绿豆遗传连锁图谱的整合

利用绿豆及其近缘种的701对SSR引物,对现有绿豆遗传连锁图谱进行补充,结果在高感豆象绿豆栽培种Berken和高抗豆象绿豆野生种ACC41两亲本间筛选到多态性SSR引物104对。群体分析后,结合其他分子数据,使用作图软件Mapmaker/Exp 3.0b,获得一张含有179个遗传标记和12个连锁群,总长1831.8cM、平均图距10.2cM的新遗传连锁图谱,包括97个SSR标记,91个来自绿豆近缘种;RFLP标记76个;RAPD标记4个;STS标记2个。对32个绿豆、小豆共用SSR标记在遗传连锁图谱的分布分析发现,二个基因组间有一定程度的同源性,共用标记在连锁群上的排列顺序基本上一致,只有部分标记显示绿豆和小豆基因组在进化过程中发生了染色体重排;利用新图谱对ACC41的抗绿豆象主效基因重新定位,仍定位于I(9)连锁群,与其相邻分子标记的距离均小于8cM,其中与右翼SSR标记C220的距离约2.7cM。与原图谱比较,新定位的抗性基因与其相邻标记的连锁更加紧密。     

Development and characterization of tomato SSR markers from genomic sequences of anchored BAC clones on chromosome 6

Simple sequence repeat motifs are abundant in plant genomes and are commonly used molecular markers in plant breeding. In tomato, currently available genetic maps possess a limited number of simple sequence repeat (SSR) markers that are not evenly distributed in the genome. This situation warrants the need for more SSRs in genomic regions lacking adequate markers. The objective of the study was to develop SSR markers pertaining to chromosome 6 from bacterial artificial chromosome (BAC) sequences available at Solanaceae Genomics Network. A total of 54 SSR primer pairs from 17 BAC clones on chromosome 6 were designed and validated. Polymorphism of these loci was evaluated in a panel of 16 genotypes comprising of Solanum lycopersicum and its wild relatives. Genetic diversity analysis based on these markers could distinguish genotypes at species level. Twenty-one SSR markers derived from 13 BAC clones were polymorphic between two closely related tomato accessions, West Virginia 700 and Hawaii 7996 and were mapped using a recombinant inbred line population derived from a cross between these two accessions. The markers were distributed throughout the chromosome spanning a total length of 117.6 cM following the order of the original BAC clones. A major QTL associated with resistance to bacterial wilt was mapped on chromosome 6 at similar location of the reported Bwr-6 locus. These chromosome 6-specific SSR markers developed in this study are useful tools for cultivar identification, genetic diversity analysis and genetic mapping in tomato.     

Detection of quantitative trait locus for leaffolder (Cnaphalocrocis medinalis (Guenée)) resistance in rice on linkage group 1 based on damage score and flag leaf width

Rice leaffolder (RLF) (Cnaphalocrocis medinalis (Guenée) is a destructive and widespread insect pest throughout the rice growing regions in Asia. The genetics of resistance to RLF in rice is very complex and not thoroughly explored. The present study was conducted to detect the quantitative trait loci (QTL) associated with RLF resistance involving 176 recombinant inbred lines (RILs) of F₈ generation derived from a cross between IR36, a leaffolder susceptible variety and TNAULFR831311, a moderately resistant indica rice culture. Simple sequence repeat (SSR) markers were used to construct specific linkage groups of rice. All the RILs were screened to assess their level of resistance to RLF by measuring the leaf area damaged. Besides this, the length and width of the flag leaf of each RIL were measured since these two parameters were considered as correlated traits to the RLF resistance in rice. All the above parameters observed across the RILs showed quantitative variation. Correlation analysis revealed that damage score based on greenhouse screening was positively correlated with length and width of the flag leaf. Out of 364 SSR markers analysed, 90 were polymorphic between the parents. Multi-point analysis carried out on segregating 69 SSR marker loci linkage group wise resulted in construction of linkage map with eleven groups of 42 SSR markers. Through single marker analysis, 19 SSR markers were found to have putative association with the three phenotypic traits studied. Of these markers, RM472 was identified as a locus having major effect on RLF resistance trait based on length of the flag leaf. Interval mapping detected two QTLs on linkage group 1. Among these QTLs, the QTL flanked by RM576–RM3412 were found to be associated with width of the flag leaf and RLF resistance. The putative SSR markers associated with leaffolder resistance identified in the present study may be one of the loci contributing resistance to RLF in rice.     

Mapping a new source of resistance to powdery mildew in mungbean

Both restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) analyses were employed to map a new source of resistance to powdery mildew in mungbean. Disease scores of an F₂ population derived from the cross between a moderately resistant breeding line VC1210A and a susceptible wild relative (Vigna radiata var. sublobata, accession TC1966) showed a continuous distribution and was treated as a quantitative trait. Although no significant quantitative trait loci (QTL) that can explain the variation was detected by QTL analysis based on the reconstructed RFLP linkage map, new marker loci associated with resistance were discovered by AFLP analysis. The RFLP loci detected by two of the cloned AFLP bands are associated with resistance and constitute a new linkage group. A major resistance quantitative trait locus was found on this linkage group that accounted for 64.9% of the variation in resistance to powdery mildew. One of the probes developed in this study has the potential to assist in breeding for powdery mildew resistance in mungbean.     

Mapping of quantitative trait loci controlling powdery mildew resistance in Mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek)

Powdery mildew disease in mungbean is caused by the fungus, Erysiphe polygoni D.C. We identified two quantitative trait loci (QTLs) controlling resistance to the disease in a RIL population of 190 F₇ lines. The population was developed from the cross between a susceptible cultivar, “Kamphaeng Saen 1” and a resistant line, “VC6468-11-1A”. Reaction to the disease was evaluated for resistance in field and greenhouse conditions. Results from analysis of variance revealed that 15 SSR loci on three linkage groups (LG) associated with the resistance. Composite interval mapping consistently identified two QTLs on two LGs, qPMR-1 and qPMR-2, conferring the resistance. qPMR-1 and qPMR-2 accounted for 20.10 and 57.81% of the total variation for plant response to the disease, respectively. Comparison based on common markers used in our and previous studies suggested that qPMR-2 is possibly the same as the major QTL reported earlier using another resistant source. The SSR markers flanking and closely linked to qPMR-1 (CEDG282 and CEDG191) and qPMR-2 (MB-SSR238 and CEDG166) are useful for marker-assisted selection for mungbean resistance to powdery mildew.     

Genetic mapping of bph20(t) and bph21(t) loci conferring brown planthopper resistance to Nilaparvata lugens St?l in rice (Oryza sativa L.)

Brown planthopper(BPH) is one of the most serious and destructive insect pests of rice in most rice growing regions of the world. In this study, two major resistance genes against BPH have been identified in an Oryza rufipogon (Griff.) introgression rice line, RBPH54. Inheritance of the BPH resistance in RBPH54 was studied by screening the resistance in parents, F1, F2 and BC1 generations against BPH biotype 2. A population of BC3F2 lines was developed and SSR markers were employed for the gene mapping, and new markers were designed for fine mapping of the resistance genes, while sequence information of BAC/PAC clones was used to construct physical maps of the genes. The results showed that the BPH resistance in RBPH54 was governed by recessive alleles at two loci, tentatively designated as bph20(t) and bph21(t). The locus bph20(t) was fine mapped to the short arm of chromosome 6 about 2.7 cM to the upper marker RM435 and 2.5 cM to lower marker RM540 and in a 2.5 cM region flanked by two new SSR markers BYL7 and BYL8 which were developed in the present study. The other BPH resistance locus bph21(t) was initially mapped to a region 7.9 cM to upper marker RM222 and 4.0 cM to lower marker RM244 on the short arm of chromosome 10. For physical mapping, the bph20(t)-linked markers were landed on BAC/PAC clones of the reference cv., Nipponbare, released by the International Rice Genome Sequencing Project. The bph20(t) locus was physically defined to an interval of about 75 kb with clone P0514G1. Identification and location of these two genes in the present study have diversified the BPH resistance gene pool, which give benefit to the development of resistant rice cultivars, and the linkage PCR-based SSR markers for the bph20(t) and bph21(t) genes would help realize the application of the genes in rice breeding through marker-assisted selection.     

The major quantitative trait locus for mungbean yellow mosaic Indian virus resistance is tightly linked in repulsion phase to the major bruchid resistance locus in a cross between mungbean [Vigna radiata (L.) Wilczek] and its wild relative Vigna radiata ssp. sublobata

Mungbean yellow mosaic Indian virus (MYMIV) and bruchid infestation are severe production constraints of mungbean in South Asia, a major global mungbean production area. Marker-assisted selection for resistance against these disorders while maintaining or even improving agronomic traits is an important step toward breeding elite mungbean varieties. This study employed recombinant inbred lines (F12) derived from a cross between MYMIV-tolerant Vigna radiata NM92 and bruchid-resistant V. radiata ssp. sublobata TC1966 to identify chromosomal locations associated with disease and insect pest resistance and seed traits. A linkage map comprising 11 linkage groups was constructed with random amplified polymorphic DNA (RAPD), sequence characterized amplified regions (SCAR), cleaved amplified polymorphic DNA (CAP), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Quantitative trait loci (QTLs) for MYMIV and bruchid resistance, 100 seed weight and seed germination rate were identified. Three major QTLs for MYMIV and one major bruchid resistance locus were mapped on LG 9. The resistance alleles were contributed by the MYMIV tolerant parent NM92 and the bruchid resistant parent TC1966 respectively. One of the MYMIV QTLs was tightly linked in repulsion phase to the bruchid resistance locus. In addition, three minor QTLs for MYMIV resistance were found, where the resistance alleles were contributed by TC1966. Lines combining MYMV resistance alleles from both parents have greater resistance to MYMIV than the tolerant parent. Two minor bruchid resistance QTLs were identified in TC1966. Furthermore, three QTLs each for 100 seed weight and germination rate were detected. The markers defining the QTLs identified in this study will be useful in marker-assisted breeding of improved mungbean varieties in the future.     

Map- vs. homology-based cloning for the recessive gene ol-2 conferring resistance to tomato powdery mildew

The recessive gene ol-2 confers papilla-associated and race-non-specific resistance to tomato powdery mildew caused by Oidium neolycopersici. In order to facilitate marker assisted selection (MAS) in practical breeding programmes, we identified two simple sequence repeat (SSR) markers and one cleaved amplified polymorphic sequence (CAPS) marker which are linked to the resistance locus and co-dominantly inherited. Aiming to provide a base for ol-2 positional cloning, we used a large segregating F₂ population to merge these markers with all the ol-2 linked amplified fragment length polymorphism (AFLP^®) markers previously identified in an integrated genetic map. By screening a tomato bacterial artificial chromosome (BAC) library, we detected two BAC clones containing two expressed sequence tags (ESTs) homologous to the gene mlo, responsible for powdery mildew resistance in barley, as well as an ol-2-linked marker. Chromosomal mapping by Fluorescence in situ Hybridization (FISH) revealed major signals of the two BAC DNAs in the pericentromeric heterochromatin of the short arm of chromosome 4, in the same region where the ol-2 gene was previously mapped. The genetic and cytogenetic co-localisation between ol-2 and tomato mlo-homologue(s), in addition to the similarity of ol-2 and mlo resistances for both genetic and phytopathological characteristics, suggests that ol-2 is likely a mlo-homologue. Thus, a homology-based cloning approach could be more suitable than positional cloning for ol-2 isolation.     

籼稻多蘖矮半矮秆基因的遗传分析和基因定位

对籼稻标记基因系材料多蘖矮的遗传分析表明, 其矮生性状是由2对隐性半矮秆基因控制的,分别为sd1和一个新的半矮秆基因,该基因初步定名为sdt3。以多蘖矮与南京6号杂交F₂的分离群体为基础,应用SSR标记进行连锁分析,将半矮秆基因sdt3定位于第11染色体的SSR标记SSR98和SSR35之间,分别相距0.06 cM、0.13 cM,二者之间的物理距离约为93kb。以南京6号为轮回亲本与多蘖矮进行回交和自交获得由半矮秆基因sdt3控制的近等基因系（新多蘖矮）,以赤霉素处理表明由sdt3控制的半矮秆系新多蘖矮对赤霉素不敏感。     

SSR markers associated for late leaf spot disease resistance by bulked segregant analysis in groundnut (Arachis hypogaea L.)

Late leaf spot (LLS) caused by Phaeoisariopsis personata is the major foliar disease that reduces the pod yield and severely affects the fodder and seed quality in groundnut. Molecular markers linked with LLS can improve the process of identification of resistant genotypes. In the present study, a LLS susceptible genotype (TMV 2) and the LLS resistant genotype (COG 0437) were crossed and their F₂ population was used for marker analysis. The phenotypic mean data on F_2:3 progenies were used as phenotype. Parents were surveyed with 77 SSR (Simple Sequence Repeat) primers to identify polymorphic markers. Among SSR markers, nine primers were found polymorphic between the parents TMV 2 and COG 0437. These markers were utilized for bulked segregant analysis (BSA). Among the polymorphic SSR markers, three primers viz., PM 375₁₆₂, pPGPseq5D5₂₂₀ and PM 384₁₀₀ were able to distinguish the resistant and susceptible bulks and individuals for LLS. In single marker analysis, the markers PM 375, PM 384, pPGPseq5D5, PM 137, PM 3, PMc 588 and Ah 4-26 were linked with LLS severity score. The phenotypic variation explained by these markers ranged from 32 to 59?%. The markers identified through BSA were also confirmed with single marker analysis. While validating the three primers over a set of resistant and susceptible genotypes, the primer PM 384₁₀₀ allele had association with resistance. Hence PM 384 could be utilized in the marker assisted breeding programme over a wide range of genetic background.     

小豆SSR引物在绿豆基因组中的通用性分析

分析了187对小豆SSR引物在绿豆基因组中的可转移性,以期为绿豆分子遗传育种研究提供分析工具.结果表明,约75%的小豆SSR引物可在绿豆中有效扩增,但不同小豆连锁群SSR引物的可转移率存在差异.多态性分析发现80对引物中有28对在60份绿豆种质中可以检测到多态性,等位变异数从2～7不等,平均为2.9,PIC指数从0.02～0.69,平均为0.36.UPGMA聚类及主坐标分析表明,尽管同一省份的种质不能紧密聚在一起,但大多在聚类图上成簇状分布,说明相同来源的绿豆种质具有相似的遗传背景;此外,国外及我国边远地区的绿豆种质在遗传背景上与内部省份间存在明显差异.这些多态性SSR不仅可以有效用于绿豆分子遗传学研究,还可以用于不同来源绿豆种质资源的辅助鉴别.     

Mapping of EST-derived CAPS markers in Italian ryegrass (Lolium multiflorum Lam.)

New molecular markers derived from expressed sequence tag (EST) sequences were mapped on linkage maps of Italian ryegrass by a two-way pseudo-testcross strategy. cDNA sequences were obtained from various tissues of Italian ryegrass ( Lolium multiflorum ) and converted into cleaved amplified polymorphic sequence (CAPS) markers. Of 260 EST primer pairs that amplified a single band, 74 generated bands that showed clear polymorphisms among individuals of an F₁ mapping family. Of the 74 polymorphic marker loci, 69 were mapped on an Italian ryegrass linkage map previously constructed using amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR) markers. The newly-developed EST-CAPS markers would be useful as an efficient tool to identify genetic markers and to identify candidate genes for quantitative trait loci (QTLs) associated with important traits in Italian ryegrass.     

Linkage analysis of RFLP markers for clubroot resistance and pigmentation in Chinese cabbage (Brassica rapa ssp. pekinensis)

A restriction fragment length polymorphism (RFLP) – based linkage map of Chinese cabbage (Brassica rapa ssp. pekinensis) (2n=20) including two agronomic traits, clubroot resistance and orange-yellow pigmentation, was constructed using doubled haploid parents. The total linkage distance was 735 cM; 63 loci were distributed into ten linkage groups. Clubroot resistance of the parental line T136-8 to the current pathotype, race 2, was predominantly controlled by a single dominant gene that originated from European turnip. The locus for clubroot resistance by the dominant major gene (CRa) was mapped on linkage group 3, and RFLP loci HC352b and HC181 were located 3 cM and 12 cM from it, respectively. The locus HC352b was identified by a 4.4 Kb Eco R I fragment, which segregated for null allele. The absence of an allelic fragment in HC352b could be interpreted by deletion in the resistance source; homozygotes for CRa could be efficiently selected by detecting null types for the marker. Orange-yellow pigmentation expressed in head inner leaves and petals was governed by a single recessive gene. The locus (Oy) for the pigmentation was mapped on linkage group 1, being located 17–19 cM from three RFLP loci that were closely linked to each other. The linkage analysis for clubroot resistance and unique pigmentation revealed some informative RFLP markers. Identification of molecular markers for clubroot resistance and other agronomically important traits would provide useful information in breeding programs of Chinese cabbage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Stability of seed oil quality traits in high and mid-oleic acid sunflower hybrids

Powdery mildew caused by Podosphaera xanthii is an important disease of melon, and race 2F is the predominant race in most areas of China. Resistance to P. xanthii race 2F in melon K7-1 was controlled by a dominant gene, designated Pm-2F, in a 106-member population of recombinant inbred lines derived from K7-1× susceptible K7-2. Using bulked segregant analysis with molecular markers, we have identified two polymorphic simple sequence repeats (SSR) to determine that Pm-2F is located on linkage group II. Comparative genomic analyses using mapped SSR markers and the cucumber genome sequence showed that the melon chromosomal region carrying Pm-2F is homologous to a 288,223 bp genomic region on cucumber chromosome (chr) 1. The SSR markers on chr 1 of cucumber, SSR02734, SSR02733 and CS27 were found linked with Pm-2F. Comparative mapping showed that two SSR markers (SSR02734 and CMBR8) flanked the Pm-2F locus and two nucleotide binding site-leucine-rich repeat resistance genes were identified in the collinear region of cucumber. A cleaved amplified polymorphic sequence (CAPS) marker was developed from the sequence of resistance genes and it delimits the genomic region carrying Pm-2F to 0.8 cM. The evaluation of 165 melon accessions and 13 race differential lines showed that the newly developed CAPS (CAPS-Dde I) marker can be used as a universal marker for effective marker assisted selection in melon powdery mildew resistance breeding. The putative resistance gene cluster provides a potential target site for further fine mapping and cloning of Pm-2F.     

Comparisons of RFLP and PCR-based markers to detect polymorphism between wheat cultivars

Previously chromosome 3A of wheat (Triticum aestivum L.) was reported to carry genes influencing yield, yield components, plant height, and anthesis date. The objective of current study was to survey various molecular marker systems for their ability to detect polymorphism between wheat cultivars Cheyenne(CNN) and Wichita (WI), particularly for chromosome3A. Seventy-seven `sequence tagged site' (STS), 10simple sequence repeat (SSR), 40 randomly amplified polymorphic DNA (RAPD) markers, and 52 restriction fragment length polymorphism (RFLP) probes for wheat homoeologous group 3 chromosomes, were investigated. Three (3.9%) STS-PCR primer sets amplified polymorphic fragments for the two cultivars, of which one was polymorphic for chromosome 3A. Sixty percent of SSR markers detected polymorphism between CNN and WI of which 50% were polymorphic for chromosome 3A. Twenty percent of RAPD markers detected polymorphism between CNN and WI in general, but none of these detected polymorphism for chromosome 3A. Of the fifty-two RFLP probes, 78.8% detected polymorphism between CNN and WI for group 3 chromosomes with one or more of seven restriction enzymes and 42% of the polymorphic fragements were for chromosome 3A. These high levels of RFLP and SSR polymorphisms between two related wheat cultivars could be used to map and tag genes influencing important agronomic traits. It may also be important to reconsider RFLP as the most suitable marker system at least for anchor maps of closely related wheat cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

Abundance, polymorphism and genetic mapping of microsatellites in oilseed rape (Brassica napus L.)

The objective of the present study was to estimate the abundance and degree of polymorphism of simple sequence repeat (SSR) markers in rapeseed. By screening about 45000 clones of a small inserts library of rapeseed total DNA the abundances of GA/TC and CA/TG simple sequence repeats in the rapeseed genome were estimated to be approximately one repeat every 100 kb and 400 kb, respectively. After sequencing 13 positive clones, primer pairs could be designed for 11 microsatellite loci. Seven of these primer pairs produced reproducible amplification products in a set of 31 rapeseed genotypes, with one pair amplifying two independent products, giving a total of eight amplified loci. The different microsatellite loci displayed between one and three visible alleles. At four loci, additional null alleles were observed. With up to four alleles, polymorphic microsatellite markers show significantly higher allele numbers in rapeseed than restriction fragment length polymorphism (RFLP) markers. Four of the eight microsatellite markers could be mapped on four different linkage groups of an RFLP map of the rapeseed genome.     

Mapping and validation of QTLs for resistance to an Indian isolate of Ascochyta blight pathogen in chickpea

Ascochyta blight (AB) caused by Ascochyta rabiei, is globally the most important foliar disease that limits the productivity of chickpea (Cicer arietinum L.). An intraspecific linkage map of cultivated chickpea was constructed using an F₂ population derived from a cross between an AB susceptible parent ICC 4991 (Pb 7) and an AB resistant parent ICCV 04516. The resultant map consisted of 82 simple sequence repeat (SSR) markers and 2 expressed sequence tag (EST) markers covering 10 linkage groups, spanning a distance of 724.4 cM with an average marker density of 1 marker per 8.6 cM. Three quantitative trait loci (QTLs) were identified that contributed to resistance to an Indian isolate of AB, based on the seedling and adult plant reaction. QTL1 was mapped to LG3 linked to marker TR58 and explained 18.6% of the phenotypic variance (R ²) for AB resistance at the adult plant stage. QTL2 and QTL3 were both mapped to LG4 close to four SSR markers and accounted for 7.7% and 9.3%, respectively, of the total phenotypic variance for AB resistance at seedling stage. The SSR markers which flanked the AB QTLs were validated in a half-sib population derived from the same resistant parent ICCV 04516. Markers TA146 and TR20, linked to QTL2 were shown to be significantly associated with AB resistance at the seedling stage in this half-sib population. The markers linked to these QTLs can be utilized in marker-assisted breeding for AB resistance in chickpea.     

Molecular tagging of Asiatic soybean rust resistance in exotic genotype EC 241780 reveals complementation of two genes

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi severely reduces seed yield in soybean. Molecular tagging of ASR resistance can help in the process of resistance breeding. In this study, an F₂ population of cross (susceptible cultivar ‘NRC 7’ × resistant exotic genotype EC 241780) was used for bulked segregant analysis (BSA) with 25 SSR (simple sequence repeat) primers linked with six Rpp genes. Among them, five polymorphic SSR markers, viz., Sct 187, SSR 1859, Satt 191 (Rpp1b like loci) and Satt 215, Sat_361 (Rpp2 loci) distinguished the ASR resistant and susceptible bulks and individuals. In combined marker analysis, the markers Satt 191 (Rpp1b like loci) and Satt 215 (Rpp2 loci) were linked with ASR severity score and were also confirmed in individual 110 F₂ segregants. Hence, these markers could be utilized in the marker assisted rust resistance breeding of Rpp1b like and Rpp2 genes. In silico candidate gene analysis for hypersensitive response revealed that Satt 191 linked region was rich in genes encoding apoptotic ATPase having leucine‐rich repeat (LRR) domain.     

陆地棉分子遗传图谱的构建

利用SSR引物对具有抗黄萎病性状的F2群体进行分子遗传图谱构建研究。结果表明：2000对SSR引物在对新陆中10号和新陆早7号的的多态性筛选中,共筛选到89个稳定的SSR多态性位点,其中共显性标记为74个,显性标记15个;利用Mapmaker/EXP（version 3.0b）对89个标记构建了连锁群,其中65个标记位点被分配到19个不同的连锁群上,24个标记位点没有分配到连锁群上;19个连锁群连锁群总的长度962.2cM,覆盖率为17.49%。