Molecular fingerprinting of hybrids and assessment of genetic purity of hybrid seeds in rice using microsatellite markers

Microsatellite markers were used for fingerprinting of hybrids, assessing variation within parental lines and testing the genetic purity of hybrid seed lot in rice. Ten sequence tagged microsatellite sites (STMS) markers were employed for fingerprinting 11 rice hybrids and their parental lines. Nine STMS markers were found polymorphic across the hybrids and produced unique fingerprint for the 11 hybrids. A set of four markers (RM 206, RM 216, RM 258 and RM 263) differentiated all the hybrids from each other, which can be used as referral markers for unambiguous identification and protection of these hybrids. Cluster analysis based on Jaccard's similarity coefficient using UPGMA grouped the hybrids into three clusters. Within the cluster all the hybrids shared a common cytoplasmic male sterile line as female parent. The genetic similarity between the hybrids ranged from 0.33 to 0.92 with an average similarity index of 0.63. The analysis of plant-to-plant variation within the parental lines of the hybrid Pusa RH 10, using informative markers indicated residual heterozygosity at two marker loci. This highlights the importance of STMS markers in maintaining the genetic purity of the parental lines. The unique value of the restorer gene linked marker for testing the genetic purity of hybrid seeds is demonstrated for the first time.     

利用RAPD标记鉴定大白菜杂交种纯度的研究

应用随机扩增多态性ＤＮＡ（ＲＡＰＤ）标记鉴定大白菜杂种商品种子的纯度。用５０个随机引物检测了２个杂交种北京５７号和北京１０６号及其亲本,引物ＯＰＥ－２０在北京５７号杂交种中产生了有别于双亲的特殊标记,引物ＯＰＨ－０６及ＯＰＨ－０７在北京１０６杂交种中产生了有别于双亲的特殊标记,能清楚地区分杂交种及其双亲,并将这些引物应用在北京５７号和北京１０６号杂交种样品的纯度检测中。这个结果显示了ＲＡＰＤ标记在大白菜杂交种商品种子纯度检测上的实际用途。     

The use of AFLP markers for the identification of carrot breeding lines and F1 hybrids

Four inbred lines of carrot (cytoplasmic male‐steriles and corresponding maintainers) and eight of their F₁ hybrids were studied with the amplified fragment length polymorphism (AFLP) technique to examine their genetic relationship and produce markers useful for testing hybrid seed purity. Eighty‐six polymorphic amplicons were identified in bulked DNA samples using eight primer pair combinations. Genetic distance was estimated on the basis of the presence or absence of polymorphic bands. The dendrogram plotted on the basis of the AFLP data closely represented the pedigree relationships of the lines and their hybrids. From one to six amplicons specific for a breeding line were identified. Most of them were also present in the DNA bulks of respective F₁ hybrids. However, screening performed on individual plants of two parental lines and the corresponding hybrid indicated insufficient uniformity of parental lines, limiting the applicability of AFLP markers for testing hybrid seed purity.     

A simple method to control the seed purity of japonica hybrid rice varieties using PCR-based markers

Cytoplasmic male sterility (CMS) by the cms‐bo cytoplasm and its restoration by the nuclear restorer gene, Rf‐1, are used for seed production of japonica hybrid rice varieties. To produce pure hybrid seeds, a prerequisite is to properly manage the seed purity of parental lines, especially CMS lines. In this study, three dominant polymerase chain reaction (PCR)‐based markers (M1, M2 and M3) were developed to detect mutual contamination in seed batches of CMS lines, maintainer lines, restorer lines and hybrids. M1 detected the mitochondrial sequence that was present in the cytoplasm of common japonica varieties and absent in the cms‐bo cytoplasm. M2 and M3 detected the chromosomal sequence related to the Rf‐1 allele in restorer lines and the rf‐1 allele in common japonica varieties, respectively. By the strategic use of these markers, japonica hybrids and their parental lines could be efficiently distinguished from each other. Furthermore, sensitivity tests for the three markers with a series of crude DNA samples prepared from polished grains demonstrated that these markers could detect one contaminating grain among 500 or 1000 grains. Therefore, the bulk PCR analyses with the markers developed here probably make it possible to control the seed purity of japonica hybrids properly by selecting appropriate seed batches of their parental lines quickly and efficiently.     

玉米杂交种及其亲本自交系的生化指纹鉴定

本试验以浙单9号等五个玉米杂交组合及其亲本自交系为材料,进行种子盐溶蛋白聚丙烯酰胺凝胶电泳等多种电泳鉴定方法的研究,以揭示玉米杂交种及其亲本自交系的“生化指纹”(biochenucal fingerprint),以及筛选出适合于玉米杂交种及其亲本自交系真实性和纯度鉴定的方法。结果表明,各供试玉米杂交种及其亲本自交系都具有相应的、唯一的种子盐溶蛋白聚丙烯酰胺凝胶电泳所显现的生化指纹。对于有些组合。玉米芽鞘和叶片绿色组织过氧化物酶同工酶电泳图谱存在阴极第4、第5酶带差异,因这两条酶带的差异稳定,并且重现性好,故能用过氧化物酶同工酶技术对其进行有效地鉴定。上述两种方法,尤其是前者,因技术要求不高,费用低,快速及重现性好等特点,能满足我国目前种子检验室日常玉米品种纯度快速测定工作的要求,具育良好的应用前景。     

Prediction of heterosis for grain yield in rice using 'key' informative EST-SSR markers

This study was undertaken to assess the comparative potential of 25 Expressed Sequence Tag derived simple sequence repeats (EST-SSRs) and 25 genomic SSRs in the prediction of grain yield heterosis using a set of nine cytoplasmic male sterile (CMS) lines and 32 restorer lines of rice. EST-SSRs and genomic SSRs exhibited an average Polymorphism Information Content value of 0.37 and 0.45, respectively. The coefficient of marker polymorphism among parental lines with respect to a set of hypervariable EST and genomic SSRs was correlated with standard heterosis for grain yield of six public bred rice hybrids. EST-SSRs gave a better correlation (r = 0.75) as compared with genomic SSRs (r = 0.09). When 10 'key' informative EST-SSR markers which showed a higher positive correlation with grain yield heterosis were validated in a new set of 14 experimental hybrids, the markers exhibited a higher correlation (r = 0.79), indicating the predictive value of these EST-SSRs. We recommend these 10 'key' informative EST-SSR markers for analysis of genetic diversity of parental lines and prediction of heterosis in hybrid rice breeding programmes.     

Electrophoretic variation as a tool for determining seed purity and for breeding hybrid varieties of Brassica oleracea

Summary Isozyme phenotypes at four known genetic loci were determined in parental inbreds and corresponding F₁ hybrid seed lots of four commercial broccoli, two cauliflower, and two cabbage varieties to determine seed purity. Most inbred lines were completely homozygous at all four loci but differed with respect to alleles at one to three loci. Several parental inbreds of the cabbage hybrids were segregating at two to three of the loci. Models were developed to estimate seed purity in cases where parents were either fixed or segregating at diagnostic loci. Estimates of contamination ranged from 1.5 to 40.1%. These estimates were comparable with those from commercial grow-outs with a tendency for the former estimates to be higher. It was concluded that more stable SI alleles or genetic male sterility should be used to reduce contamination. Electrophoretic variation was further discussed as a tool for selecting homozygous plants and for strong self-incompatibility.     

Identification of informative SSR markers capable of distinguishing hybrid rice parental lines and their utilization in seed purity assessment

With the objective of identifying SSR markers that can distinguish parental lines of rice hybrids, we characterized 10 each of cytoplasmic male sterile (CMS) and restorer (R) lines along with 10 popular Indian rice varieties using a set of 48 hyperpolymorphic SSRs distributed uniformly across the rice genome. All the SSR markers were polymorphic, amplifying a total of 163 alleles, with an average of 3.36 ± 1.3 allelic variants per locus. Twenty-seven SSR markers showed amplification of an allele, which was very specific and unique to a particular parental line and not amplified in any other rice genotype tested. Through multiplex PCR, SSR marker combinations that were unique to a particular parental line or hybrid were also identified. With a set of 10 SSR markers, all the public bred Indian rice hybrids along with their parental lines could be clearly distinguished. To utilize these SSR markers effectively for detection of impurities in parental lines, a two dimensional bulked DNA sampling strategy involving a 20 × 20 grow-out matrix has been designed and used for detection of contaminants in a seed-lot of the popular CMS line IR58025A. We have also designed a multiplex PCR strategy involving single tube analysis using 2–3 markers for hybrid seed purity assessments and demonstrate its superiority over single marker analysis in accurate detection of impurities in hybrids. Implications of parental and hybrid specific SSR markers and strategies to utilize the informative SSR markers for detection of contaminants in a cost effective manner are discussed.     

Identification of ‘Sib’ plants in hybrid cauliflowers using microsatellite markers

Hybrid cauliflowers have been developed to exploit heterosis and to improve uniformity of production. Two breeding systems are commonly employed, self-incompatibility (SI) and cytoplasmic male sterility (CMS). Sibs, assumed to be self-inbred, often contaminate hybrid seed lots in the SI system and whilst self-inbreeding is not possible in the CMS system, plants that look like sibs occur. The objective of this study was to develop microsatellite markers for male and female cauliflower parent lines of both SI and CMS systems and to use them to screen sibs and aberrant plants in F₁ hybrids. Fifty six pairs of microsatellite primers were screened and 8 primer pairs produced co-dominant markers in parent plants and two pairs of markers were chosen for purity testing of F₁ hybrid seeds. Controlled pollinations were conducted in the glasshouse to produce hybrid and selfed-seeds. These seeds were grown in a field trial to identify morphologically normal and sib plants and to assess the reliability of microsatellite markers in detecting sib plants. Microsatellite analysis of morphological sib plants from the SI system revealed that these were not always self-inbred, in contrast, most self-inbred plants showed normal growth. Similarly, all morphological sibs from the CMS system showed hybrid bands. This suggests that morphological sibs were not always due to selfing but possibly to an interaction between genetic and environmental factors and this requires further investigation.     

利用SSR技术快速准确鉴定杂交玉米种子纯度

利用一种快速、廉价的DNA提取方法,提取了两个玉米杂交种及其亲本的DNA用于SSR分子标记分析。分析结果显示,10对被分析的引物中有4对在两个杂交种双亲之间显示出多态性,可以用于相应的杂交种纯度分析。另外,分别利用同工酶技术和SSR分子标记技术同时分析了来自这两个杂交种的种子样品的纯度,分析发现,对于一个杂交种,两种方法都能可以鉴定,并且两者检测的结果是一致的,但是对于另外一个杂交种,由于其父母本非常接近,同工酶不能将其区分。因此,这些结果显示SSR分子标记技术可以很好地用于杂交玉米种子的纯度鉴定,即使是这个杂交种是来自两个非常接近的自交系的。     

酸性PAGE鉴定杂交水稻品种纯度的研究

研究了水稻水溶蛋白、盐溶蛋白、醇溶蛋白的提取方法和酸性电泳系统中的胶联度、离子强度、染色方法，探索出了一套适宜水稻品种纯度和真实性鉴定的酸性聚丙烯酰胺凝胶电泳系统。应用该方法对当地推广的11个杂交水稻组合（包括12个亲本和11个杂交F1代）进行了分析。结果表明，该技术较为可靠、稳定、经济、快速，鉴别亲缘关系较近的材料具有较好效果。     

甘蓝型油菜隐性核不育系ZWAB临保三交种

利用甘蓝型油菜隐性核不育系ZWAB及临保系和恢复系分别配制了临保三交种、单交种进行组合的杂种优势比较。结果表明：临保三变种在抗性方面优于单交种，在经济性状及产量上与单交种相当。临保三交种完全可以替代单交种，从而节省了两系制种过程中初花期拔除50％可育株的环节，降低了劳动强度和成本，提高了制种产量和种子纯度。     

甘蓝型油菜隐性核不育系ZWAB临保三交种与单交种的比较

利用甘蓝型油菜隐性核不育系ZWAB及临保系和恢复系分别配制了临保三交种、单交种进行组合的杂种优势比较,结果表明:临保三交种在抗性方面优于单交种,在经济性状及产量上与单交种相当。临保三交种完全可以替代单交种,从而节省了两系制种过程中初花期拔除50%可育株的环节,降低了劳动强度和成本,提高了制种产量和种子纯度。     

甘蓝型油菜中油杂8号种子纯度的SSR鉴定

（中国农业科学院油料作物研究所/国家油料作物改良中心/农业部油料作物遗传改良重点实验室,湖北武汉 430062）     

Transferability of non-genic microsatellite and gene-based sunflower markers to safflower

Safflower (Carthamus tinctorius L.) DNA marker resources are currently very limited. The objective of this study was to determine the feasibility of transferring non-genic microsatellite (SSR) markers and gene-based markers, including intron fragment length polymorphism (IFLP) and resistance gene candidates (RGC)-based markers from sunflower (Helianthus annuus L.) to safflower, both species belonging to the Asteraceae family. Cross-species amplification of 119 non-genic SSRs, 48 IFLPs, and 19 RGC-based sunflower markers in 22 lines and germplasm accessions of safflower was evaluated. Additionally, 69 EST-SSR markers previously reported to amplify in safflower were tested. The results showed that 17.6% of the non-genic SSR, 56.2% of the IFLP, and 73.7% of the RGC-based markers were transferable to safflower. The percentage of transferable markers showing polymorphic loci was 66.6% for non-genic SSR, 70.6% for EST-SSR, 55.5% for IFLP, and 71.4% for RGC-based markers. The highest polymorphism levels were found for non-genic SSR. The average number of alleles per polymorphic locus and mean heterozygosity values were 2.9 and 0.46, respectively, for non-genic SSR, 2.2 and 0.35 for EST-SSR, 2.1 and 0.24 for IFLP, and 2.0 and 0.34 for RGC-based markers. The results of this study revealed a low rate of transferability for non-genic SSR sunflower markers and a better rate of transferability for IFLP and RGC-based markers. Transferable genic and non-genic sunflower markers can have utility for trait and comparative mapping studies in safflower.     

运用SRAP分子标记鉴定辣椒杂交种纯度

摘要：以辣椒品种航椒4号和航椒5号及其亲本H09-4、H09-2、WS-3、B-2-2为试验材料,运用SRAP分子标记技术研究了辣椒杂种与其亲本之间 扩增条带的多态性,鉴定和分析了航椒4号辣椒品种的真实性。结果表明,所试验的18对SRAP引物中有13对引物分别在2个辣椒杂交种和其亲本之间存在扩增条带的多态性,平均每个引物组合扩增的清晰条带数为23.5条,多态性比率为58.89%,其中有3条偏母型引物,2条偏父型引物, 2条互补型引物。用互补型引物DC1-EM9对航椒4号和航椒5号进行了各100粒种子SRAP鉴定,所测纯度分别为100%和97.3%,与田间纯度100%和98.9%非常接近,表明了SRAP分子标记技术是鉴定辣椒一代杂种纯度的有效方法,具有准确、可靠、快速的特点,在辣椒杂交种子纯度室内快速检测中有很大的应用前景。     

利用清蛋白多态性鉴定玉米种子纯度研究

利用改进的聚丙烯酰胺凝胶电泳技术,对生产上应用的53个玉米杂交种及亲本自交系干种子清蛋白(白蛋白Albumin)进行了电泳分析。结果表明,改进的聚丙烯酰胺凝胶电泳技术分辨率高、鉴别能力强、准确;不仅能鉴别杂交种及亲本自交系,对血缘关系较近的材料,如同母异父单交种、含有同一血缘的不同衍生系也能区分开。利用该技术     

Genetic linkage between male sterility and non‐spiny trait in safflower (Carthamus tinctorius L.)

Genetic male sterility (GMS) exists naturally in safflower (Carthamus tinctorius L.). In the existing safflower GMS lines, sterile and fertile plants are distinguishable at flowering. This causes delay in fertile plants rouging and reduction in hybrid purity. In this investigation, a cross between a spiny GMS parent 13‐137 and a spiny non‐GMS parent ‘A1’ was effected. One sib cross, SC‐67, producing non‐parental‐type non‐spiny sterile and spiny fertile plants in F₃ was advanced to F₉ through sib crossing between non‐spiny sterile and spiny fertile plants. Mendelian digenic segregation was not observed for non‐spiny trait and male sterility. The results revealed strong linkage between these traits. The linkage was confirmed in F₂ generations of crosses between a non‐spiny marker‐linked GMS line (MGMS) and five elite lines. Male sterility–linked non‐spiny trait could distinguish sterile and fertile plants at elongation stage. The MGMS would be useful in production of pure F₁ hybrid seed and development of elite populations.     

同工酶差异位点分析在蔬菜杂交种纯度检测中的应用

用10种同工酶和蛋白质分析体系，分析了7种重要蔬菜的71个杂交种与其亲本之间的差异位点，以及这些差异位点用于杂交种纯度检测的可能性和存在的问题。试验表明，作物的不同种类，杂交种与亲本之间的亲缘关系以及作物各类的遗传多态性，都会影响同工酶差异位点产生的多寡，从而影响到这一技术是否能用于此种作物的纯度检测。分析了不同同工酶在不同蔬菜中的多态性以及在蔬菜种子纯度检测中的表现。     

Assessment of DNA markers for seed contamination testing and selection of disease resistance in cabbage

Cabbage (Brassica oleracea L. var. capitata) is an important vegetable worldwide. Most Japanese commercial cultivars of cabbage use an F₁ hybrid seed production system. The purity of F₁ hybrid seeds is important and the assessment of purity based on DNA markers can be highly accurate. In addition, selection of agronomically important traits such as disease resistance based on DNA markers is useful for breeding of cabbage. The aim of this study is to demonstrate the effectiveness of DNA marker-assisted selection in cabbage. In this study we distinguished the parental S haplotypes in 35 F₁ hybrid cultivars by combining several linked DNA markers. Thirty-one highly polymorphic simple sequence repeats (SSR) markers were screened from 175 reported SSR markers, which are useful for assessment of the purity of F₁ hybrid seeds. We examined the relationship between the DNA marker based genotype and the phenotype by an inoculation test of clubroot disease. A co-dominant PCR–RFLP marker was developed for selection of Fusarium yellows resistance and the genotypes using this marker were consistent with inoculation test in all tested samples.