玉米自交系及單交種之蠟質基因序列與子粒品質之相關性研究

Abstract

為瞭解台灣廣泛栽培之玉米單交種之蠟質基因DNA序列變異與其和澱粉組成相關性狀之關聯性，本研究以2個白玉米、4個糯玉米、4個甜玉米及4個飼料玉米等共14個單交種品種，以及10種子粒透光度具有差異的白玉米種原為材料，探討蠟質基因與澱粉組成性狀之關係。為區分單交種異結合對偶基因之序列，本研究採用延長電泳時間、針對InDel上游區域SNP設計專一性引子及PCR產物之TA-分子選殖等3種方法，由結果顯示以TA-分子選殖之區分能力為最佳。澱粉組成相關性狀調查包括澱粉含量、直鏈性澱粉含量、白度(whiteness)及色度，結果得知甜玉米具有最低的澱粉含量，糯玉米具有最低的直鏈性澱粉含量，而白玉米與飼料玉米均具有高澱粉與直鏈性澱粉含量；而白度與色度分析結果顯示直鏈性澱粉含量越高，則白度越低，而色度分析結果則和果皮顏色具有高度相關。將各品種之蠟質基因DNA序列變異與外表型比較後顯示exon區域有18個變異，而intron區域則有64個變異；就變異比率而言，intron區域約為exon區域的4.5倍，且蠟質基因序列exon 12區域下游具有較大之變異比率；其中推測影響蠟質基因功能之變異位點為位於exon 7之30 bp缺失，以及位於exon 14中4380 bp(G/A)、4387 bp(G/C)與4472 bp(G/A)等共3個SNP，此4個變異皆造成蠟質基因產物之胺基酸缺失或改變。由NCBI搜尋包含玉米、大麥、水稻、小米及高粱等6個單子葉物種，和馬鈴薯、木薯、甘藷及豌豆等4個雙子葉物種之蠟質基因DNA序列後，由於物種間DNA序列變異過大，難以劃分保留區域，經單子葉/雙子葉進行分群後，發現單子葉作物群內變異較小，保留區域片段較大，而雙子葉作物則序列變異較大，保留區域片段較小，而單子葉與雙子葉作物群間則具有3個小片段之保留區，由此結果可知單子葉作物間蠟質基因演化速度較慢。本研究分析玉米單交種蠟質基因DNA序列變異性與澱粉組成相關性之結果，在不同用途之玉米品種改良上具應用潛力。

In order to understand the association between DNA sequence variation of waxy genes, and kernel appearance traits related to starch composition of maize hybrids grown in Taiwan, two white corns, four waxy corns, four sweet corns, four feed corns and 10 waxy inbreds with various whiteness were used as materials. In this study, extension of electrophoresis time, designation of SNP-specific primer, and TA-cloning of PCR product were used to identify the alleles of hybrid F1. The result indicates that the TA-cloning is the most efficient way to identify hybrid alleles. To determine the kernel appearance traits, total starch content, amylose content, whiteness and L, a, b value were investigated. It was found that sweet corns had the least starch content and waxy corns had the least amylase content, but the most starch and amylose content were produced in the kernel of white corns and feed corns. Results from whiteness and color scale determination showed that high amylose lines had lower whiteness, and the L, a, b values were highly correlated with the pericarp. After comparing and analyzing the DNA sequences and kernel phenotypes, 18 variation sites in exon region, and 64 variation sites in intron region were observed. The sequence variation proportion is approximately 3.5 times higher in intron than in exon, and the down-stream region of exon 12 has the highest rate of variation. Four functional sequence variations, including 30 bp deletion in exon 7, SNPs on 4380 bp(G/A), 4387 bp(G/C) and 4472 bp(G/A), which caused amino acid substitution or deletion in waxy protein, were identified. The waxy gene sequences from 6 monocot species (including maize, barley, rice, millet and sorghum), and 6 dicot species (including potato, cassava, sweet potato and pea) extracted from NCBI database were aligned and analyzed. Only three conserved segments in waxy DNA sequences were identified because of the highly difference among the surveyed sequences. However, conserved regions in monocot crops are larger than in dicot ones. These results revealed the slower evolution rate in waxy genes of monocot species. The result in this study involved in the relationship between DNA sequence variation of waxy allele and kernel starch composition have application potential for breeding corns with different utilizations.