(yùn)含著豐富的S基因資源。為完善梨S基因鑒定的技術(shù)體系,加快梨品種S基因資源挖掘與梨品種S基因型鑒定的進(jìn)程,本文制備了梨S-RNase基因cDNA芯片并對芯片系統(tǒng)檢測條件進(jìn)行了優(yōu)化,利用S-RNase基因cDNA芯片并行檢測的特點(diǎn)快速準(zhǔn)確鑒定了部分梨品種S基因型、挖掘新的S基因資源并對新挖掘的S基因資源進(jìn)行分離鑒定和分子進(jìn)化分析。主要研究結(jié)果如下:(1)S基因cDNA芯片制備。根據(jù)存在于東方梨品種中已知的S1-S10、S12-S13、S15-S22、S25-S32和S34-S52的序列特征及結(jié)構(gòu)特點(diǎn),以各S等位基因包含HV區(qū)的PF-PR1 mRNA序列作為cDNA探針,研制了 S基因cDNA檢測芯片;實(shí)現(xiàn)了利用S基因芯片檢測系統(tǒng)快速檢測梨品種S基因的準(zhǔn)確性和可靠性。(2)S基因cDNA芯片雜交體系優(yōu)化。獲得芯片最佳雜交條件為:最佳雜交溫度42℃,最適雜交時(shí)間8～9 h,最適PCR純化產(chǎn)物終濃度為200 ng μl-1,洗滌條件:2× SSC + 0.1%SDS、O.1× SSC + 0.1%SDS、O.1× SSC 溶液依次避光洗滌 3 min,蒸餾水沖洗2～3次(每次不超過10s),玻片甩干機(jī)離心機(jī)干燥玻片。(3)50個(gè)梨品種S基因型鑒定。利用S基因cDNA芯片優(yōu)化雜交體系完成了對50份不同種質(zhì)資源梨品種(主要是砂梨品種)S基因鑒定,確定了各品種的S基因型。優(yōu)化后的S基因芯片檢測系統(tǒng)能成功鑒定出砂梨品種所含有已知的S基因資源,同時(shí)根據(jù)芯片雜交信號可推測品種中新S基因的存在。(4)分離和鑒定了梨7個(gè)新S等位基因。對芯片系統(tǒng)鑒定只含有1個(gè)雜交信號、無雜交信號的品種,輔以酶切和DNA測序,從砂梨品種中分離獲得了PpS53(P.Pyrifolia S53)、PpS54、PpS55、PpS56、PpS57、PpS58、PpS59 等新 S等位基因的序列,均已登錄到GenBank數(shù)據(jù)庫。(5)7個(gè)新S基因資源的進(jìn)化分析。利用砂梨品種S基因和薔薇科其它植物的S基因DNA序列研究了砂梨S基因的序列多態(tài)性和分子進(jìn)化關(guān)系,確定7個(gè)新S基因間及與薔薇科其它植物的S基因間的進(jìn)化關(guān)系。(6)完善了梨S基因鑒定的技術(shù)體系。通過梨S基因芯片檢測系統(tǒng)的建立,完善了梨S基因鑒定和S基因資源研究的技術(shù)體系,為梨豐產(chǎn)栽培和新品種的選育奠定了科學(xué)基礎(chǔ)。
[Abstract]:Pear is a typical gametophyte self-incompatibility plant (gametophytic self-incompatibility,GSI), which is controlled by S-RNase complex allele (S gene) of single locus. Different S genotype pollination varieties must be used in pear cultivation in order to ensure the success of pollination. The identification of S-RNase gene in pear varieties can provide theoretical basis for the accurate allocation of pollinated varieties and the reasonable selection of parents in the course of pear breeding. The S genotypes of pear varieties in China are complex, both local cultivars and wild individuals contain abundant S gene resources. In order to perfect the technical system of pear S gene identification and accelerate the process of resource mining of pear variety S gene and identification of pear variety S genotype, the S-RNase gene cDNA chip of pear was prepared and the detection conditions of the chip system were optimized. Based on the parallel detection of S-RNase gene cDNA chip, the S-genotypes of some pear varieties were quickly and accurately identified, new S-gene resources were mined, and the newly excavated S-gene resources were isolated and identified, and the molecular evolution analysis was carried out. The main results are as follows: (1) S gene cDNA chip preparation. According to the sequence characteristics and structural characteristics of S1-S10 S12-S13 S15-S22 S25-S32 and S34-S52, the S gene cDNA detection chip was developed using the PF-PR1 mRNA sequence of each S allele including HV region as cDNA probe. The accuracy and reliability of rapid detection of S gene by using S gene chip detection system were realized. (2) the cDNA chip hybridization system of S gene was optimized. The optimum hybridization conditions were obtained as follows: the optimum hybridization temperature was 42 鈩，

本文編號：2207250