Hd1、Hd6與Ehd1基因對水稻抽穗期之影響暨野生稻DNA分子標幟之開發

Abstract

Hd1、Hd6與Ehd1基因對水稻抽穗期之影響 稉稻越光品種具有顯性的Hd1、Ehd1對光週期敏感的基因與隱性的hd6對光週期不敏感的基因，栽種於日本時具有良好的品質與產量，但在台灣因對光週期敏感而提早抽穗，降低產量與品質。台農67號為台灣優良栽培品種，帶有隱性的hd1、ehd1對光週期不敏感的基因與顯性的Hd6對光週期敏感的基因。本研究利用分子輔助選種 (marker assisted selection, MAS) 將台農67號三個抽穗期基因hd1、Hd6和ehd1導入越光中，建立了衍生自編號為＃286-85-A4、＃286-14-A23和＃286-85-A27三株單株之BC3F2族群。2009年二期作於田間自然光照下，三族群之抽穗期皆出現連續性分部，經分析後發現三個基因對抽穗期之影響並不一致，以Ehd1影響抽穗期效應最大，且基因型為ehd1 ehd1者有較長的抽穗期，延遲天數最長可達23.5天，其次為Hd1，以Hd6對抽穗期的影響最小。在三個基因三種不同基因型構成的27種組合中，以A27族群Hd1Hd1 Hd6 hd6 Ehd1Ehd1組合抽穗期的69.4天最早；A4族群hd1hd1 Hd6Hd6 ehd1ehd1之組合的109天抽穗期最晚，大多數組合的抽穗期則介於80 ∼ 90天左右。比較越光與台農67之平均抽穗期後顯示透過抽穗期基因的改變，可達延遲越光抽穗的目的；不同的基因型組合對抽穗期造成不同程度的延遲。透過MAS可育成帶有不同抽穗期基因之越光近似同源系，評估基因型對抽穗期的影響，選育出有最適抽穗期的品種，以適應不同的栽種環境與制度。

野生稻DNA分子標幟之開發 水稻經由人類之馴化、育種培育出許多優良的栽培品種，造就龐大的經濟價值，然造成栽培種的遺傳歧異度越趨一致，為了提高栽培種的遺傳變異性，除了對地方種種原的再利用外，亦可由不同的野生種獲得新的遺傳資源，用以因應詭譎多變的氣候變遷與世界糧食需求激增等挑戰。開發多型性分子標幟及建立連鎖圖譜，將有助於探勘野生稻有用之基因，如耐生物逆境和非生物逆境基因，及分子標幟輔助選種之實行 (Gupta et al., 2002) ，以加速水稻之育種。本研究最終目的為將野生稻O. officinalis (CC基因體) 和O. australiensis (EE基因體) 之重要基因導入於稻O. sativa (AA基因體) 之稉稻台農67號 (TN G67) 和秈稻台中秈10號 (TCS 10)，現階段之目標為開發多型性分子標幟。 篩選249個水稻 SSR（simple sequence repeat又稱為Rice Microsatellite，微衛星）分子標幟，只有72 (72%) 個和71 (71%) 個分子標幟能在O. officinalis vs. TCS 10和O. officinalis vs. TNG 67間呈現多型性；而只有51 (76%)個和53 (79%) 個能在O. australiensis vs. TCS 10和O. australiensis vs. TNG 67間呈現多型性。因SSR分子標幟是以栽培稻種O. sativa進行設計引子對，不同基因體間基因染色體結構體歧異度大，造成SSR引子序列於野生稻之鍊合成功率低。為能更有效率的取得具多型性的分子標幟，以生物資訊方法進行栽培種與野生種的基因體序列比較，依據插入/缺失（indel）DNA片段，一共設計了121個分子標幟，在 O. officinalis vs. TCS 10和O. officinalis vs. TNG 67間呈現多型性的有79 (72%) 個與82 (75%) 個分子標幟；在O. australiensis vs. TCS 10和O. australiensis vs. TNG 67間呈現多型性的分別有64 (62%) 個和65 (63%) 個分子標幟。此策略能較有效率的獲得能在野生種與栽培種間呈現多型性的分子標幟，進而進行基因圖譜分析或以分子標幟輔助選種 (marker assisted selection, MAS) 建立帶有野生稻染色體片段的部分染色體置換系族群 (chromosome segment substitution lines, CSSLs)，供研究或育種使用，提高野生種原的利用效率。 以多型性分子標幟進行O. officinalis × TNG 67衍生之BC1F1、BC2F1世代之染色體結構分析。因著野生稻與栽培稻種因親源與染色體結構上之差異，二物種間染色體不易發生重組，二個回交世代之染色體中，異型合子比例由86.7％ (BC1F1) 下降為18.1％ (BC2F1)，而TNG 67同型合子比例則大幅提升，顯示野生稻之染色體片段迅速的自子代染色體中剔除，因此不易建立完整的部分染色體片段置換系。

The Effects of Hd1, Hd6, and Ehd1on Rice Heading Date TNG 67 was an elite cultivar in Taiwan. This variety carried hd1, hd6 and ehd1 three recessive genes and insensitive to photoperiod. The Japanes elite cultivar, Ko-shihikari, possessing good quality, is very popular not only in Japan but also in Taiwan. However, Koshihikari heads early in Taiwan because of 3 photoperiodic genes Hd1, Hd6 and Ehd1. One of elite cultivar in Taiwan, Tainung 67 is photoperiod insensitive. Through marker assisted selection (MAS), in these three heading date genes of TNG 67 had been introgressed to Koshihikari, and three BC3F2 populations derived from three different BC3F1 individuals, ＃286-85-A4、＃286-14-A23 and ＃286-85-A27. In second crop season in 2009, those BC3F2 populations were planted under natural lightness’ field. The heading dates for each population displayed continuous distribution. After analysis the effect cause by three genes, Ehd1 with the largest effect on heading date. Recessive homozygotes ehd1 could posponed 23.5 days to head, significantly. Hd1 was the second effective gene, Hd6 had the minor effect for heading date delay. Total of 27 genotypes composed by combination of three heading date genes. The earliest heading time was 69.4 days found in A27 population, which genotype was Hd1Hd1 Hd6 hd6 Ehd1Ehd1. The lasted heading time was 109 days. Discovered in A4 population, which genotype were hd1hd1 Hd6Hd6 ehd1ehd1. The heading dates for most of the genotypic combinations in three populations were be-tween 80 to 90 days and transgressive segregants were found. Thus heading date could be adjusit by different genotype combination. By using MAS, near isolation lines of Koshihikari carring different heading date genes could be developed. After evaluation of field trials under different environment, several varieties derived from NILs of Koshihikari possessing different heafing dates could be promoted to growal different location and different crop seasons. Development of molecular markers of wild rice Through human domestication and breeding program, people have developed a lots of elite cultivars and received huge economical reward, but it let the genetic di-versity of the rice gene pool much narrower than before. Recovering the land race and wild rice material is one way to improve the genetic diversity of cultivar gene pool searching for polymorphic markers is essential. Through this method, researchers have discoveried lots of genes, which can endow with elite cultivar resistance of biotic and abiotic stress, developed a complete linkage map to posit the genes; breeders can breed by marker assisted selection (MAS) a more efficient way to utilized wild genetic resource to improve better cultivar traits (Gupta et al., 2002). The final goal for this research is to develop a complete chromosome segment substitution (CSSLs), each carry chromosome from O. officinalis (CC genome) and O. australiensis with O. sativa sub-species (AA genome) TCS 10 and TNG 67 background, respectively. This research was trying to figure out a new way to received polymorphic DNA markers between wild and cultivar rice. 249 SSR (simple sequences repeat; rice microsatellite) had been selected, but only 100 (40%) and 67 (27%) of markers could amplify in O. officinalis and O. aus-traliensis successfully. Due to all SSR markers are developed based on sequence of O. sativa, the sequence variation between difference genomes, the regions for primers may lose in the others genome. Few SSR markers can be transferred into others ge-nome. To more effencially acquire the polymorphic markers; we compare the sequence of wild and cultivar rice by bioinformatics software, search for indel regions. Finally 123 markers had been developed. 109 (89%) and103 (84%) markers can amplify in two wild species successfully, and 79 (72%) and 82 (68%) of markers can present polymorphic between O. officinalis . TCS 10 and O. officinalis vs. TNG 67; 64 (62%) and 65 (63%) of markers can present polymorphic between O. australiensis vs. TCS 10 and O. australiensis vs. TNG 67. This is an efficient way to receive polymorphic markers for linkage analysis, develop such as CSSLs for research and breeding program, to improve the use efficiency of wild material. From the result of genotying BC1F1 and BC2F1 populations, derived from O. of-ficinalis × TNG 67, the ratios of heterozygous change from 86.7% in the (BC1F1) to 18.1% (BC2F1), the chromosome segment of wild rice be eliminated from progenies, rapidly. So it is almost impossible to develop a complete CSSLs population, which carried the chromosome segment from different genome of wild species into O. sativa background.