西北地区甜菜品系遗传多样性的SRAP分析

为给甜菜杂交育种的亲本选择、分子标记辅助选择育种、种质资源引进等提供理论依据,采用SRAP分子标记方法对西北地区的81份甜菜材料和9份外国品种进行遗传多样性分析。利用33对有效引物组合共得到592条扩增带,其中多态性条带有324条,平均多态性条带的比率为54.7%,平均遗传距离为0.3723,平均遗传相似系数为0.6891。遗传相似系数平均值大小为单胚品系0.8364>国外品种 0.7528>多胚四倍体品系0.7059>多胚二倍体品系0.6970。利用MEGA3.1软件,在遗传距离0.20处,可将供试材料分为三大类群。结果显示,西北产区甜菜供试材料遗传多样性较丰富。利用POPGEN32软件分析遗传多样性各项参数,表明供试的西北品系与外国品种的遗传基础有明显差异。田间生物学性状调查结果表明,西北产区供试材料主要特性为根产量高,抗丛根病性中等。     

A high-throughput DNA extraction method for barley seed

A non-destructive, quick DNA extraction method for barley seed is described. The method is simple and consists of drilling out a sample from the seed, adding sodium hydroxide, heating in a microwave oven and neutralizing with Tris-HCl. The seed DNA extract can be used directly for PCR with extra cycles added to the PCR programme compared to PCR programmes used for leaf extracts. This protocol was developed in particular for a micro satellite marker genetically linked to barley yellow mosaic virus resistance, but it can be applied toother markers of interest for barley breeding. The quick seed extraction protocol makes it possible to handle thousands of samples per day. Extraction of DNA from seed also facilitates transfer of plant material compared to the long-distance transfer of leaf samples. This revised version was published online in July 2006 with corrections to the Cover Date.     

水稻耐冷性研究Ⅲ.特定颖花结实率作为耐冷性指标的分子依据

以云南稻种耐冷性资源昆明小白谷及弱耐冷性的日本品种十和田配制的杂交F2代为材料,采用159个RFLP及SSR分子标记构建的连锁图和STATISTIC等分析软件,以单株结实率作为耐冷性评价指标.分析结果显示单株结实率与单株特定颖花结实率之间的相关系数(r)为0.8364;与耐冷性(以单株结实率为指标)相关的分子标记有43个,分布在8条染色体     

基于黄麻转录组序列SNP位点的CAPS标记开发与验证

黄麻CAPS分子标记的开发,可以为黄麻遗传多样性分析、种质资源鉴定和分子标记辅助选择等研究提供有效方法。本试验以黄麻179和爱店野生种为材料,采用IlluminaHiSeq4000测序技术进行转录组测序,对其SNP位点进行分析,设计了与木质素合成基因4CL、COMT及转录因子MYB相兲的SNP引物,在此基础上,应用dCAPS Finder2.0软件开发了CAPS标记,幵对其有效性进行了验证。结果表明:(1)组装后的黄麻unigene序列共72,674条,序列总长度为29,705,997 bp,检测到的SNP位点总数为67,567个,平均每440 bp有1个SNP。(2)获得了39对分别与4CL、COMT和MYB相兲的SNP引物,从中筛选获得26对CAPS标记引物,开发成功率为66.7%,其中11对CAPS标记具有多态性,多态性比例为43.2%。(3)开发的CAPS标记能较好地将12份不同类型的黄麻种质区分开来,表明CAPS是适用于黄麻研究的较理想的分子标记方法,为黄麻遗传基础研究提供了可靠工具。     

118份春性甘蓝型油菜恢复系的桔红花色位点鉴定

甘蓝型油菜桔红花色恢复系在杂交制种过程中起到指示作用,可去除杂株,因此培育出桔红花色优良恢复系是十分有必要的。本研究利用已开发的与桔红花色位点Bnpc1和Bnpc2紧密连锁的4个分子标记对118份春性甘蓝型油菜恢复系的桔红花色位点基因型进行鉴定,从中筛选出Bnpc1位点为显性,Bnpc2位点为隐性(BnPC1 BnPC1 Bnpc2 Bnpc2)或Bnpc1位点为隐性,Bnpc2位点为显性(Bnpc1 Bnpc1 BnPC2 BnPC2)的纯合黄花资源,从而可以利用这两种基因型恢复系相互杂交,选育出桔红花色优良恢复系。这为培育出桔红花色优良恢复系提供了一种简单有效的方法,同时也为花色选育提供了优良桔红花色遗传资源。     

小麦株高相关性状与SNP标记全基因组关联分析

株高是影响小麦产量和控制倒伏的重要因素,研究小麦株高相关性状的遗传机制对高产育种具有指导意义。以205份中国冬麦区小麦品种(系)为材料,利用分布于小麦全基因组的24 355个单核苷酸多态性(SNP)标记对株高相关性状进行关联分析。共发现38个与株高相关性状显著关联(P0.0001)的SNP,分布在1B、2A、2B、3A、3B、3D、4A、4B、5A和6D染色体上。其中,11个位点至少在2个环境中稳定表达,可用于开发CAPS标记。同时,发掘了一批株高性状相关基因的优异等位变异,如降低株高的等位变异Bob White_c48009_52,平均降低株高12.9 cm;控制穗下节间长的等位变异BS00039422_51-C和IAAV1698-A,分别调控穗下节间长5.9 cm和6.6 cm。本研究发掘的控制小麦株高基因位点为在分子水平上研究小麦株高复杂性状提供了有价值的参考。     

Construction of high‐throughput genotyped chromosome segment substitution lines in rice (Oryza sativa L.) and QTL mapping for heading date

Chromosome segment substitution lines (CSSLs) provide ideal materials for quantitative trait loci (QTLs) mapping and genetic dissection of complex traits. In this study, we developed a set of CSSL population consisting of 175 lines, which were derived between the recipient ‘Guangluai 4’ and the donor ‘Nipponbare’. Based on 260 molecular markers, we firstly constructed a physical map of core 97 lines. Then, these 97 lines were further genotyped based on resequencing data, and a resequencing‐based physical map was constructed. Compared with the molecular marker‐based physical map, the resequencing‐based physical map of 97 lines contained 367 substituted segments with 252 newly discovered segments. The total size of the 367 substituted segments was 1,074 Mb, which was 2.81 times the size of rice genome. Using the 97 CSSLs as materials, we identified nine QTLs for heading date and three of them were firstly reported. All the QTLs had positive additive effects, ranging from 9.50 to 16.50 days. These CSSLs may greatly help forge a new resource for functional genomics studies and molecular breeding in rice.     

Phenotypic and marker‐assisted pyramiding of genes for resistance to zucchini yellow mosaic virus in oilseed pumpkin (Cucurbita pepo)

We report on the identification of phenotypic and molecular markers for genes introgressed into oilseed pumpkin Cucurbita pepo from C. moschata germplasm originating in Nigeria, Portugal and Puerto Rico, which provide resistance against zucchini yellow mosaic virus (ZYMV) and on pyramiding these genes for improved and long‐lasting field protection of oilseed pumpkins. One SCAR and two SSR markers have been found for three dominant resistance genes, Zym‐0, Zym‐1 and Zym‐2. Characteristic reactions to ZYMV inoculation of plants carrying the recessive genes for resistance zym‐4* and zym‐6 have been defined. Described are procedures and results of pyramiding various combinations of these genes in oilseed pumpkin using the three markers and the specific phenotypic reactions to infection of some of these genes. The putative combination of all six resistance genes in one genotype resulted in a resistance that appeared to be at least as strong as or even stronger than that of the resistance source germplasm in C. moschata.     

Development of two new molecular markers specific to cytoplasmic male sterility in tuber mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tumida</Emphasis> Tsen et Lee)

A novel and stable cytoplasmic male sterility CMS line of tuber mustard has been bred by subsequent backcrosses for 10 years. Two specific markers atpA and orf220 were cloned and partially characterized in our previous study (Zhang et al. 2003). In this study, two new molecular markers, orf256 and orf305/orf324, have been isolated and identified. The orf256 gene size was found to be 825 bp in CMS line and a 1,357 bp in its maintainer line. Sequence analysis indicated that the orf256 gene was an entire coding sequence and downstream of the cox1 gene. Interestingly, the 906 bp fragment, which contains part of the sequence of orf222, nad5 and orf139 genes, was found to be inserted from the 451st bp of 5′-flank of the 1,357 bp fragment. In the same way, the orf324 gene was isolated from CMS line and orf305 gene from its maintainer line. Both of them are entire coding sequences, upstream from nad3 and rps12 gene, and co-transcribed with the nad3 and rps12 genes. In addition, two molecular markers, orf256 and orf324/orf305, have been successfully converted into the SCAR markers. Subsequently, ORF256, ORF324, ORF305 protein and ORF256-M-431 fragment are predicated to contain signal peptide sequences, and ORF220 was predicated to contain signal anchor sequence. RFLP analysis results revealed that all of the molecular markers exhibited polymorphisms. Northern blot analysis indicated that the expression level of these genes in CMS line is higher than that of the maintainer line. In the mass, all of these genes are expressed lower in the leaf than that of floral organs between the CMS line and its maintainer line. The difference in expression pattern of different mitochondrial specific marker genes suggests that the abundance of mitochondrial proteins is differentially regulated in the organ/tissue development in tuber mustard. Results of this study also provide some novel and useful clues to explore the biological function of these specific marker genes in the tuber mustard.     

Development and application of SCAR markers for sex identification in the dioecious species <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

There is an urgent need for early sex identification to support field planting in Ginkgo biloba L., due to the different economic and medicinal values between male and female trees. An easy, rapid and reliable molecular method for sex type determination of G. biloba was reported in the paper. Random amplification of polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) were used to search for specific molecular markers linked to the sex locus. A total of 48 primers were used for screening of specific RAPD markers in six male and three female samples. Only one primer, S10, showed different amplification band patterns associated with sex types. Then the sex-specific bands, S10-BandA and S10-BandB, were cloned and sequenced. Based on the sequences two pairs of SCAR primers, GBA and GBB, were designed. The GBA primers amplify a single 571 bp band in male samples but not in female samples, and DNA amplification using GBB primers could generate a 688 bp band only in the female individuals. Finally, the SCAR primers were used to test 16 sex-unknown samples. SCAR primers developed in this paper can be used as effective, convenient and reliable molecular markers for sex identification in G. biloba.