小麦区试品系DUS测试的分子标记

Molecular Markers for Estimating Distinctness,Uniformity,and Stability of Wheat Lines in Regional Trials

为了确定测试小麦(Triticum aestivumL.)区试品系特异性、一致性、稳定性(DUS)的分子标记,采用156个来自我国不同麦区的品种对SSR、EST-SSR和AFLP-SCAR标记的1334对引物进行筛选,根据在染色体上分布均匀、多态性信息指数较高、带型清晰、不同等位变异的带型易于区分及PCR产物稳定的原则,筛选出105对小麦品种DUS测试的分子标记引物,包括63对SSR引物、21对EST-SSR引物和21对AFLP-SCAR引物,可以检测122个位点的754个等位变异,平均每条染色体上被检测位点5.8个,平均每个位点包含7.2个等位变异。根据DUS测试的需求、引物的染色体分布、PIC值大小和带型特点,将105对引物分为21对核心引物、29对一级备用引物和55对二级备用引物。核心引物分辨力较高,可以完成约80%品系的特异性检测,约95%品系的种子纯度检测和约60%品系的一致性、稳定性检测;备用引物用于确定品系DNA位点纯合率和相似品种(系)之间的遗传相似系数,以判断DNA指纹相同或相似的品种(系)之间的相似性和特异性,评价核心标记中具有非纯位点的品系的DNA位点纯合度,同时完成核心引物未能完成的少数品系的种子纯度检测。通过在2006—2007、2007—2008、2008—2009年度对464个冬小麦区试品系DUS测试中的应用,证明105对引物具有很好的代表性和实用性,可以完成90%以上参试品系的DUS检测。

To determine the molecular markers for testing the distinctness, uniformity, and stability （DUS） of wheat （Triticum aestivum L.） lines in regional trials, 156 wheat cultivars were used to screen 1 334 pairs of primers of SSR, EST-SSR, and AFLP-SCAR markers. According to the even distribution of molecular markers on 21 wheat chromosomes, the higher polymorphism information content （PIC）, the clear PCR bands, the easy discrimination of different alleles, and the stable PCR amplification, 105 pairs of primers, including 63 pairs of SSR primers, 21 pairs of EST-SSR primers, and 21 pairs of AFLP-SCAR primers, were selected for wheat cultivar （lines） DUS testing. A total of 754 alleles at 122 loci can be identified with 105 pairs of primers, with 5.8 loci per chromosome and 7.2 alleles per locus on average. The three kinds of molecular markers can amplify the DNA fragments that are inside/outside of genes and repeative/non-repetitive sequences. The 105 pairs of primers were classified into 21 pairs of core primers and 84 pairs of standby primers based on their pattern definition, distribution on 21 chromosomes, and PIC value. The 21 core primers distributing on 21 chromosomes could determine the distinctness test for about 80% of total cultivars （lines）, the seed purity test for 95%, and the uniformity and stability test for 60%. The 84 standby primers were used as the first grade （29 pairs） and the second grade （55 pairs） to the homozygous DNA locus ratio of lines with no-homozygous loci of core markers and the genetic similarities or the distinctness of similar cultivars （lines） pairs revealed by the core primers. At the same time, we determined the seed purity for the other 5% of lines. These 105 pairs of primers could finish DUS test for more than 90% of lines in the winter wheat regional trials in 3 cropping seasons from 2006 to 2009. Although less than 10% of lines should be evaluated in combination with phenotypic identification in the field, the effectiveness and efficiency of the 105 pairs of primers are satisfactory in bulk screening for wheat DUS test.