Development of cultivar-specific DNA markers based on retrotransposon-based insertional polymorphism in Japanese pear

We developed retrotransposon-based insertional polymorphism (RBIP) markers based on the long terminal repeat (LTR) sequences of copia-like retrotransposon Ppcrt4 and flanking genome sequences, which were derived from 454 sequencing data from Japanese pear (Pyrus pyrifolia) ‘Hosui’. Out of 40 sequences including both LTR and flanking genome regions, we developed 22 RBIP markers and used them for DNA profiling of 80 pear cultivars: 64 Japanese, 10 Chinese (Pyrus ussuriensis) and 6 European (Pyrus communis). Three RBIP markers were enough to differentiate ‘Hosui’ from the other Japanese pear cultivars. The 22 RBIP markers could also distinguish 61 of the 64 Japanese pear cultivars. European pears showed almost no amplification of the 22 RBIP markers, which might suggest that retrotransposons had transposed during Asian pear evolution or reflect the genetic relationship between Asian and European pears. Sixteen of the RBIP markers could be positioned on a genetic linkage map of ‘Hosui’. The RBIP loci were distributed in 10 linkage groups, and some loci were very closely located within the same linkage group. The information obtained will be applicable to developing cultivar-specific RBIP marker sets in plants.     

Agronomic traits and gene containment capability of cleistogamous rice lines with the superwoman1-cleistogamy mutation

Pollen-mediated transgene flow is a major concern for the production of genetically modified (GM) rice. Cleistogamy is a useful tool for preventing this form of gene flow. We previously identified the cleistogamous rice mutant superwoman1-cleistogamy (spw1-cls) and determined its molecular genetic mechanism. In the present study, we cultivated spw1-cls over five years to examine effects of cleistogamy on agronomic traits. Simultaneously, we cultivated cleistogamous backcross lines created by continuous backcrossing with “Yumeaoba” (a japonica cultivar) as the recurrent parent and by application of a DNA marker. In these experimental cultivations, spw1-cls and its backcross lines showed almost equal or slightly lower, but acceptable, agronomic traits compared with each control line. We also conducted natural crossing tests in paddy fields to assess the gene containment capability of spw1-cls. In a series of field experiments, there was no natural crossing between spw1-cls (pollen donor) and pollen recipient lines, but the wild-type donor and recipient lines were crossed. Thus, the cleistogamy of the spw1-cls mutation is able to inhibit natural crossing effectively, without significant loss of commercial benefits, such as yield. We conclude that spw1-cls cleistogamy is a practical tool for gene containment in GM rice cultivation.     

Estimation of the outcrossing rate for annual Asian wild rice under field conditions

The Asian wild rice, Oryza rufipogon, has partial outcrossing behavior and shows high levels of genetic variation. To estimate an accurate outcrossing rate of annual form of O. rufipogon, two backcross lines (Lines R1 and R2) between Oryza sativa Nipponbare and O. rufipogon W630 were examined under field conditions. A chromosome survey confirmed that these lines had wild chromosomal segments at more than 92% of marker loci. As for the traits of glume, stamen and pistil, Line R1 showed similar floral morphology as that of O. rufipogon W630, whereas Line R2 had larger glumes. In 2005, 2006 and 2008, a total of 22 backcross plants were planted in the middle of wild rice plots. The successive progenies of each plant were examined using microsatellite markers that could clearly detect self-pollination and outcrossing. The outcrossing rates of Line R1 plants ranged from 4.04% to 25.50% with an average of 10.20%. This indicates that cross-pollination of wild rice is a chance event affected by many environmental factors. The outcrossing rates of Line R2 plants also varied, however, no significant difference was observed between the averages of Lines R1 and R2, suggesting that the glume sizes are not critical for outcrossing ability.     

青藏高原老芒麦和垂穗披碱草SSR分子标记鉴别

垂穗披碱草(Elymus nutans)和老芒麦(E.sibiricus)是青藏高原高寒区广泛种植的优质牧草草种,由于生态适应性变异,在对一些野生种质资源鉴定中,二者常易混淆。为了区别鉴定垂穗披碱草和老芒麦,本研究结合细胞学鉴定,利用不同来源的老芒麦和垂穗披碱草种质材料,对源于普通小麦(Triticum aestivum)的42对SSR引物进行筛选。结果表明,筛选出的小麦EST SSR引物Xcwem38c在垂穗披碱草中的多态性标记可以有效区分垂穗披碱草和老芒麦。     

Myoglobin as a prognostic indicator for outcome in dogs with gastric dilatation-volvulus

Objective – To determine whether myoglobin (Mb) is a useful prognostic indicator for outcome and to investigate any relationship between Mb and mortality in dogs with gastric dilatation‐volvulus (GDV). Design – Prospective study. Setting – Veterinary teaching hospital. Animals – Seventy‐two dogs with GDV. Interventions – Blood sampling. Measurements and Main Results – Mb levels were measured at the time of diagnosis (Mbt₀), 24 hours (Mbt₁), and 48 hours (Mbt₂) after signs of GDV were recognized. Fifty‐seven dogs survived (group I) and 15 dogs did not survive (group II). Mb t₀ differed significantly between groups (P=0.04). Mbt₀ in group I ranged from <30 to >700 ng/mL (n=57, median 74 ng/mL), and in group II from 34 to >700 ng/mL (n=15, median 238 ng/mL). Analysis of a receiver operating characteristic curve of Mbt₀ suggested that the best single cutpoint would be 168 ng/mL (sensitivity 60.0%, specificity 84.2%). Fifty percent of dogs with Mbt₀>168 ng/mL were euthanized, while 88.9% with Mbt₀<168 ng/mL survived. Mbt₁ and Mbt₂ differed significantly between groups I and II. Mbt₁ in group I ranged from 32 to >700 ng/mL (n=55, median 123 ng/mL), and Mbt₁ in group II ranged from 131 to 643 ng/mL (n=7, median 343 ng/mL) (P=0.006). Mbt₂ in group I ranged from 30 to 597 ng/mL (n=54, median 101 ng/mL), and in group II from 141 to >700 ng/mL (n=8, median 203 ng/mL) (P=0.02). Conclusions – In this study, Mbt₀ is a moderately sensitive and specific prognostic indicator. Almost 90% of the dogs below the cutpoint survived to discharge, whereas 50% with Mbt₀ above the cutpoint did not survive.     

云南大河猪保种群的微卫星检测

作者采用世界粮农组织（FAO）和国际动物遗传学会（ISAG)联合推荐的27对微卫星DNA引物中的20对引物,检测了云南大河猪保种群的83个个体的基因型。通过计算等位基因频率、遗传杂合度（H）、多态信息含量(PIC)、有效等位基因数,从而分析大河猪保种群的遗传多样性。结果表明,大河猪保种群在20个微卫星座位上拥有的等位基因较为丰富,20个微卫星座位上共有137个等位基因,平均6.85个,群体有效等位基因数共104个,平均5.2个;大河猪保种群的遗传多样性较为丰富,杂合度为0.792,多态信息含量为0.765。     

~(60)Co γ射线辐照对海雀稗愈伤组织植株再生的影响

以海雀稗品种Adalay(Paspalum vaginatum Sw.cv.Adalay)的颗粒状胚性愈伤组织为材料,在离体培养条件下研究不同剂量的60Coγ射线辐照对愈伤组织绿苗分化和植株再生的影响。结果显示,辐照剂量在20～50Gy时,辐照处理的愈伤组织分化绿苗的频率从94%左右降至85%;剂量在60～80Gy时,愈伤组织的绿苗分化率从76%左右降至30%左右,相对植株再生率从44.5%降至8.7%。60Gy为辐照处理海雀稗颗粒状愈伤组织的适宜剂量,辐照处理的愈伤组织在附加BAP2.0mg/L的愈伤组织分化培养基上,绿苗分化率在76%左右,分化的绿苗转到附加NAA0.5mg/L的生根培养基上,相对植株再生率为44.5%。SRAP分子标记测定结果显示,特异性SRAP标记与体细胞辐照诱变突变体相关。本研究建立起海雀稗体细胞60Coγ射线辐照诱变技术,为在细胞水平上开展海雀稗辐照诱变育种提供条件。     

大刍草(Zea mexicana schard)×自交系K169选系的核型和SSR分析

本研究以玉米自交系K169及其与大刍草(Zea mexicana schard)远缘杂交而成的5个选系为研究材料,对它们进行核型和SSR分析,以期从细胞学和分子水平的角度为玉米远缘杂交育种提供理论依据。结果表明,5个远缘选系和轮回亲本K169体细胞染色体数目均为2n=20,但在核型结构上却存在较大差异,其中自交系K169和远缘选系1191在第6染色体的短臂上有随体,1183和1189在第8染色体的短臂上有随体,1193和1195未发现带随体的染色体;选系1183、1193和1195的染色体长度比(最长/最短)与K169之间存在较大差异;选系1183和1195的核型类型与K169不一致。96对SSR引物在5个选系和轮回亲本K169中共检测出27个多态性位点,位点多态信息量(PIC)的平均值为0.724;基因型多样性(H′)的平均值为1.400,它们之间的遗传相似系数平均值为0.5791。综合分析表明,这些选系与其轮回亲本K169之间存在真实的遗传差异,是一批新的玉米种质资源。     

利用生物信息学方法进行基于表达序列标签的玉 米单核苷酸多态性标记的开发

针对简单重复序列（SSR）标记密度不足、单核苷酸多态性（SNP）标记开发一般只基于2种基因型的序列差异,用以检测其他基因型的材料时多态性不高,不能满足玉米基因精细定位需要的现状,本研究从公共数据库下载来自不同遗传背景的玉米表达序列标签（EST）,结合运用各种生物信息学软件,开发基于EST序列的高多态性SNP标记。通过对2018530条EST序列的比对分析,拼接发掘出遍布全基因组的80363个SNP位点。在SNP位点两侧保守序列上设计PCR引物,开发出12388 个SNP标记（www.sicau.edu.cn/web/yms/snp/snp.html）,包含34721个SNP位点。其中,6008个标记只含单一SNP位点,12762个位点的多态性信息含量（PIC）大于04,具有高度多态性。