以次世代定序定位一個水稻粒長基因

Abstract

隨著氣候變遷及世界人口成長快速，糧食供應逐漸成為重要議題，水稻為世界重要的糧食作物，因此，水稻產量提升為現今育種之重要目標。穀粒外型為產量構成要素之一，本研究從Taiwan Rice InsertionalMutants (TRIM) 族群中挑出一株具高株、長穀粒之T-DNA插入突變品系TALL，針對穀粒外型進行研究。前人分析結果顯示長穀粒性狀與T-DNA插入不具相關性，因此本研究利用TALL與台稉9號建立1000株F2族群，並以次世代定序 (next-generation sequencing) 為基礎之分群分析法 (bulked segregant analysis) 定位穀粒長基因。針對定位族群進行外表型鑑定後，本研究以五組F2樣本進行次世代定序及後續資料分析，分別為混合20株、40株及兩個單株長穀粒F2個體及混合20株短穀粒F2個體。結果顯示四筆長穀粒F2樣本於第三條染色體近中節處存在一段非重組區域，其遺傳背景大多由TALL所組成；然而，混合20株短榖粒樣本於此處能正常分離。隨後比較混合20株、40株及兩者合併之混合60株長穀粒F2樣本，可將候選區間縮小至15-17.5 Mb。針對假定SNP頻率為1之位點於此區間進行基因註解後，顯示共有172個錯義突變 (missense mutation) 及108個沉默突變 (silent mutation)。第17365441個由C變成A的突變位點，導致此區間內唯一一個基因提早出現終止密碼子 (early stop codon)，此基因為已發表之穀粒長基因座GS3，由於此突變位點基因型及位置皆與前人研究相同，因此我們推測GS3極有可能為目標基因。相較於傳統雙親定位法，本研究使用少量F2個體並以次世代定序輔助，便能快速定位目標基因，故此新型分析法可應用於定位其他重要農藝性狀基因座。此外本研究亦發現台農67號與TALL於第三條染色體之候選區間內存在超過5200個同結合SNP，此種變異可能來自體細胞變異 (somaclonal variation)，但其原因仍有待日後進行更深入研究。

With rapid population growth and climate changes, sufficient food production is a major challenge. Rice is one of major crops in the world, and the major goal is to increase grain yield. Rice grain length is an important component of grain yield, so we examined the T-DNA mutant line TALL,with agronomically important long grain length and tall height. The mutant line does not have the recessive phenotype cosegregated with its T-DNA insertion, so we used bulked segregant analysis (BSA) with next-generation sequencing (NGS) to isolate the gene controlling the important traits. We crossed Tall163 and an elite rice cultivar, TK9, showing normal grain length, and generated an F2 population of 1000 individuals. After phenotyping, we divided 62 long-grain and 20 normal-grain plants into 5 samples:2 bulked samples of 20 and 40 long-grain plants and 1 of 20 normal-grain plants, and 2 samples of 1 long-grain plant each. The 4 long-grain samples had a non-recombinant region close to the centromere region of chromosome 3 and showed the same genotype as TALL. By combining the2 mutant bulked samples into a sample of 60 long-grain plants and comparing with the 2 mutant bulked samplesof 20 and 40 mutant plants, the location of the candidate gene could be narrowed down to a 2.5-Mb region. Annotating each position in the candidate region with putative SNP frequency of 1 revealed 172 missense mutations and 108 silent mutations.Futhermore, the only mutationwith an early stop codon was caused by a C-A mutation in position 17365441, which is the published gene, GS3. The mutant genotype and position were verified with previous studies, so GS3may be the candidate gene. With few F2 plants and a short time, we identified the candidate gene by NGS-based BSA, which may be useful in identifying other agronomical traits.TALLshows more than 5200 homozygous SNPs in the candidate region of chromosome 3, possibly caused by somaclonal variations, as compared with its parent line TNG67, but further studies are needed to identify the mechanism of the somaclonal variations.