應用雙限制酶切位點標定法定位番椒稔性恢復基因

Construction of a High Density Linkage Map with Double Digest RADseq and Mapping of Restorer-of-Fertility Gene in Pepper (Capsicum annuum L.)

高驥(Chi Kao)

指導教授 ： 胡凱康

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201610013

摘要

番椒 (Capsicum annuum L.) 屬茄科 (Solanaceae) 番椒屬，為重要經濟蔬菜作物。目前商業上主要以細胞質與細胞核雄不稔系統 (Cytoplasmic-Genetic Male Sterility, CMS) 來降低雜交種子之成本，而稔性恢復基因 (Restorer-of-fertility, Rf) 為此系統之核心基因。承2012年龔以SSR分子標誌分析204株番椒F2找尋Rf基因連鎖SSR之研究成果，本研究挑選其中80株F2材料與兩親本使用雙限制酶切位點標定法 (double digest Restriction Associated DNA sequencing, ddRAD)，簡稱雙重酶切系統，建立具有條碼標示的定序文庫 (library)。次世代定序 (Next generation sequencing, NGS) 採用Illumina HiSeq平台，定序結果以Stacks作為核心程式進行分析，獲得2096組具有多型性且缺值小於10%之SNP分子標誌。結合先前研究之SSR分子標誌，建立同番椒染色體數目12個連鎖群，1277個分子標誌，總長為1623.25 cM之連鎖圖譜。SNP分子標誌在連鎖圖譜上的分布較物理圖譜平均，染色體中段每圖譜單位的序列長度較兩端長，與番椒染色體上異染色質的分布一致；並且在這些區域中所獲得的SNP分子標誌相對較少，顯示本研究中所採用對於甲基化敏感的PstI限制酶可有效避開異染色質區域，使開發獲得的分子標誌能均勻分布於連鎖圖譜。本次研究找尋Rf基因兩端連鎖距離範圍內分子標誌組合，從定序資料設計ASP (Allele-specific PCR)、TaqMan等SNP分析平台引子，驗證204株F2基因型，將Rf基因定位於分子標誌32081與27394之間，遺傳距離分別為5.8 cM與9.5 cM。將此SNP組合用於Rf基因分子輔助回交選種，於回交4代時，仍可確保98%之植株保留Rf基因，與傳統育種使用後裔檢定相比可大幅提升育種之效率。本研究結果顯示雙重酶切系統對於建立複雜基因組物種的高密度連鎖圖譜與其遺傳相關研究是一個有效的工具。

關鍵字

番椒 ； 雙限制酶切位點標定法 ； 連鎖圖譜 ； 稔性回復基因

並列摘要

Pepper (Capsicum annuum L.) is an important vegetable crop worldwide as well as in Taiwan. Production of commercial hybrid seeds relies on cytoplasmic-genetic male sterility (CMS) system governed jointly by a nuclear Restorer-of-fertility (Rf) gene and male-sterile cytoplasm. Linked molecular markers are useful tool to monitor the transferring of Rf gene during backcross breeding. In this study, a barcode tagged sequencing library was constructed following the double digest Restriction Associated DNA sequencing (ddRAD) scheme with 80 plants randomly selected from a previously mapped F2 population as well as both parents. Sequencing was conducted on an Illumina HiSeq platform and the obtained reads were analyzed by the Stack program, resulting 2096 polymorphic SNP markers with call rate larger than 90%. Combining with the previously mapped SSR markers, a linkage map of 1623.25 cM, consisted 1277 markers on 12 linkage groups was constructed. SNP markers on the linkage map were more uniformly distributed than their locations on the physical map. Drastic increase of base pairs per map unit toward the center regions of chromosomes consistent with the distribution of heterochromatin on pepper chromosomes. The relatively low frequencies of SNP markers discovered in these regions suggested that the use of methylation-sensitive restriction enzyme PstI as the rare cutter in ddRAD may be an effective way for methylation filtration. Positions of SNP markers surrounding Rf gene were validated using 204 F2 plants of the previously SSR mapped population. The Rf gene was located between markers 32081 and 27394, with genetic distances of 5.8 cM and 9.5 cM, respectively. With the aid of these SNP markers, the probability of retaining Rf gene after 4 generations of backcross is increased to 98%.

並列關鍵字

pepper ； ddRAD ； linkage map ； Restorer-of-fertility

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