Study on construction method of microcore germplasm of Populus tomentosa
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摘要:目的 探讨构建微核心种质的方法,旨在为精准选择毛白杨杂交亲本或研究材料提供科学依据,为其他物种构建微核心种质提供参考。方法 本研究利用荧光SSR分子标记对272份毛白杨样本进行分子基因型检测,计算每个样本对总体遗传多样性的贡献值,从大到小全部排序,计算新排序的前25%、20%、15%、10%、5%、2.5%、2%和1.5%共8个取样比例的平均有效等位基因数(Ne)、平均Shannon信息指数(I),平均期望杂合度(He)和平均多态信息指数(PIC)值等,分析微核心种质的代表性,通过与前面的取样比例以及原始种质的相应参数进行比较,确定合适的微核心种质取样比例。利用t检验进行统计学验证。根据所有引物的峰值,构建每份微核心种质的指纹图谱。基于指纹图谱的差异,分析挖掘特异等位基因。结果 (1)当取样比例由25%逐渐降为2%时,Ne、He和PIC这3个重要的遗传多样性参数分别逐渐达到最大值,而I在取样比例为10%时达到最大值。(2)当取样比例为2%时,Ne、I、He和PIC值分别是3.513、1.254、0.643和0.597,均大于272份原始种质的相应值2.075、0.825、0.432和0.364,表明构建的微核心种质有效去除了遗传冗余,具有丰富的遗传多样性。(3)t检验结果表明:所构建的微核心种质与原始种质的遗传多样性无显著差异,具有可靠的代表性,可以作为微核心种质。(4)不论10%、5%还是2%取样比例,杂交种质所占比例均大于50%。(5)218号样品在11号位点拥有特异等位基因,261号样品在16号位点拥有特异等位基因,263号样品在7号位点拥有特异等位基因。结论 毛白杨微核心种质最为科学、可信、简约和高效的取样比例为2% ~ 10%,其中最精准的取样比例为2%。如果种质资源数目较大,可以根据遗传多样性适当降低取样比例;如果基数较小,可以适当升高。建议以后构建核心种质或者微核心种质时,不要先人为进行分组,而是先根据每个样品对总体遗传多样性的贡献从大到小排序。本研究从分子水平证明杂交种质在种质资源保存和林木遗传育种中占有重要地位,将为其他物种微核心种质构建提供参考。Abstract:Objective The method of constructing microcore germplasm was discussed to provide scientific basis for precise selection of hybrid parents or research materials of P. tomentosa, and to provide reference for other species to construct microcore germplasm.Method In this study, molecular genotypes of 272 P. tomentosa samples were detected by fluorescent SSR molecular markers. The contribution value of each sample to the overall genetic diversity was calculated and sorted from large to small. Then, the representativity of microcore germplasm was analyzed and the appropriate sampling proportion was determined by comparing the average effective number of alleles Ne, the average Shannon’s information index I, the average expected heterogenicity He and the average polymorphic information content PIC values obtained by sampling proportion of top 25%, 20%, 15%, 10%, 5%, 2.5%, 2% and 1.5%. Finally, t test was used for statistical verification. According to the band-shaped results of all SSR primers, fingerprints of each microcore germplasm were formed. According to the difference of fingerprint, the specific alleles were analyzed and excavated.Result When the sampling proportion gradually decreased from 25% to 2%, three important genetic diversity parameters of Ne, He and PIC gradually reached the maximum values, respectively, while I reached the maximum value when the sampling proportion was 10%. When the sampling ratio was 2%, the values of Ne, I, He and PIC were 3.513, 1.254, 0.643 and 0.597, respectively, which were all greater than 2.075, 0.825, 0.432 and 0.364 of the original germplasm, indicating that we had effectively removed the genetic redundancy, and the constructed microcore germplasm had abundant genetic diversity. The t test results showed that there was no significant difference in genetic diversity between the constructed microcore germplasm and the original germplasm, which was reliable and representative and could be used as the microcore germplasm. The proportion of hybrid germplasm was more than 50% regardless of the sampling proportion of 10%, 5% or 2%. Sample 218 had a specific allele at locus 11, sample 261 had a specific allele at locus 16, and sample 263 had a specific allele at locus 7.Conclusion When the sampling ratio of microcore germplasm of poplar is 2%−10%, it is the most scientific, reliable, simple and efficient, among which the most accurate sampling ratio is 2%. If the number of germplasm resources is large, it can be appropriately reduced according to the genetic diversity, and on the contrary, it can be increased appropriately. It is suggested that when constructing core germplasm or microcore germplasm in the future, rather than grouping, each sample is firstly ranked from largest to smallest according to its contribution to overall genetic diversity. It is proved from the molecular level that hybrid germplasm plays an important role in the conservation of germplasm resources and the genetic breeding of trees. At the same time, this study also provides reference for the construction of microcore germplasm of similar species.
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表 1 产地样本数及无性系编号
Table 1 Sample number from provenances and clone No.
产地
Provenance样本数
Sample number无性系编号
Clone No.北京 Beijing 25 1 ~ 25 河北 Hebei 56 26 ~ 81 山东 Shandong 19 82、83、221、222、232、234、235、237、242 ~ 249、251、254、255 河南 Henan 41 84 ~ 114、223、225 ~ 230、257 ~ 259 山西 Shanxi 44 115 ~ 157、260 陕西 Shaanxi 49 158 ~ 203、238、239、261 甘肃 Gansu 5 204 ~ 208 安徽 Anhui 9 209 ~ 217 江苏 Jiangsu 4 218 ~ 220、263 表 2 毛白杨核心种质排序表
Table 2 Rank of core germplasm of P. tomentosa
顺序
Order无性系编号
Clone No.顺序
Order无性系编号
Clone No.顺序
Order无性系编号
Clone No.顺序
Order无性系编号
Clone No.1 156 18 265 35 227 52 161 2 258 19 263 36 264 53 113 3 236 20 233 37 211 54 160 4 253 21 225 38 196 55 259 5 266 22 267 39 208 56 203 6 262 23 218 40 176 57 158 7 261 24 222 41 195 58 110 8 231 25 270 42 138 59 155 9 34 26 197 43 193 60 272 10 241 27 256 44 152 61 202 11 271 28 269 45 184 62 154 12 242 29 220 46 180 63 255 13 252 30 250 47 181 64 151 14 186 31 191 48 89 65 201 15 240 32 219 49 26 66 47 16 237 33 72 50 178 67 147 17 268 34 215 51 126 68 109 表 3 不同取样比例种质总数及不同来源份数
Table 3 Total number of germplasms with different sampling ratios and the number of samples from different sources
比例
Ratio/%北京
Beijing河北
Hebei山东
Shandong河南
Henan山西
Shanxi陕西
Shaanxi甘肃
Gansu安徽
Anhui江苏
Jiangsu新疆
Xinjiang杂种
Hybrid总计
Total100.0 25 56 19 41 44 49 5 9 4 1 19 272 25.0 0 4 4 8 8 18 1 2 4 1 18 68 20.0 0 3 3 6 4 14 1 2 4 1 17 55 15.0 0 2 3 3 1 7 1 2 4 1 17 41 10.0 0 1 3 2 1 3 0 0 2 1 14 27 5.0 0 1 1 1 1 2 0 0 0 0 8 14 2.5 0 0 0 1 1 1 0 0 0 0 4 7 2.0 0 0 0 1 1 0 0 0 0 0 3 5 1.5 0 0 0 1 1 0 0 0 0 0 2 4 表 4 不同取样比例微核心种质遗传多样性分析
Table 4 Genetic diversity analysis of microcore germplasm with different sampling ratios
样本量
Sample
number (N)取样比例
Sampling ratio/%等位基因数
Allele
number (Na)有效等位基因数
Effective number
of allele (Ne)Shannon信息指数
Shannon
information
index (I)观测杂合度
Observed
heterozygosity
(Ho)期望杂合度
Expected
heterozygosity
(He)平均多态信息指数
Average polymorphic
information index (PIC)272 100.0 6.625 2.075 0.825 0.561 0.432 0.364 68 25.0 6.063 2.761 1.094 0.605 0.539 0.499 55 20.0 6.063 2.916 1.141 0.605 0.557 0.519 41 15.0 5.875 3.145 1.205 0.601 0.584 0.547 27 10.0 5.813 3.317 1.271 0.581 0.614 0.578 14 5.0 4.938 3.385 1.234 0.589 0.616 0.576 7 2.5 4.625 3.404 1.254 0.554 0.638 0.594 5 2.0 4.438 3.513 1.254 0.550 0.643 0.597 4 1.5 3.625 3.003 1.048 0.625 0.563 0.512 表 5 微核心种质(10%)与初始种质的遗传多样性比较
Table 5 Comparison of genetic diversity between microcore germplasm (10%) and primary germplasm
群体 Population N Na Ne I Ho He PIC 初始种质 Primary germplasm 272 6.625 2.075 0.825 0.561 0.432 0.364 微核心种质 Microcore germplasm 27 5.813 3.317 1.271 0.581 0.614 0.578 保留率 Retention rate/% 10 87.743 159.855 154.061 103.565 142.130 158.791 t 0.993 8 P 0.358 7 > 0.05 表 6 微核心种质(2%)与初始种质的遗传多样性比较
Table 6 Comparison of genetic diversity between the microcore germplasm (2%) and primary germplasm
群体 Population N Na Ne I Ho He PIC 初始种质 Primary germplasm 272 6.625 2.075 0.825 0.561 0.432 0.364 微核心种质 Microcore germplasm 5 4.438 3.513 1.254 0.550 0.643 0.597 保留率 Retention rate/% 2 66.989 169.301 152.000 98.039 148.843 164.011 t 0.999 4 P 0.356 2 > 0.05 表 7 微核心种质(15%)与初始种质的遗传多样性比较
Table 7 Comparison of genetic diversity between the microcore germplasm (15%) and primary germplasm
群体 Population N Na Ne I Ho He PIC 初始种质 Primary germplasm 272 6.625 2.075 0.825 0.561 0.432 0.364 微核心种质 Microcore germplasm 41 5.875 3.145 1.205 0.601 0.584 0.547 保留率 Retention rate/% 15 88.679 151.566 146.061 107.130 135.185 150.275 t 0.994 6 P 0.358 4 > 0.05 表 8 微核心种质(1.5%)与初始种质遗传多样性比较
Table 8 Comparison of genetic diversity between the microcore germplasm (1.5%) and primary germplasm
群体 Population N Na Ne I Ho He PIC 初始种质 Primary germplasm 272 6.625 2.075 0.825 0.561 0.432 0.364 微核心种质 Microcore germplasm 4 3.625 3.003 1.048 0.625 0.563 0.512 保留率 Retention rate/% 1.5 54.717 144.723 127.030 111.408 130.324 140.659 t 1.006 5 P 0.353 0 > 0.05 表 9 16个SSR位点27份微核心种质的多态性分析
Table 9 Polymorphism analysis of 27 accessions of microcore germplasm at 16 SSR locus
引物
Primer位点
LocusN Na Ne I Ho He
PIC1 Ptr_1_SSR2 27 5.000 3.291 1.300 0.593 0.696 0.640 2 Ptr_7_SSR14 27 7.000 4.500 1.649 0.815 0.778 0.744 3 Ptr_1_SSR1 27 3.000 1.573 0.655 0.333 0.364 0.327 4 Ptr_9_SSR3 27 4.000 2.282 1.040 0.222 0.562 0.512 5 Ptr_11_SSR1 27 7.000 4.084 1.595 0.556 0.755 0.718 6 Ptr_10_SSR1 27 4.000 2.809 1.136 0.852 0.644 0.574 7 Ptr_11_SSR8 27 5.000 1.421 0.653 0.148 0.296 0.283 8 Ptr_14_SSR12 27 7.000 4.166 1.636 0.852 0.760 0.726 9 Ptr_14_SSR7 27 5.000 3.827 1.465 0.852 0.739 0.698 10 Ptr_16_SSR3 27 6.000 1.369 0.651 0.111 0.270 0.263 11 Ptr_3_SSR13 27 7.000 3.044 1.364 0.259 0.671 0.615 12 Ptr_14_SSR11 27 6.000 2.553 1.239 0.778 0.608 0.566 13 Ptr_18_SSR17 27 10.000 6.568 2.032 0.815 0.848 0.830 14 Ptr_7_SSR15 27 9.000 6.688 2.015 0.926 0.850 0.833 15 Ptr_13_SSR6 27 5.000 3.539 1.401 0.889 0.717 0.672 16 Ptr_19_SSR1 27 3.000 1.353 0.503 0.296 0.261 0.241 平均值 Mean 27 5.813 3.317 1.271 0.581 0.614 0.578 表 10 前8对引物构建的27份毛白杨微核心种质指纹图谱
Table 10 Fingerprint map of 27 microcore germplasms of P. tomentosa constructed using the top 8 pairs of primers
bp 顺序
Order无性系编号
Clone No.引物 Primer 1 2 3 4 5 6 7 8 1 156 378/378 393/396 185/185 369/372 271/281 241/244 332/332 333/345 2 258 382/390 363/366/399 185/187 372/372 269/283 238/241/244 332/332 337/355 3 236 374/382/390 360/363/399 185/185 369/369 269/283 238/241 332/332 335/337 4 253 378/378 360/396 185/185 372/372 271/287 238/241 294/294 333/337 5 266 390/390 357/363 183/183 269/269 275/275 238/238 330/330 333/333 6 262 374/378 360/396 185/185 372/372 271/283 241/244 332/332 333/333 7 261 382/390 363/396 185/187 269/269 269/269 238/241/250 332/332 337/337 8 231 376/378 393/396 185/185 369/372 271/271 241/244 332/332 333/345 9 34 382/390 363/366/399 185/187 372/372 269/283 238/241/244 332/332 337/355 10 241 378/378 393/396 185/185 372/372 271/271 238/244 332/332 333/353 11 271 374/382/390 360/363/399 185/185 369/369 269/269 238/241 332/332 333/337 12 242 382/390 363/366/399 185/187 372/372 269/283 238/241/244 332/332 337/355 13 252 378/378 396/396 185/185 369/372 271/281 241/244 332/332 333/345 14 186 382/390 363/396 185/187 369/369 269/269 238/241/250 332/332 335/337 15 240 378/378 393/396 185/185 372/372 271/271 238/244 332/332 333/353 16 237 382/390 363/366/399 185/187 372/372 269/283 238/241/244 332/332 337/355 17 268 378/378 360/396 185/185 369/372 271/271 241/244 332/332 333/345 18 265 378/382 360/399 185/185 372/372 283/287 238/241 294/332 333/337 19 263 382/390 360/363 185/185 372/372 269/281 238/250 332/338 333/337 20 233 378/378 396/396 185/185 366/369 271/271 241/241 332/332 331/333 21 225 382/390 363/366/399 185/187 372/372 269/283 238/241/244 332/332 337/355 22 267 390/390 363/363 185/187 269/269 285/285 238/238 330/334 333/333 23 218 378/378 360/360 185/185 366/366 271/271 241/241 332/332 331/353 24 222 382/390 363/366/399 185/187 372/372 269/283 238/241/244 332/332 337/355 25 270 378/378 396/396 185/185 369/372 271/271 241/244 332/332 333/345 26 197 382/390 360/363 185/185 372/372 269/281 238/250 332/332 333/337 27 256 378/382/390 360/363/399 185/185 372/372 271/283 238/241 294/332 335/337 表 11 后8对引物构建的27份毛白杨微核心种质指纹图谱
Table 11 Fingerprint map of 27 microcore germplasms of P. tomentosa constructed using the last 8 pairs of primers
bp 顺序
Order无性系编号
Clone No.引物 Primer 9 10 11 12 13 14 15 16 1 156 253/262 356/356 373/373 327/348 177/179 347/355 339/345 289/292 2 258 250/259 356/356 353/373 327/336 175/179 345/361/363 339/342/348 289/289 3 236 259/259 356/356 353/353 327/327 171/181 355/361/363 342/345 280/289 4 253 253/259 356/356 373/373 327/336 173/177 339/365 339/339 289/289 5 266 259/259 340/340 371/371 321/330 193/203 363/363 336/342 289/289 6 262 262/262 356/356 353/353 327/327 179/181 339/365 336/345 280/289 7 261 256/259 356/356 353/353 327/336 171/173/175 359/363 339/342 280/289 8 231 253/262 356/356 373/373 327/348 177/181 347/355 339/345 289/292 9 34 250/259 356/356 353/373 327/336 175/179 345/361/363 339/348 289/289 10 241 253/259 356/356 375/375 306/327 173/173 347/373 336/342 289/289 11 271 256/262 356/356 353/353 327/327 171/171 355/361/363 342/345 280/289 12 242 250/259 356/356 353/373 327/336 175/179 345/361/363 339/342/348 289/289 13 252 253/262 356/356 373/373 327/348 177/179 347/355 339/345 289/289 14 186 256/259 356/356 353/353 327/336 171/173/175 359/363 339/342 280/289 15 240 253/259 356/356 375/375 306/327 173/173 347/373 336/342 289/289 16 237 250/259 356/356 353/373 327/336 175/179 345/361/363 339/342/348 289/289 17 268 253/262 356/356 373/373 327/348 177/177 347/355 342/345 280/289 18 265 259/262 356/356 387/387 327/327 173/179 361/365 336/339 289/289 19 263 256/262 356/356 353/353 327/336 173/191 361/363 339/342 289/289 20 233 259/262 326/334 373/373 327/327 179/181 347/355 339/345 289/289 21 225 250/259 356/356 353/373 327/336 175/179 345/361/363 339/342/348 289/289 22 267 259/262 316/328 369/369 321/330 201/201 363/365 342/348 289/289 23 218 259/259 326/334 353/377 327/327 177/179 355/355 339/339 289/289 24 222 250/259 356/356 353/373 327/336 175/179 345/361/363 339/342/348 289/289 25 270 253/262 356/356 373/373 327/348 177/179 347/355 339/345 289/289 26 197 256/262 356/356 353/353 327/336 173/191 361/363 339/342 289/289 27 256 259/262 356/356 353/353 327/336 171/173 361/363/365 339/339 289/289 -
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