Phylogenetic Relationships in Genus Arachis Based on SSR and AFLP Markers

Fourteen wild species of different sections in the genus Arachis and 24 accessions of the AABB allotetraploid A. hypogaea （cultivated peanut） from several countries which belong to different botanical varieties, were analyzed by SSR and AFLP marker systems. The assay-units per system needed to distinguish among all the tested accessions were at least five for SSR or two for AFLP. The genetic distance detected by the SSR markers ranged from 0.09 to 0.95, and the mean was 0.73; and the genetic distance detected by the AFLP markers ranged from 0.01 to 0.79 with an average of 0.42. All the tested peanut SSR primer pairs were multilocus ones, and the amplified fragments per SSR marker in each peanut genome ranged from 2 to 15 with the mean of 4.77. The peanut cultivars were closely related to each other, and shared a large numbers of SSR and AFLP fragments. In contrast, the species in the genus Arachis shared few fragments. The results indicated that the cultivated peanut （A. hypogaea L.） varieties could be partitioned into two main groups and four subgroups at the molecular level, and that A. duranensis is one of the wild ancestors of A. hypogaea. The lowest genetic variation was detected between A. cardenasii and A. batizocoi, and the highest was detected between A. pintoi and the species in the section Arachis. The relationships among the botanical varieties in the cultivated peanut （A. hypogaea L.） and among wild species accessions in section Arachis and those in other sections in the genus Arachis were discussed.     

Application of Molecular Markers Linking to Cytoplasmic Male Sterile Loci to Assist Maintainer Line Selection and Their Selection Efficiency in Welsh Onion （Allium fistulosum L.）

Cytoplasmic male sterility exists widely in most natural populations of welsh onion （Alliumfistulosum L.）, which makes it possible to breed out many male sterile lines for heterosis utilization. Unfortunately, the breeding of cytoplasmic male sterility in welsh onion has a little progress due to the limitation of its biological characteristic and traditional selection approach. To study the feasibility and the efficiency of utilizing marker assisted selection for male sterile lines in welsh onion, one SCAR marker, SCS13, and one RAPD marker, S2002400, which could distinguish between N and S cytoplasm in several welsh onion cultivars, were identified. The two markers were then confirmed by Southern blotting, and used to screen the N or S cytoplasm of individual plants in seven welsh onion cultivars in this study. Male sterile and fertile plants were evaluated by aceto-carmine dying. The frequency of N-cytoplasmic plants and maintainer genotype was calculated in the seven open populations of welsh onion. The minimum number of plants needed to identify a maintainer was evaluated to be 95% reliable. Results showed that 20 to 80% decrease of crosses and self-crosses for identifying a maintainer genotype could be achieved by the marker-assisted selection compared with traditional selection method. It was proved that the molecular markers could precisely identify cytoplasmic types individually, performed by one generation of cross and two generations of testcrosses and self-crosses. Finally, several maintainer genotype plants were selected with the help of the two markers in the seven cultivars. The screened markers could assist and accelerate sterile and maintainer lines selection with less labor and cost.     

Molecular Characterization of Atlas 66-Derived Wheat Near-lsogenic Lines Contrasting in Aluminum （Al） Tolerance

Aluminum （Al） toxicity is the major limiting factor for wheat growth in acidic soils. Genetic improvement of Al tolerance is one of the most cost-effective solutions to improve wheat productivity. The objective of this study was to characterize near isogenic lines （NILs） contrasting in Al tolerance derived from Atlas 66 in the backgrounds of Al-sensitive cultivars Chisholm and Century using amplified fragment length polymorphism （AFLP） and simple sequence repeat （SSR）. A total of 200 AFLP and 88 SSR primer pairs were screened and 12 markers （11 AFLPs and one SSR） were associated with Al-tolerance in NILs of at least one recurrent parental background. Among them, nine were linked to A1 tolerance in the Chisholm-derived NILs, seven were associated with Al-tolerance in the Century-derived NILs, and three AFLPs derived from the primer combinations of pAG/mGCAG, pCAG/mAGC and pGTG/mGCG, and one SSR, Xwmc331 on chromosome 4D, associated with A1 tolerance in NILs of both recurrent parental backgrounds. Those common markers across two backgrounds may be the major marker loci associated with Al-tolerance in Atlas 66 and could be useful for marker-assisted breeding to improve Al tolerance in wheat. In addition, evaluation of Al tolerance among different genotypes using hematoxylin stain and relative root growth revealed that Atlas 66 was more tolerant to Al stress than the NILs, therefore suggested that the Al-tolerant NILs might not carry all Al-tolerance loci from Arias 66 and inheritance of Al tolerance in Arias 66 is more likely multigenic.     

Development of Sequence Characterized Amplified Region （SCAR） Primers for the Detection of Resistance to Sporisorium reiliana in Maize

Head smut of maize （Zea mays L.）, which was caused by Sporisorium reiliana, occurred in most of the maize growing areas of the world. The purpose of this study was to develop SCAR markers for map-based cloning of resistance genes and MAS. Two sets of BC3 progenies, one （BC3Q） derived from the cross Qi319 （resistance）×Huangzao 4 （susceptible）, the other （BC3M） from Mol7 （resistance）× Huangzao 4 （susceptible）, were generated. Huangzao 4 was the recurrent parent in both progenies. A combination of BSA （bulked segregant analysis） with AFLP （amplified fragment length polymorphism） method was applied to map the genes involving the resistance to S. reiliana, and corresponding resistant and susceptible bulks and their parental lines were used for screening polymorphic AFLP primer pairs. One fragment of PI3M61-152 was converted into SCAR （sequence charactered amplified fragment） marker S130. The marker was mapped at chromosome bin 2.09, the interval of a major QTL region previously reported to contribute to S. reiliana resistance. Furthermore, S130 was highly and facilitate map-based cloni associated with resistance to S. reiliana, and could be useful for marker-assisted selection ng of resistance genes.     

Inheritance of Powdery Mildew Resistance in Cucumber （Cucumis sativus L.） and Development of an AFLP Marker for Resistance Detection

Cucumber powdery mildew is one of the most destructive diseases of cucumber throughout the world. In the present study, inheritance of powdery mildew resistance in three crosses, and linkage of resistance with amplified fragment length polymorphism （AFLP） markers are studied to formulate efficient strategies for breeding cultivars resistant to powdery mildew. The joint analysis of multiple generations and AFLP technique has been applied in this study. The best model is the one with two major genes, additive, dominant, and epistatic effects, plus polygenes with additive, dominant, and epistatic effects （E-l-0 model）. The heritabilities of the major genes varied from 64.26% to 97.82%, and susceptibility was incompletely dominant for the two major genes in the three crosses studied. The additive effects of the two major genes and the dominant effect of the second major gene were high, and the epistatic effect of the additive-dominant between the two major genes was the highest in cross I . In cross II, the absolute value of the additive effect, dominant effect, and potential ratio of the first major gene were far higher than those of the second major gene, and the epistatic effect of the additive-additive was the highest. The genetic parameters of the two major genes in cross III were similar to those in cross II. Correlation and regression analyses showed that marker E25/M63-103 was linked to a susceptible gene controlling powdery mildew resistance. The marker could account for 19.98% of the phenotypic variation. When the marker was tested on a diverse set of 29 cucumber lines, the correlation between phenotype and genotype was not significant, which suggested cultivar specialty of gene expression or different methods of resistance to powdery mildew. The target DNA fragment was 103 bp in length, and only a small part was found to be homologous to DNA in the other species evaluated, which indicated that it was unique to the cucumber genome.     

Molecular Identification and Cultivar Fingerprints of Prunus persica （L.） Batsch Germplasms

[Objective] The aim was to study the molecular identification and cultivar fingerprints of Prunus persica （L.） Batsch germplasms.[Method] Sixty peach genotypes,representing China common local cultivars and European samples were screened by microsatellites （simple sequence repeats,SSRs） and Inter-Simple Sequence Repeat （ISSR） markers.[Result] 26 reproducible bands were amplified by Nine SSR primers,and 24 of which were polymorphic; 236 bands were amplified by 30 ISSR primers,and 113 of which were polymorphic.31 genotypes were discriminated with 1-3 distinct polymorphic bands generated from the primers ISSR and SSR.Seven cultivar-specific ISSR fragments and two SSR unique alleles obtained from this study were available to be converted into Sequence Characterized Amplified Region （SCAR） markers.The genetic similarity coefficient （GS） estimated from these molecular data averaged were 0.939 （ranged from 0.856 to 0.983） for ISSR and 0.646 （ranged from 0.240 to 1.000） for SSR,respectively.The combined grouping association indicated that most local Chinese peach cultivars and exotic accessions were clustered together.This could be related to the mode of introduction and maintenance of the peach cultivars involving limited foundation germplasm,exchange of cultivars between plantations,and periodic development of new recombinant cultivars following sexual reproduction.[Conclusion] The results obtained in this work would help to improve the conservation,molecular identification and management of peach germplasm in breeding.     

Assessment of the Genetic Relationship and Diversity of Mango and Its Relatives by cplSSR Marker

Chloroplast inter-simple sequence repeat markers in mango were developed and used to analyze the genetic relationship and diversity of mango and its relatives. Thirty-six mango cultivars （Mangifera indica L.） and its relative species collected from the fruit germplasm collection in the Guangxi Academy of Agricultural Sciences, China, were examined by ISSR-PCR with chloroplast DNA （cpDNA）. Eight better primers for chloroplast DNA that provided reproducible, polymorphic DNA amplification patterns were screened from 50 ISSR primers and used for UPGMA analysis. According to the band patterns with 8 primers for chloroplast DNA, all cultivars tested were distinguished from each other and these showed ample genetic diversity; the average percentage of polymorphism was 77.2%. The 36 samples could be clustered into four groups by UPGMA analysis at the coefficient 0.74. The results indicated that the cplSSR marker was a new powerful tool for the identification of mango cultivars or its relative species, and their genetic relationship analysis and diversity evaluation.     

Development of an STS Marker Linked to Powdery Mildew Resistance Genes PmLK906 and Pm4a by Gene Chip Hybridization

Wheat （Triticum aestivum L.） line Lankao 90（6） carries a recessive powdery mildew resistance gene temporarily named PmLK906 on chromosome 2AL. Near PmLK906 there is another known powdery mildew resistance gene locus Pm4. To track the two powdery mildew resistance genes in wheat breeding program by marker assisted selection （MAS）, a linked molecular marker was developed in this study. Wheat gene chip hybridization combined with bulked segregant analysis （BSA） was used to develop an sequence-tagged sites （STS） marker for PmLK906 and Pm4. A new 2 125 bp full-length cDNA clone （GenBank accession no. EU082094） similar to csAtPR5 ofAegilops tauschii was isolated from Lankao 90（6） 21-12, and temporarily named TaAetPR5. Specific products could be amplified from cultivars or lines possessing Pm4a, Pm4b and PmLK906 with primers p9-7pl and p9-7p2 derived from TaAetPR5. TaAetPR5 was linked to PmLK906 at a genetic distance of 7.62 cM, and cosegregated with Pm4a. The p9-7p1 and p9-7p2 could be used as an STS marker for these resistance genes in wheat breeding. Because this marker was cosegregated with Pm4a, it can be used in map-based cloning of the alleles at Pm4 locus also.     

Soil Aggregation and Its Relationship with Organic Carbon of Purple Soils in the Sichuan Basin, China

The interaction of soil aggregate dynamics with soil organic carbon is complex with varied spatio-temporal processes in macro-and micro-aggregates. This paper is to determine the aggregation of soil aggregates in purple soils （Regosols in FAO Taxonomy or Entisols in USDA Taxonomy） for four types of land use, cropland [corn （Zea mays L.）], orchard （citrus）, forestland （bamboo or cypress）, and barren land （wild grass）, and to explore their relationship with soil organic carbon in the Sichuan basin of southwestern China. Procedures and methods, including manual dry sieving procedure, Yoder＇s wet sieving procedure, pyrophosphates solution method, and Kachisky method, are used to acquire dry, wet, and chemically stable aggregates, and microaggregates. Light and heavy fractions of soil organic carbon were separated using 2.0 g·mL^-1 HgI2-KI mixed solution. The loosely, stably, and tightly combined organic carbon in heavy fraction were separated by extraction with 0.1 M NaOH and 0.1 M NaOH-0.1M Na4P2O7 mixed solution （pH 13）. The results show that the contents of dry and wet macroaggregates 〉0.25 mm in diameter were 974.1 and 900.0 g·kg^-1 highest in red brown purple soils under forestland, while 889.6 and 350.6 g·kg^-1 lowest in dark purple soil and lowest in grey brown purple soils under cropland, respectively. The chemical stability of macroaggregates was lowest in grey brown purple soil with 8.47% under cropland, and highest in red brown purple soil with 69.34% under barren land. The content of microaggregates in dark purple soils was 587g·kg^-1 higher than brown purple soils, while 655g·kg^-1 in red brown purple soils was similar to grey brown purple soils （651g·kg^-1）. Cropland conditions, only 38.4% of organic carbon was of the combined form, and 61.6% of that existed in light fraction. Forestland conditions, 90.7% of organic carbon in red brown purple soil was complexed with minerals as a form of humic substances. The contents and stability of wet aggregates 〉 0.25 mm, con     

Genetic Relation Analysis on Ramie [Boehmeria nivea （L.） Gaud.] Inbred Lines by SRAP Markers

The objective of this article is to reveal the variations of ramie inbred lines in DNA level and discuss their molecular background to provide a theoretical basis for ramie cross breeding. In the present study, the genetic relationships among 33 inbred line accessions and two wild types that originated from China and Brazil were estimated using sequence-related amplified polymorphism （SRAP） markers. The results showed that 33 out of 81 primer combinations turned out to be polymorphic and 332 polymorphism bands were obtained. On the basis of the appearance of the markers, the genetic relationships were analyzed using unweighted pair-group method of arithmetic average cluster analysis （UPGMA）, and the genetic Jaccard similarity coefficients were calculated. The inbred-lines originating from China and Brazil formed a cluster suggesting a possibility that the Brazilian cultivars could have developed from cultivars introduced from China. Within ramie inbred-lines, the groupings also indicated that the greatest genetic relationship among cultivars was correlated to the region of origin of cultivars. The results provided the evidence that SRAP was an efficient approach, suitable for taxonomic analysis of ramie inbred lines, To the authors＇ knowledge, this is the first application of SRAP marker on the systematics of ramie inbred lines.     

与茄子果皮颜色相关联的AFLP及SCAR标记

 【目的】寻找与茄子皮色或果皮花青素合成相关基因连锁的分子标记,检验混合品系法（bulked lines analysis）在筛选分子标记中的应用。【方法】采用AFLP技术,以具有不同果皮颜色的茄子及近缘种为材料,采用单株检测和混合品系法分别筛选与茄子果皮颜色相关基因连锁的分子标记。【结果】对58份高代自交系材料进行单株检测（亲缘关系分析）,筛选到了1个与茄子紫红、紫黑果色相关的共显性标记。测序结果表明,片段长度分别为107和108 bp,为发生插入/缺失突变的同源序列;将其转化为SCAR标记,对136份茄子材料进行验证,在其中111份紫红、紫黑皮色材料中,相关符合率为90%,在白色及绿色材料中表现为不规律分布。最后以混合品系法,在两池内进一步筛选到了6个多态性标记,对6个标记进行单株验证,确定了它们与紫红、紫黑果色相关。【结论】本试验所获得的SCAR标记、AFLP标记可以用于茄子果色分子标记辅助育种。     

西南牡丹品种起源的ISSR研究

【目的】通过对西南牡丹21个品种、中原牡丹18个品种、江南牡丹1个品种和1个野生种之间的亲缘关系进行研究,了解西南牡丹品种群的遗传背景,为品种选育奠定基础。【方法】通过改良的CTAB法对41份材料抽提总基因组DNA,采用ISSR分子标记的方法,通过优化的ISSR-PCR反应体系,从60个哥伦比亚大学（UBC）开发的通用引物中筛选27个进行PCR扩增差异比较,利用POPGENE软件计算多态位点百分率（%）;依据Nei’s遗传距离,运用NTSYSPC进行UPGMA聚类构建不同品种群和品种间亲缘关系。【结果】利用27个ISSR引物对41个牡丹样本扩增,共获得的317个条带,其中304个为多态性条带,多态性条带比率为95.41%,平均每个引物扩增条带为11.74;41个样本的遗传相似性系数为0.483-0.811,云南牡丹‘丽江紫5’和‘大关粉4’的遗传相似性系数最大,为0.811;云南牡丹‘大关粉4’和中原牡丹‘彩绘’的遗传相似性系数最小,为0.483;UPGMA聚类结果显示41个样本在阈值为0.625时,聚为四支：第一支由19份样本组成,其中包括5个中原牡丹品种和14个西南牡丹品种,其中,云南牡丹‘丽江紫5’与‘大关粉4’亲缘关系最近,而云南牡丹‘丽江紫5’与中原牡丹‘首案红’的亲缘关系最远,其遗传相似性系数仅为0.609;第二支包括10份样本：有‘乌龙捧盛’、‘豆绿’等5个中原品种和‘四川粉紫’、‘昭通粉’、‘丽江粉1’等5个西南牡丹品种,其中天彭牡丹‘紫金荷2’与‘四川粉紫’的亲缘关系最近,遗传相似性系数最大为0.770;天彭牡丹‘紫金荷2’与中原牡丹‘罗婺现瑞’亲缘关系最远,其遗传相似性系数为0.625;第三支包括11个样本：1个江南品种,8个中原牡丹品种;3个西南牡丹品种。亲缘关系最近的是中原牡丹‘菱花湛露’与‘朱砂垒’,其遗传相似性系数为0.779;亲缘关系最远的是‘朱砂垒’和‘凤丹白’;第四支为黄牡丹单独聚为一支。多数花色相同的供试中原品种表现出近缘关系,红色系的天彭牡丹‘胭脂楼’与紫红色系的中原牡丹品种有着较近的亲缘关系,而供试的紫红色系的天彭牡丹分别紫红色、粉色、紫色和红色的中原品种有近缘关系;云南紫牡丹品种与紫色、紫红色系的中原品种有一定关系,粉牡丹则与紫红、浅红、浅紫红、浅紫色等不同色系的中原牡丹和天彭牡丹都有一定的亲缘关系。天彭牡丹总是先与中原牡丹品种相聚,再与云南牡丹相聚;云南牡丹品种除‘狮山皇冠’、‘香玉板’外,不同产地、株型相似和花色相同的云南牡丹品种间遗传相似性较高,总是先聚为一分支后,才与其他中原品种相聚。【结论】西南牡丹品种栽培起源较复杂,天彭牡丹比云南牡丹与中原牡丹有着较近缘关系,云南牡丹不可能是天彭牡丹直接引种驯化产物,推测云南牡丹品种可能是由几个祖先品种演化的产物,但本地黄牡丹参与起源的可能性较小。     

SSR分子标记分析彩色马铃薯品种间的遗传关系

利用SSR分子标记对来源不同的30份彩色马铃薯种质材料进行遗传多样性研究.结果表明,41个SSR引物扩增30个供试品种得到152条带,其中128个为多态性带,平均每个引物扩增3.12个多态性条带,其中包括一些品种特异性的SSR位点.UPGMA法进行聚类分析,在简单遗传相似系数SM=0.65处可将供试材料分成两大类群:类群Ⅰ为来自北美和欧洲的彩色马铃薯品种,在SM=0.72处可分为紫色马铃薯亚群,红色马铃薯亚群和白色马铃薯亚群;类群Ⅱ主要是不同来源的彩色马铃薯种质资源,包括二倍体原始栽培种,二倍体野生种和四倍体栽培种安弟斯亚种资源.聚类遗传关系表明类群Ⅰ和类群Ⅱ遗传关系较远,性状差异较大,可以作为极好的遗传资源进行杂交育种,扩展和丰富彩色马铃薯种质遗传背景     

利用AFLP方法筛选中华绒螯蟹性别相关标记

利用AFLP(amplified fragment length polymorphism)技术筛选中华绒螯蟹性别相关标记,共使用192对引物组合检测中华绒螯蟹雌、雄和雌雄混合3个基因组池的多态性,共扩增出5 376条多态性条带,平均每对引物组合扩增出28条多态性条带,获得88条雌雄差异条带。利用雌雄各10个个体再次进行AFLP验证,共62条差异条带具有性别差异,包括两种情况:(1)有49条条带在雌性的4～6个个体中存在,在雄性全部个体中缺失;(2)有13条条带在雄性4～6个个体中存在,在所有的雌性个体中缺失;其余的26条没有性别差异性。差异条带经回收、克隆、测序,结果发现有些条带包含多个序列,共得到77条DNA序列,其中31条序列与鸟类、两栖类和哺乳类等脊椎动物性染色体DNA部分片段具有同源性,相似区间在22～212 bp不等,其中6条DNA序列与命中序列之间具有生物学相关性。设计特异性的引物在基因组中检测,没有获得性别特异SCAR标记。     

分子标记技术及其在大豆疫霉根腐病研究中的应用

随着遗传学的不断发展,遗传标记的种类和数量也不断增加。与其他标记技术相比,分子标记技术具有许多明显的优越性,因而应用也日趋广泛。综述了RFLP、RAPD、SSR、AFLP、SCAR、SNP、SRAP等几种主要DNA分子标记技术原理和方法及其在大豆疫霉根腐病研究中的应用。     

茶树的RAPD标记向SCAR标记转化的研究

SCAR标记是在RAPD技术的基础上发展起来的一种新型分子标记技术,相对其他分子标记,它具有开发成本低,稳定性高,单位点多态等特点,将为茶树分子育种开辟一条新的有效途径。通过将随机引物在不同茶树品种基因组DNA中扩增得到的特异性片段,经回收、克隆、测序后重新设计1对特异性引物,将RAPD标记成功转化成SCAR标记,提高了分子鉴定的稳定性。     

几种茄属植物基因组DNA提取与RAPD分析

研究了适于茄子及其近缘野生种基因组ＤＮＡ的提取方法，并对ＤＮＡ初步进行了ＲＡＰＤ分析。结果表明：ＳＤＳ－氯仿－异戊醇法不适于茄属植物ＤＮＡ提纯，而改进的ＣＴＡＢ－酚－氯仿－异戊醇对其成株叶／芽即可获得较高产率及纯度的ＤＮＡ；ＰＣＲ反应扩增出不同片段，引物ＯＰＧ—１１扩增片段在栽培品种与野茄中无差异，而在红茄与龙葵中呈现出一定的多态性。     

赤芝菌株的SCAR标记鉴别

赤芝是种重要的药用真菌,品种繁多,由于目前食用菌管理制度不完善,为稳定菌株质量迫切需要建立起快速鉴定赤芝菌株的有效办法。在对赤芝（Ganoderma lucidum）菌株进行SRAP多态性分析基础上,将属于某几个菌株的SRAP特异片段转化为稳定性较高的SCAR标记,共获得17个SCAR标记。聚类分析显示,供试的23个赤芝菌株在遗传距离0.63下分为4类,其中19个菌株在遗传距离0.50下聚成一类。将其中的9个SCAR标记线性组合,建立起赤芝菌株的多标记综合鉴别法,可对供试的23个菌株进行有效鉴别。由此可见,SCAR分子标记能很好地解释赤芝菌株间的亲缘关系,是种快速、稳定、准确鉴别赤芝菌株的方法。     

玉米抗甘蔗花叶病毒SCAR分子标记开发

 【目的】玉米矮花叶病（由甘蔗花叶病毒引起）是中国和欧洲玉米产区的重要病害,开展分子标记辅助育种可以明显提高抗病育种效率。【方法】本文采用改进的BSA法（bulked segregant analysis,BSA）构建玉米抗感自交系DNA池,结合AFLP技术筛选多态性标记,转化后获得实用性强的SCAR标记,并借助100份自交系抗性鉴定结果对SCAR标记进行相关验证。【结果】获得了2个多态性稳定的AFLP标记P66M38-220和P55M51-240,其中将P66M38-220转化为SCAR112标记,此标记与甘蔗花叶病毒抗性高度相关。【结论】改良BSA法是一种发掘性状基因紧密连锁标记的有效方法;开发的SCAR112 标记可以用于玉米抗甘蔗花叶病毒的分子标记辅助选择。     

水稻AFLP标记的多态性、分布及分离的研究

采用５个ＰｓｔⅠ／ＭｓｅⅠ引物组合对来自２个籼稻品种Ｈ３５９和Ａｃｃ８５５８的重组自交系（ＲＩ,Ｆ７代,１３１个系）群体进行了扩增片断长度多态性（ＡＦＬＰ）分析,并对ＡＦＬＰ的多态性程度、ＡＦＬＰ标记的染色体分布及分离比例进行了研究．在ＡＦＬＰ分析中,共检测到９８个多态性带,其中７８个定位到连锁图中,并分布在所有的１２条染色体上,表明ＡＦＬＰ是一种高效的分子标记,可与ＲＦＬＰ标记结合应用于遗传图谱的构建和基因定位．