Doudrick R L
Southern Institute of Forest Genetics, USDA Forest Service Saucier, Mississippi, 39574-9344, USA.
Symp Soc Exp Biol. 1996;50:53-60.
Slash pine is native to the southeastern USA, but is commercially valuable world-wide as a timber-, fiber- and resin-producing species. Breeding objectives emphasize selection for fusiform rust disease resistance. Identification of markers linked to genetic factors conditioning specificity should expand our knowledge of disease development. Towards this end, random amplified polymorphic DNA (RAPD) markers were identified and mapped in a tree hypothesized to be homozygous dominant for resistance at one locus and homozygous recessive at another. Because the DNA prepared for analysis was from haploid maternally-inherited, megagametophyte tissue of seeds, RAPD markers were observed as either present or absent. The analysis revealed 13 linkage groups of three or more loci, ranging in size from 28 to 68 cM, and nine linked pairs. The 22 groups and pairs included 73 RAPD markers and covered a genetic map distance of approximately 782 cM. Genome size estimates, based on linkage data, range from 2,880 to 3,360 cM, and equal 6.0-6.9 x 10(6) bp/cM (physical size > 20,000 Mbp). Using a 30 cM map scale and including unlinked markers, ends of linkage groups, and linked pairs, the RAPD markers account for approximately 2,160 cM or 64-75% of the genome. Mapping 80 additional RAPD markers placed 131 loci total in 20 linkage groups of three or more loci, nearly doubling the coverage in the groups to a genetic map distance of approximately 1,347 cM. Two other slash pine trees also have been RAPD mapped. DNA-DNA in situ hybridization and cytochemical staining are being used to integrate the genetic recombinational maps. A karyotype and ideogram have been prepared for slash pine (2n = 2x = 24); metaphase chromosome preparations show 11 pairs of long metacentric chromosomes and one shorter pair of submetacentric chromosomes. Patterns of fluorescence in situ hybridization to genes for the large and small rRNA subunits and fluorochrome banding patterns using the GC-base-specific chromomycin A3 (CMA) and AT-base-specific 4',6-diamidino-2-phenylindole (DAPI) allowed all twelve pairs of chromosomes to be identified and a standard karyotype established. A family of sequences associated with (TTTAGGG)n related repeats has been identified in slash pine using a labeled synthetic oligonucleotide probe. Fluorescence in situ hybridization shows a weak signal at telomeres and significantly stronger intensity at non-telomeric sites. The most common non-telomeric location was in the pericentric regions of chromosomes; interstitial sites of hybridization were relatively common. Microsatellite DNAs, an abundant retrotransposon-like element, and total genomic in situ hybridization and species and chromosome specific DNAs are being evaluated for analyses of interspecific hybrids and chromosome evolution between related species. Interest in low and single copy sequences is increasing.
湿地松原产于美国东南部，但作为一种木材、纤维和树脂生产树种，在全球具有商业价值。育种目标强调选择对梭形锈病具有抗性的品种。鉴定与决定特异性的遗传因素相关的标记，应能扩展我们对疾病发展的认识。为此，在一棵假定在一个位点对抗性为纯合显性而在另一个位点为纯合隐性的树上，鉴定并绘制了随机扩增多态性DNA（RAPD）标记。由于用于分析的DNA来自种子的单倍体母系遗传的大配子体组织，RAPD标记表现为存在或不存在。分析揭示了13个由三个或更多位点组成的连锁群，大小从28到68厘摩不等，以及9个连锁对。这22个群和对包括73个RAPD标记，覆盖的遗传图谱距离约为782厘摩。基于连锁数据估计的基因组大小范围为2880至3360厘摩，相当于6.0 - 6.9×10⁶碱基对/厘摩（物理大小>20000兆碱基对）。使用30厘摩的图谱比例尺并包括未连锁标记、连锁群末端和连锁对，RAPD标记约占基因组的2160厘摩或64 - 75%。另外绘制80个RAPD标记后，总共131个位点位于20个由三个或更多位点组成的连锁群中，使这些群中的覆盖范围几乎翻倍，达到约1347厘摩的遗传图谱距离。另外两棵湿地松也已进行RAPD图谱绘制。正在使用DNA - DNA原位杂交和细胞化学染色来整合遗传重组图谱。已为湿地松制备了核型和染色体模式图（2n = 2x = 24）；中期染色体标本显示有11对长的中着丝粒染色体和一对较短的亚中着丝粒染色体。利用针对大、小rRNA亚基基因的荧光原位杂交模式以及使用GC碱基特异性的放线菌素A3（CMA）和AT碱基特异性的4',6 - 二脒基-2 -苯基吲哚（DAPI）的荧光染色模式，能够识别所有12对染色体并建立标准核型。使用标记的合成寡核苷酸探针在湿地松中鉴定出了一个与（TTTAGGG）n相关重复序列相关的序列家族。荧光原位杂交显示在端粒处信号较弱，而在非端粒位点强度明显更强。最常见的非端粒位置在染色体着丝粒周围区域；杂交的间隙位点相对常见。微卫星DNA、一种丰富的类反转录转座子元件以及全基因组原位杂交以及物种和染色体特异性DNA正在用于分析相关物种间的种间杂种和染色体进化。对低拷贝和单拷贝序列的兴趣正在增加。
