Cloning of a Potato Proteinase Inhibitor Gene PINII-2x from Diploid Potato(Solanum phurejia L.) and Transgenic Investigation of Its Potential to Confer Insect Resistance in Rice

Both cDNA and a genomic DNA fragment encoding a new potato proteinase inhibitor Ⅱ were isolated from a diploid potato IVP101 （Solanum phurejia L.） and named PINII-2x. Nucleotlde sequencing confirmed that the DNA fragment of PINll-2xwas 580 bp, including a 115-bp intron and two exons. The deduced PINII-2x proteln contained an Intact signal peptide and two active sites. The PINII-2x gene and its deduced PINII-2x protein had 88% and 93% homology with another tetraploid potato proteinase inhibitor Ⅱ, respectively. Northern blotting analysis Indicated that the mRNA of PINII-2x gene was wound induced in potato leaves. Binary vector pNAR301 and pNAR302 were constructed for rice transformation, in which the PINII-2x cDNA was driven, respectively, by rice actin I promoter （Actl） and maize ubiquitin promoter （Ubll）. Via an Agrobacteriummediated method, these two constructs were transferred into japonica rice cv. Xiushui 63. PCR and Southern blotUng analysis for transgenic rice revealed the integration of the PINII-2x gene. Northern blotting analysis also confirmed transcripts of the PINII.2x gene in transgenic rice plants. Insect bloassays using stripe stem borer （Chilo auppressalis Walker） demonstrated that the average weight and body length of larvae In transgenic plants were only nearly 50% and 61% of those of larvae in control plants, respectively. These results Indicate that the PINII-2x gene should be an effective insect-resistance gene and could be valuable for application in crop breeding for insect resistance.

Developing a new model system for potato genetics by androgenesis

High heterozygosity and tetrasomic inheritance complicate studies of asexually propagated polyploids,such as potato.Reverse genetics approaches,especially mutant library construction,can be an ideal choice if a proper mutagenesis genotype is available.Here,we aimed to generate a model system for potato research using anther cultures of Solanum verrucosum,a self-compatible diploid potato with strong late blight resistance.Six of the 23 regenerants obtained(SVA4,SVA7,SVA22,SVA23,SVA32,and SVA33)were diploids,and their homozygosity was estimated to be>99.99%with 22 polymorphic In Del makers.Two lines-SVA4 and SVA32-had reduced stature(plant height≤80 cm),high seed yield(>1,000 seeds/plant),and good tuber set(>30 tubers/plant).We further confirmed the full homozygosity of SVA4 and SVA32 using wholegenome resequencing.These two regenerants possess all the characteristics of a model plant:diploidy,100%homozygosity,self-compatibility,and amenability to transgenesis.Thus,we have successfully generated two lines,SVA4 and SVA32,which can potentially be used for mutagenesis and as model plants to rejuvenate current methods of conducting potato research.

二倍体马铃薯栽培品种遗传多样性的SSR标记分析

二倍体马铃薯基因组相对简单,借助二倍体进行育种可以加速马铃薯的育种进程,因此评价二倍体马铃薯种质的遗传多样性,挖掘和有效利用优良性状显得非常必要。为了筛选多态性的SSR标记,用55对SSR引物扩增39个遗传关系相对较远的二倍体马铃薯材料。选取分布在12条染色体上的12个具有高多态性的SSR标记评价192份二倍体马铃薯栽培品种的遗传多样性,共检测到98个等位位点,其中97个为多态性位点;每对SSR引物扩增出的等位位点为6-18个,平均8.2个。用非加权配对算术平均法（UPGMA）进行聚类,显示出所有供试材料的遗传关系：12对SSR引物可以将192份材料中的186份区分开;这192份材料被划分为11个组群,其中第一个组群包含了83.3%的材料。

二倍体彩色马铃薯杂交群体的蒸食品质遗传多样性评价

二倍体育种是近年马铃薯育种热点。为了筛选蒸食品质优良的二倍体彩色马铃薯亲本,本研究以11个天然结实群体和15个人工杂交组合的309个二倍体彩色马铃薯后代为研究对象,对其薯块蒸食品质的质地、变色、酶促褐变程度、香味和口感这5个方面进行鉴定与评分,并比较天然结实和人工杂交组合后代薯块蒸食品质各指标的辛普森指数多样性的差异。研究结果表明:综合评价指标口感等级评分中,共有24份材料平均口感评分在8.00分以上,其中10份来自天然结实的群体,其余14份来自人工杂交组合。在蒸食评价质地方面,天然结实组合后代平均多样性指数高于人工杂交组合后代;在蒸后变色和酶促褐变方面,天然结实和人工杂交后代差别很小;在香味和口感评分等级方面,人工杂交后代多样性略高于天然结实后代。本研究中所筛选出来的蒸食品质优良的材料可作为二倍体亲本,通过杂交筛选二倍体彩色马铃薯品种。