CN110060739B - A method for evaluating the genetic diversity of waterfowl PCM - Google Patents
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Abstract
本发明公开了一种水禽PCM遗传多样性评价方法，该方法是通过对水禽(鸭/鹅)初生重(g)、成年体重(g)、72周龄产蛋量(个)；1号染色体(％)、Z染色体(％)、W染色体(％)的相对长度；以及群体有效等位基因数、杂合度、多态信息含量进行测定，进一步采用主成分分析法对上述9个指标进行多指标综合评价，最终完成水禽遗传资源的多样性综合评价。相对于现有技术，本发明方法有效解决了单一指标难以评估水禽遗传多样性的技术难题，全面展现了水禽遗传多样性的全貌，为水禽(鸭/鹅)合理保种和科学选育提供有效的技术支撑，具有良好的应用前景。The invention discloses a method for evaluating the genetic diversity of waterfowl PCM. (%), relative lengths of Z chromosomes (%), W chromosomes (%); and the number of effective alleles, heterozygosity, and polymorphism information content in the population. Comprehensive evaluation of indicators, and finally complete the comprehensive evaluation of the diversity of waterfowl genetic resources. Compared with the prior art, the method of the invention effectively solves the technical problem that it is difficult to evaluate the genetic diversity of waterfowl with a single index, comprehensively shows the whole picture of the genetic diversity of waterfowl, and provides effective protection for rational conservation and scientific selection of waterfowl (duck/geese). It has a good application prospect.
Description
Technical Field
The invention relates to a waterfowl PCM genetic diversity evaluation method, belonging to the technical field of waterfowl PCM genetic diversity evaluation.
Background
Agriculture is the foundation of human development, animal husbandry is an essential component of agriculture, and livestock and poultry germplasm resources are the cornerstone of animal husbandry development. China is a large country for breeding and consuming waterfowls, and in recent years, the slaughtering amount of meat ducks is about 30 hundred million per year, and the slaughtering amount of geese is 5.34 hundred million. China is also a water fowl resource big country, and 31 goose genetic resources and 34 duck genetic resources are listed in the Chinese livestock and poultry genetic resource book-poultry book. The excellent local genetic resources have distinctive physical and appearance features, have obvious advantages in the aspects of adaptability, disease resistance, flavor quality and the like, contain huge genetic diversity and selection potential, and are the fundamental guarantee of the inexhaustible spring for innovation of high-quality meat quality and egg quality varieties and the continuous development of industry. Protecting waterfowl germ plasm resources, namely protecting the future waterfowl industry. How to protect waterfowl resources while developing and utilizing is a problem to be urgently solved by waterfowl workers in China in recent years. The key point is to develop a waterfowl genetic diversity evaluation method, which is an important technical means for effectively protecting waterfowl resources.
At present, the genetic diversity of Chinese waterfowl is mostly evaluated by a microsatellite detection method in the research of the genetic resource diversity of the waterfowl, such as Jiwenlin and Shaotai-san et al, (the genetic diversity detection of seven domestic duck groups in the national waterfowl gene bank, Jiwenlin, etc., Chinese poultry 2016,28 (24): 72-74; the genetic diversity of duck varieties, Shaotai-san et al, 2009, 20 (1): 190-. But only a single microsatellite index is used, so that the diversity of waterfowl resources cannot be comprehensively evaluated.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the technical problems, the invention provides a waterfowl PCM genetic diversity evaluation method which is simple, convenient and reliable and provides scientific basis for waterfowl genetic diversity evaluation.
The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a waterfowl PCM (P: phenotype; C: genetics marker; M: molecular marker) genetic diversity evaluation method comprises the following steps:
(1) determining the relative lengths of the primary weight, the adult weight, the egg yield of 72 weeks, the No. 1 chromosome, the Z chromosome and the W chromosome of the waterfowl, the effective allele factors, the heterozygosity and the polymorphic information content of a population, performing multi-index comprehensive evaluation and weight coefficient determination on the 9 indexes by adopting a principal component analysis method, and establishing a PCM genetic diversity comprehensive evaluation model y (d) azp of the waterfowl1+bzp2+czp3+dzc1+ezc2+fzc3+gzm1+hzm2+izm3Wherein: zp1, zp2, zp3, zc1, zc2, zc3, zm1, zm2 and zm3 represent normalized birth weight, adult body weight, 72-week-old egg production, chromosome 1, chromosome Z, relative length of chromosome W, population effective allele factor, heterozygosity and polymorphism information content measurement values, respectively; a. b, c, d, e, f, g, h and i are respectively related weight coefficients;
(2) feeding different breeds of waterfowls to 72 weeks of age, and measuring birth weight (p1), adult weight (p2) and egg yield (p3) at 72 weeks of age;
(3) preparing waterfowl chromosomes by adopting a peripheral blood culture method, and measuring the relative lengths of a chromosome 1 (c1), a chromosome Z (c2) and a chromosome W (c 3);
(4) performing waterfowl STR typing by adopting a fluorescence-multiplex PCR technology, respectively counting population gene frequency distribution, and respectively counting effective allele factors (m1), heterozygosity (m2) and polymorphic information content (m3) of the population;
(5) the measured or calculated values (p1, p2, p3, c1, c2, c3, p1, p2, p3) are calculated according to the formula
Data was normalized (where X is the measured value of the trait,
is the mean value of the trait, and S is the standard deviation of the trait);
(6) substituting the results calculated in the step (5) into the PCM genetic diversity comprehensive assessment model y (d) ═ azp in the step (1)1+bzp2+czp3+dzc1+ezc2+fzc3+gzm1+hzm2+izm3And calculating the comprehensive score of the waterfowl (duck/goose) PCM genetic diversity, and sequencing according to the comprehensive score to determine the genetic diversity of different waterfowl varieties.
Preferably, the method comprises the following steps:
the waterfowl comprises ducks or geese.
The duck species include Gaoyou duck, Gy duck, Jingjiang duck, Liancheng white duck, Jinding duck, Linwu duck, Jian red hair duck and Beijing duck.
The goose variety includes lion head goose, Wanxi white goose, Zhedong white goose, Sichuan white goose, Taihu lake goose, gapping goose and Wugang copper goose.
The determination condition in the step (2) is ground flat culture, the weight determination quantity of each waterfowl is 50-70, and each half of male and female birds are half of the weight determination quantity; and (5) egg yield populations of 100 and 300 eggs are counted, and egg yields of the 72-week-old populations are counted.
Feeding the waterfowls to be more than 10 weeks old, collecting more than 10 peripheral blood from the wing veins, separating lymphocytes, preparing waterfowl chromosomes by adopting a peripheral blood lymph culture method, selecting 5 chromosome metaphase mitotic phases with good spreading and clear periphery for each waterfowl to observe, calculating the relative length of 10 pairs of macrochromosomes before the mitotic phase according to a formula,
the relative lengths of chromosome 1 and Z, W were incorporated into cytogenetics markers (c1, c2, and c 3).
Feeding the water poultry to the age of more than 10 weeks, collecting more than 60 peripheral blood by the wing veins and collecting each half of the male and female blood, extracting blood genome DNA, adopting a fluorescence-multiplex PCR technology, taking an ABI 3730XL DNA full-automatic gene analyzer as a platform, carrying out STR typing, and respectively counting:
effective allelic factor of the population (m1)
Wherein, Pij: frequency of the jth allele at the ith locus; m: allelic factors for the jth locus; and (3) Nei: an effective number of alleles at position i; n: the total number of loci determined;
heterozygosity (m2)
Wherein k is the allelic base factor; pi is the frequency of the ith allele;
polymorphic information content (m3)
Wherein, k: the number of alleles; pi: frequency of the ith allele; pj: frequency of the j-th allele.
In order to comprehensively evaluate the waterfowl genetic diversity, the waterfowl (duck/goose) PCM genetic diversity evaluation method not only is used for evaluating the initial weight (g), the adult weight (g) and the egg yield (egg yield) of 72 weeks old waterfowls (ducks/geese) from the aspects of phenotype, cell and molecule; relative lengths of chromosome 1 (%), chromosome Z (%), chromosome W (%); and determining the effective allele factors, heterozygosity and polymorphic information content of the population, and further performing multi-index comprehensive evaluation on the 9 indexes by adopting a principal component analysis method to finally complete the comprehensive evaluation of the diversity of waterfowl resources, thereby providing a theoretical basis for the protection and utilization of the genetic resources of the waterfowl in China.
The technical effects are as follows: compared with the prior art, the method effectively solves the technical problem that the genetic diversity of the waterfowl is difficult to evaluate by a single index, comprehensively shows the complete picture of the genetic diversity of the waterfowl for the first time, provides effective technical support for the reasonable seed conservation and scientific breeding of the waterfowl (duck/goose), and has good application prospect.
Detailed Description
The technical solution of the present invention is further described in detail by the following specific examples.
Example 1
The early-stage method comprises the following steps:
the determination conditions are that the ducks are raised on the ground and on the net, the determination quantity of each duck is 300, and the duck varieties (Gaoyou ducks, Gyun sheldrake, Jingjiang sheldrake, Liancheng white ducks, Jinding ducks, Linwu ducks, Jian red hair ducks and Beijing ducks) commonly used in the current production are subjected to primary weight (g), adult weight (g) and egg yield (one) of 72 weeks old; relative lengths of chromosome 1 (%), chromosome Z (%), chromosome W (%); and determining 9 indexes such as group effective allelic base factor, heterozygosity and polymorphic information content, performing multi-index comprehensive evaluation and weight coefficient determination on the 9 indexes by adopting a principal component analysis method, and establishing a duck PCM genetic diversity comprehensive evaluation modelType y ═ 0.054zp1+0.094zp2-0.045czp3 +0.024zc1+0.003zc2+0.024zc3+0.021zm1+0.075zm2+0.062zm3In the formula: zp1, zp2, zp3, zc1, zc2, zc3, zm1, zm2 and zm3 represent normalized birth weight, adult body weight, 72-week-old egg production, chromosome 1, chromosome Z, relative length of chromosome W, population effective allele factor, heterozygosity, and polymorphism information content measurement values, respectively.
On the basis of obtaining the comprehensive evaluation model of the duck PCM genetic diversity, the duck genetic diversity is comprehensively evaluated by the following method:
(2) taking 300 ducklings of 1 day old Gaoyou duck and Jinding duck, feeding to 72 weeks old, randomly selecting 30 male ducks and 30 female ducks, accurately measuring the determined initial weight (g) and the weight (g) of 43 weeks old ducks by an electronic balance, and respectively setting the average values of the initial weights of the male ducks and the female ducks as follows: 47.5g, 47.8 g; the average weight values of the 43-week-old animals are 2653.8g and 1796.0g respectively; and the egg production of 72-week-old populations is counted, and the egg production is 210 and 288 respectively.
(3) Feeding Gaoyou duck and Jinding duck for 70 days, randomly selecting 10 male and 10 female of each variety, collecting blood from wing vein, gradient centrifuging to separate lymphocyte, preparing duck or goose chromosome by peripheral hemolymph culture method, culturing in RMPI1640 culture medium for 72h, harvesting cell, Gimsa dyeing, selecting 5 well-spread and clear-periphery chromosome metaphase for microscopic observation, and calculating relative length of 10 pairs of macrochromosomes in front of division phase according to formula
The average relative lengths of the Gaoyou duck, the Jinding duck No. 1 chromosome and the Z, W chromosome are respectively 24.3%, 9.66% and 4.87%; 25.98%, 10.06% and 4.81%.
(4) Raising Gaoyou duck and Jinding duck to 70 days, randomly selecting 10 male, 10 female and wing veins for blood sampling, extracting blood genome DNA, adopting fluorescence-multiplex PCR technology (primer is shown in table 1), using ABI 3730XL DNA full-automatic gene analyzer as platform, carrying out STR typing, respectively counting effective allelic gene factors of the group (m1)
(wherein Pij: frequency of j 'th allele at i' th locus; M: allele number at j 'th locus; Nei: effective number of alleles at i' th locus; n: total number of loci determined.); heterozygosity (m2)
(where k is the allele number; Pi is the frequency of the ith allele.) polymorphic information content (m3)
(where k is the number of alleles; Pi is the frequency of the ith allele; Pj is the frequency of the jth allele). According to statistics, the effective allelic base factors, the heterozygosity and the polymorphic information content of the Gaoyou duck and the Jinding duck are respectively 5.92, 0.475 and 0.534; 7.67, 0.416, 0.560.
TABLE 1 combination of duck STR typing fluorescent primers and optimized reaction conditions
(5) The measured value or the calculated value is calculated according to
Data was normalized (where X is the measured value of the trait,
is the mean value of the trait, and S is the standard deviation of the trait);
(6) according to each result calculated in the step (5), according to the duck PCM genetic diversity comprehensive evaluation model y, 0.054zp1+0.094zp2-0.045czp3+0.024zc1+0.003zc2+0.024zc3+0.021zm1+0.075zm2+0.062zm3In the formula: zp1, zp2, zp3, zc1, zc2, zc3, zm1, zm2 and zm3 respectively represent standardized initial weight, adult weight, 72-week-old egg yield, effective allele factors, heterozygosity and polymorphic information content measurement values of relative length populations of chromosome 1, chromosome Z and chromosome W, and the PCM genetic diversity comprehensive scores of the Gaoyou ducks and the Jinding ducks are respectively calculated to be 0.201 and 0.086.
Example 2
The early-stage method comprises the following steps:
the determination conditions are that the goose is raised on the ground and on the net, the determination quantity of each goose is 300, and the birth weight (g), the adult weight (g) and the egg yield (per) of 72 weeks old are determined for the goose varieties (lion head goose, Wanxi white goose, Zhedong white goose, Sichuan white goose, Taihu lake goose, gapped goose and Wugang copper goose) commonly used in the current production; relative lengths of chromosome 1 (%), chromosome Z (%), chromosome W (%); the effective allele factors, heterozygosity and polymorphic information content of the colony are determined by multi-index comprehensive evaluation and weight coefficient determination of the 9 indexes by adopting a principal component analysis method, and a goose PCM genetic diversity comprehensive evaluation model y is established to be 0.041zp1+0.036zp2-0.035czp3-0.055zc1+0.048zc2+0.027zc3+0.074zm1+ 0.100zm2+0.099zm3In the formula: zp1, zp2, zp3, zc1, zc2, zc3, zm1, zm2 and zm3 represent normalized birth weight, adult body weight, 72-week-old egg production, chromosome 1, chromosome Z, relative length of chromosome W, population effective allele factor, heterozygosity, and polymorphism information content measurement values, respectively.
On the basis of obtaining the goose PCM genetic diversity comprehensive evaluation model, the goose genetic diversity is comprehensively evaluated by the following method:
(1) taking 300 young ducks of 1 day old of Wanxi white goose and Zhedong white goose respectively, feeding to 72 weeks old, randomly selecting 30 male ducks and 30 female ducks, accurately measuring the determined initial weight (g) of the gooses by an electronic balance, and respectively measuring the average values of the initial weights (g) of the male ducks and the female ducks as follows: 103.5g and 109.0 g; the average weight values of the 43-week-old animals are 6100.0g and 4750.3g respectively; and the egg production of 72-week-old populations is counted, and the number of the populations is 34 and 36 respectively.
(2) Feeding Anhui west white goose and Zhe Dong white goose for 70 days, randomly selecting 10 male and 10 female of each variety, collecting blood from wing vein, gradient centrifuging to separate lymphocyte, preparing duck or goose chromosome by peripheral blood lymph culture method, culturing in RMPI1640 culture medium for 72h, harvesting cell, Gimsa dyeing, selecting 5 well-spread and clear-periphery chromosome metaphase for microscopic observation, and calculating relative length of 10 pairs of macrochromosomes before division phase according to formula
The average relative lengths of chromosomes of Wanxi white goose, Zhedong white goose No. 1 and Z, W are 20.29%, 11.48%, 6.97%, 22.68%, 10.68% and 6.23%, respectively.
(3) Feeding Wanxi white goose and Zhe Dong white goose for 70 days, randomly selecting 10 male and 10 female of each variety, collecting blood by wing vein, extracting blood genome DNA, performing STR typing by fluorescence-multiplex PCR technology (primers shown in Table 2) with ABI 3730XL DNA full-automatic gene analyzer as platform, and respectively counting effective allele factors of the population (m1)
(wherein Pij: frequency of j 'th allele at i' th locus; M: allele number at j 'th locus; Nei: effective number of alleles at i' th locus; n: total number of loci determined.); heterozygosity (m2)
(where k is the allele number; Pi is the frequency of the ith allele.) polymorphic information content (m3)
(where k is the number of alleles; Pi is the frequency of the ith allele; Pj is the frequency of the jth allele). After statistics, Gaoyou duck and goldDetermining the effective allelic gene factor, the heterozygosity and the polymorphic information content of the duck group to be 6.90, 0.622 and 0.563 respectively; 4.80, 0.590, 0.522.
TABLE 2 combination of goose STR typing fluorescent primers and optimized reaction conditions
(4) The measured value or the calculated value is calculated according to
Data was normalized (where X is the measured value of the trait,
is the mean value of the trait, and S is the standard deviation of the trait);
(5) according to each result calculated in the step (5), according to the goose PCM genetic diversity comprehensive evaluation model y being 0.041zp1+0.036zp2-0.035czp3-0.055zc1+0.048zc2+0.027zc3+0.074zm1+0.100zm2+0.099zm3In the formula: zp1, zp2, zp3, zc1, zc2, zc3, zm1, zm2 and zm3 respectively represent standardized birth weight, adult weight, 72-week-old egg production, chromosome 1, chromosome Z and relative length of chromosome W, population effective allele factors, heterozygosity and polymorphism information content measured values, and the comprehensive scores of PCMs of Wanxi white goose and Zhedong white goose are calculated to be 0.666 and 0.103 respectively, and the PCMs of Wanxi white goose are superior to those of Zhedong white goose in comprehensive score.
Claims (3)
1. The waterfowl PCM genetic diversity evaluation method is characterized by comprising the following steps:
(1) through measuring the primary weight, the adult weight, the egg yield of 72 weeks, the relative length of No. 1 chromosome, Z chromosome and W chromosome of waterfowls, the effective allele factor, heterozygosity and polymorphic information content of a population, carrying out multi-index comprehensive evaluation and weight coefficient determination on the 9 indexes by adopting a principal component analysis method, establishing a waterfowl PCM genetic diversity comprehensive evaluation model y (d) of azp1+ bzp2+ czp3+ dzc1+ ezc2+ fzc3+ gzm1+ hzm2+ izm3, wherein: zp1, zp2, zp3, zc1, zc2, zc3, zm1, zm2 and zm3 represent normalized birth weight, adult body weight, 72-week-old egg production, chromosome 1, chromosome Z, relative length of chromosome W, population effective allele factor, heterozygosity and polymorphism information content measurement values, respectively; a. b, c, d, e, f, g, h and i are respectively related weight coefficients;
(2) feeding different breeds of waterfowls to 72 weeks of age, and measuring the birth weight p1, the adult weight p2 and the egg yield p3 of 72 weeks of age;
(3) preparing waterfowl chromosomes by adopting a peripheral blood culture method, and measuring the relative lengths of chromosome 1 c1, chromosome z c2 and chromosome W c 3;
(4) performing waterfowl STR typing by adopting a fluorescence-multiplex PCR technology, respectively counting population gene frequency distribution, and respectively counting effective allele factors m1, heterozygosity m2 and polymorphic information content m3 of a population;
(5) the measured value or the calculated value is calculated according to
Performing data normalization, wherein X is a measured value of the trait,
is the average value of the character, and S is the standard deviation of the character;
(6) substituting the results calculated in the step (5) into the PCM genetic diversity comprehensive assessment model y (d) obtained in the step (1), wherein the result is azp1+ bzp2+ czp3+ dzc1+ ezc2+ fzc3+ gzm1+ hzm2+ izm3, calculating the PCM genetic diversity comprehensive score of waterfowls, and sequencing according to the comprehensive score to determine the genetic diversity of different waterfowl varieties;
the waterfowl is a duck;
the determination condition in the step (2) is ground flat culture, the weight determination quantity of each waterfowl is 50-70, and each half of male and female birds are half of the weight determination quantity; 100 egg yield groups and 300 egg yield groups, and the egg yield of the 72-week-old group is counted;
feeding the aquatic birds in the step (4) to the age of more than 10 weeks, collecting more than 60 peripheral blood by using the wing veins, and respectively taking half of each male parent and half of each female parent;
feeding the waterfowls to be more than 10 weeks old, collecting more than 10 peripheral blood from the wing veins, separating lymphocytes, preparing waterfowl chromosomes by adopting a peripheral blood lymph culture method, selecting 5 chromosome metaphase mitotic phases with good spreading and clear periphery for each waterfowl to observe, and calculating the relative length of 10 pairs of macrochromosomes before the mitotic phase according to a formula:
the relative lengths of chromosome 1 and Z, W are included in the index of cytogenetic evaluation.
2. The waterfowl PCM genetic diversity evaluation method according to claim 1, wherein the duck species include Gaoyou ducks, Gyue sheldrake, Jingjiang sheldrake, Liancheng white ducks, Jinding ducks, Linwu ducks, Jian red-hair ducks and Beijing ducks.
3. The waterfowl PCM genetic diversity evaluation method according to claim 1, characterized in that blood genome DNA is extracted, STR typing is performed by adopting fluorescence-multiplex PCR technology and using ABI 3730XL DNA full-automatic gene analyzer as a platform, and statistics are respectively given as follows:
effective allelic factor of a population
Wherein, Pij: frequency of the jth allele at the ith locus; m: allelic factors for the jth locus; and (3) Nei: an effective number of alleles at position i; n: the total number of loci determined;
degree of heterozygosity
Wherein, k: an allelic factor; pi: frequency of the ith allele;
polymorphic information content
Wherein, k: the number of alleles; pi: frequency of the ith allele; pj: frequency of the j-th allele.
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