CN111363785B - Construction method of maize flowering period tassel drought-resistant QTL positioning segregation population - Google Patents

Construction method of maize flowering period tassel drought-resistant QTL positioning segregation population Download PDF

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CN111363785B
CN111363785B CN202010190372.0A CN202010190372A CN111363785B CN 111363785 B CN111363785 B CN 111363785B CN 202010190372 A CN202010190372 A CN 202010190372A CN 111363785 B CN111363785 B CN 111363785B
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CN111363785A (en
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杨杰
韩登旭
郭延玲
马传禹
梁晓玲
徐明良
阿布来提·阿布拉
王业建
胡燕
杜腓利
朱彦彬
胡小明
李统中
李铭东
郗浩江
赛力汗·赛
王仙
刘强
丁新华
房世杰
樊国全
肖丽
文丽伟
帅旭明
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INSTITUTE OF CEREAL CROPS XINJIANG ACADEMY OF AGRICULTURAL SCIENCES
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Abstract

The invention discloses a construction method of maize flowering period tassel dry-resistant QTL location segregation population, belonging to the field of modern biological science research. The invention discloses a construction method of maize flowering phase tassel dry-resistant QTL location segregation population, which surrounds the trait of maize flowering phase tassel dry-resistant, and utilizes PHBA6 (P)1) Drought-sensitive series Ji 63 (P)2) And (3) assembling a basic population, and adopting a map-based cloning method to position two QTL-qMd3 and QTL-qMd9 with the positioning interval of about 13Mb on chromosomes 3 and 9 for the first time. The invention firstly utilizes the parent material PHBA6 (P) screened by the accurate identification of drought tolerance for many years1) Drought-sensitive series Ji 63 (P)2) Matched with a maize tassel drought-resistant QTL positioning basic group; and performing phenotype grading accurate identification and evaluation around the important character of tassel dry rot in the flowering period of the corn most sensitive to drought, and obtaining effective phenotype data.

Description

Construction method of maize flowering period tassel drought-resistant QTL positioning segregation population
Technical Field
The invention relates to the technical field of modern biological science research such as molecular genetics, quantitative genetics, crop breeding and the like, in particular to a construction method of a maize flowering period tassel drought-resistant QTL positioning segregation population.
Background
Corn (Zea mays L.) is a grain crop with the largest cultivation area in China and even the world, is widely applied to the fields of food, feed, industrial raw materials, biomass energy and the like, and has very important significance in guaranteeing the food and energy safety in China. Xinjiang is located in the abdominal land of the continental province of northwest Europe of China, belongs to typical arid and semiarid continental climate, and has rich photo-thermal resources and large day-night temperature difference. The large food crops in Xinjiang are mainly wheat, corn and rice, and can be planted in most areas in Xinjiang, and the sowing area accounts for more than 90% of the total area of the food crops. The accumulated temperature of more than 10 ℃ in the western regions of northern Xinjiang along Tianshan mountain and Yili river valley is stabilized at about 3600 ℃, the climate is mild, the land is fertile, the grains and wheat are more suitable for growing, and the Xinjiang granary is called as Xinjiang. This unique and superior geographical and climatic conditions are highly beneficial for corn growth and development and high yield creation. Therefore, Xinjiang is a well-recognized area with the highest potential for corn yield development in China.
However, due to the severe ecological environment and the gradual reduction and uneven distribution of precipitation worldwide, drought and high temperature extreme weather disasters occur frequently in various regions, and the great increase of the corn yield is seriously influenced.
At present, researches on phenotype identification of drought tolerance, drought tolerance related QTL positioning, GWAS analysis, miRNA sequencing, gene cloning and genetic mechanism at home and abroad mostly focus on main agronomic characters, physiological and biochemical properties, chlorophyll content, yield characters and nutritional quality in a corn seedling stage or a flowering stage. The separated cloned major drought stress response genes are relatively few, and can not meet the requirements of the improvement of the corn genetic engineering and drought-tolerant breeding at present. And the research on the aspects of leaf and tassel withering and death caused by high-temperature drought at the flowering period of the corn is not reported at home and abroad. At present, the researches on the construction of maize flowering period tassel drought-resistant QTL positioning population, the excavation of functional genes, functional verification and genetic mechanism are still lack.
Therefore, the invention provides a construction method of a maize flowering phase tassel drought-resistant QTL positioning segregation population, which aims to construct a related QTL positioning population from the forward genetics around the scientific problem of maize flowering phase tassel drought-resistant, and lays a foundation for further mining candidate genes, functional verification and backcross transformation, and creating a new maize drought-resistant and drought-resistant breeding material.
Disclosure of Invention
In view of the above, the invention provides a construction method of maize flowering stage tassel drought-resistant QTL location segregation population.
In order to achieve the purpose, the invention adopts the following technical scheme:
a construction method of maize flowering period tassel dry-resistant QTL location segregation population comprises the following specific steps:
(1) identification and selection of a population parent inbred line: after the drought tolerance identification of the field for many years, the inbred line PHBA6 with strong drought tolerance and dry-fast tassel is taken as P1The inbred line Ji 63 which is highly sensitive to drought and easy to dry tassel is taken as P2；
(2) Preparing a hybridization combination: planting P in Xinjiang1And P2And P is added during the flowering phase1And P2Bagging pollination and directional hybridization (P)1×P2) Is assembled to obtain F1Seeds; f is to be1Planting seed in Hainan area, bagging in flowering period for selfing to obtain F2The seed of (1);
(3)F2phenotypic characterization of the segregating population: selection of F2Seeds with plump seeds are planted in Xinjiang to obtain F2Group individual plants; for each F in seedling stage2Carrying out plate hanging, marking and sampling on the individual plants of the group offspring, extracting DNA, and carrying out SNP chip scanning to obtain chip data;
(4) survey of tassel dry-rot resistant phenotype: under drought stress conditions on F2Carrying out accurate identification on the phenotype of the tassel dry rot character, grading investigation, statistical analysis and data processing on a single colony plant;
(5) according to the number of SNP chipsAccording to sum of F2Constructing a genetic linkage map by using phenotype data of drought resistance of the tassels of the individual plants of the population;
(6) according to F2And carrying out QTL initial positioning on phenotype data and SNP chip data of the individual plants of the group.
Further, the soil water potential psi in the drought stress in the step (2) is-0.5-1.0 MPa.
Further, the QTL in the step (6) is initially positioned on the chromosome 3 and the chromosome 9.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects:
(1) the invention firstly carries out phenotype grading accurate identification and evaluation around the typical character of drought-sensitive blooming period tassel dry-rot of corn, and obtains effective phenotype data;
(2) the invention firstly utilizes the parent material PHBA6 (P) screened by the accurate identification of drought tolerance for many years1) Drought-sensitive series Ji 63 (P)2) Matched with a maize tassel drought-resistant QTL positioning basic group;
(3) around the trait of maize tassel dry-up in flowering period, PHBA6 (P) is utilized1) Drought-sensitive series Ji 63 (P)2) And (3) assembling a basic population, and finding two drought-resistant QTL-qMd3 and QTL-qMd9 on chromosomes 3 and 9 for the first time by using a map-based cloning method.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing showing the invention P1And P2Accurately identifying and screening drought tolerance in a field;
FIG. 2 is a drawing showing a test material P of the present invention1And P2Identifying drought tolerance in the artificial rain shelter;
FIG. 3 is a drawing ofInvention F1Hybrid combinations with F2Constructing a progeny mapping population;
FIG. 4 is a drawing showing the classification criteria of tassel dry-up investigation grade according to the present invention;
FIG. 5 is a drawing of the invention C1-C5A linkage map;
FIG. 6 is a drawing of the invention C6-C10A linkage map;
FIG. 7 is a diagram showing the result of analyzing a marker polymorphism according to the present invention;
FIG. 8 is a QTL location result of 2015 of the present invention;
FIG. 9 is a QTL location result of 2016 in the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Parent material field planting and drought stress treatment method
Under the natural drought condition of Xinjiang, 300 home and abroad maize inbred lines are planted, each inbred line is planted in a 1-row area with the row length of 4m, the average row spacing of 0.55m, the average plant spacing of 0.2m and the average planting density of 6000 plants/666.7 m2. 4-month sowing in 25 days, drip irrigation and watering for 1 time, namely, seedling emergence water (about 40 m)3) Seedling emergence is carried out about 5 months and 8 days, the seedling stage is managed as same as that of a common field, and head water is poured about No. 6 months and 10 days (about 40 m)3)。
Set up 3m wide moisture median between normal irrigation zone and arid hypochondrium district, all adopt drip irrigation under the membrane, dry seeding wet-out, the seeding is finished promptly and can be watered, guarantees to emerge neat unanimously. And water meters are installed in each area to accurately control the water filling amount. Normal irrigation treatment area: watering 10 times in the whole growth period of the normal irrigation area, and irrigating 1 time at intervals of 10d according to the weather and drought conditions, and every timeAbout 40m after secondary irrigation3Other management is consistent with the field. Drought stress treatment area: on the basis of the normal irrigation management in the early period, the irrigation is stopped 15 days before the corn is castrated, the water potential psi of the soil in the drought stress treatment area is kept at-0.5-1.0 MPa, the drought stress is continued for 30 days, and the field performance data investigation is carried out once every 10 days. And detecting the field soil water potential every 5d during the drought stress period.
The irrigation quantity, the irrigation times and the irrigation time can be properly adjusted according to the weather conditions. And managing the normal irrigation treatment area with other fields. The data in Table 1 are obtained by phenotype identification of drought tolerance, tassel drought tolerance and primary screening for 2 consecutive years.
Method for identifying and evaluating drought tolerance of parent material
The parent inbred line adopts a germplasm drought tolerance index (DTIg) method to evaluate the drought tolerance of the parent inbred line.
Calculating the formula: DTIg ═ y (Yd/Ym)/(Ymd/Ymw)
DTIg in the formula: drought tolerance index;
yd: the seed yield of the material to be detected in the drought stress treatment area is determined;
ym: the seed yield of the material to be detected in the normal irrigation water treatment area is determined;
ymw: the seed yield of all materials in a normal irrigation water treatment area is increased;
ymd: grain yield in drought stress treated areas of all materials.
Note: the material to be detected and all the materials are each material in 300 parts of inbred lines, the material to be detected is to calculate the drought tolerance index of a certain material, and the Yd value of which material is the cell yield of the material in a drought area; ymw and Ymd are the sums of the plot yields of all materials (300 inbred lines) in the water and drought regions, all parameters determined to calculate the drought tolerance index for each material.
The drought tolerance of the corn germplasm resources is divided into 5 grades according to the drought tolerance index, and the evaluation standard is as follows:
level 1: the drought resistance is strong, and the drought resistance index is more than or equal to X +1.5 sigma;
and 2, stage: the drought tolerance is strong, and the drought tolerance index is more than or equal to X +0.5 sigma when X +1.5 sigma is more than or equal to X +0.5 sigma;
and 3, level: in drought tolerance, the drought tolerance index is more than or equal to X-0.5 sigma when X +0.5 sigma is more than or equal to X-0.5 sigma;
4, level: the drought tolerance is weak, and the drought tolerance index is more than or equal to X-1.5 sigma when the drought tolerance index is more than or equal to X-0.5 sigma;
and 5, stage: the drought tolerance is extremely weak, and the drought tolerance index is less than or equal to X-1.5 sigma.
Wherein X is the average drought tolerance index; σ is the standard deviation of drought tolerance index.
Drought resistance coefficient is the yield of drought stress treatment area/the yield of normal irrigation treatment area.
TABLE 1 field phenotype accurate identification screening promotion-grade statistic data table of drought tolerance of strong drought tolerance and drought tolerance materials
Name of Material Yield per mu in dry area Yield per mu in water area Drought tolerance index Drought tolerance coefficient Grade of arid region Drought tolerance
P1(PHBA6) 362.85 406.59 1.38 0.9 Level 1 Extremely strong
AB-88♂ 119.2 141.52 1.28 0.83 Stage 2 High strength
Heddle 31 111.12 140.61 1.24 0.8 Stage 2 High strength
New jade 47 female 413.15 536.49 1.19 0.77 Stage 2 High strength
PH6WC 277.49 368.91 1.16 0.75 Stage 2 High strength
502 219.51 295.37 1.15 0.74 Stage 2 High strength
B73 169 233.14 1.12 0.73 Stage 2 High strength
New jade 47 (good jade) 303.05 429.21 1.09 0.7 Grade 3 In
Dan 340 95.66 139.1 1.07 0.7 Grade 3 In
Zheng 58 162.23 240.32 1.06 0.68 Grade 3 In
New disclosure 588 190.72 281.33 1.05 0.68 Grade 3 In
Ji 853 128.79 192.53 1.04 0.67 Grade 3 In
Tuck 478 91.32 151.62 0.93 0.6 Grade 3 In
PH4CV 263.68 470.93 0.87 0.56 Grade 3 In
Chang 7-2 74.75 142.63 0.82 0.53 4 stage Weak (weak)
Yellow morning four 136.27 257.08 0.82 0.53 4 stage Weak (weak)
P2(Ji63) 74.25 200.01 0.58 0.38 4 stage Weak (weak)
Yellow C 33.74 163.75 0.31 0.2 Grade 5 Extremely weak
Selecting 1 part of American inbred line (PHBA6 is named as P) with high yield, strong drought resistance, dry-fast resistance of tassel and high combining ability according to the data in Table 11) A material; meanwhile, 1 part of domestic self-bred inbred line which is far from a P1 blood related line, has large genetic background difference, is highly sensitive to drought, is easy to dry tassels, is inactivated and dead by pollen and cannot normally pollinate and fruit (Ji 63 is named as P: P)2)；P1And P2The precise identification and screening of drought tolerance in the field is shown in figure 1.
1 2Accurate identification and difference analysis of P and P tassels of two parent inbred lines and main agronomic characters
The agronomic traits of the two parental inbred lines P1 and P2 were further characterized in an artificial rain shelter. The test was divided into a normal irrigation treatment area and a drought stress treatment area. Set up 3 meters wide moisture median between normal irrigation zone and the arid hypochondriac area, all adopt drip irrigation under the membrane, dry seeding is wet out, and the seeding is finished promptly and can be watered, guarantees to emerge neat unanimously, and each district installation water gauge carries out accurate control to the volume of watering. Normal irrigation treatment area: watering 10 times in normal irrigation area in whole growth period, and irrigating 1 time every 10 days according to drought conditions, wherein each time is about 40m3Other management in the field is consistent with that in the field. Drought stress treatment area: on the basis of the previous normal irrigation management, the irrigation is stopped 15d before the corn is castrated, the water potential psi of the soil in the drought stress treatment area is kept at-0.5-1.0 MPa, the drought stress is continuously carried out for 30d, and the field performance data investigation is carried out once every 10 d. And detecting the field soil water potential every 5d during the drought stress period.
Continuous soil drought stress causes curling, wilting and even severe withering of maize leaves and tassels to death, and the results are shown in fig. 2. For reference material P1And P2Major agronomic performances such as plant height, spike position, tassel branch number, tassel length, spike-related characters, leaf curliness, withering symptoms and the like under drought stress and normal irrigation conditions are investigated, and the results are shown in table 2.
TABLE 2 analysis of differences in major agronomic traits of test materials under drought stress treatment
Figure BDA0002415662150000071
Note: at "/" is the drought region P2The row number and the grain number of the ears result in poor fruit setting due to drought, the ears and grains are irregular, and the total ears and the seeds can be 100 grains without statistics.
Table 2 the results show that:drought-enduring line P1Drought sensitive line P with comprehensive agronomic characters2Is strong.
For reference material P1And P2The yields of (2) were investigated and analyzed, and the results are shown in Table 3.
TABLE 3 significance analysis of yield differences under drought stress treatment
Figure BDA0002415662150000072
The results in Table 3 show that the drought line P is1The yield difference is small under normal irrigation and drought stress, and the strong drought tolerance is shown; and P is2Is extremely sensitive to drought and has large yield difference.
Example 2
F1Hybrid combinations with F2Construction of progeny mapping populations, see fig. 3: respectively adding P1And P2Performing directional hybridization under normal irrigation and drought stress conditions, and obtaining F1Seed, F1Further selfing and generation-adding in Hainan to obtain F2The seed of (1).
Selection of F2500 full seeds are obtained in the offspring, and the selected 500 seeds are sown in Xinjiang to obtain F2The group is single plant, and the seedling stage is managed in the same general field. On the basis of the normal irrigation management in the early period, the irrigation is stopped 15 days before the corn is castrated, the water potential psi of the soil in the drought stress treatment area is kept at-0.5-1.0 MPa, the drought stress is continued for 30 days, and the field performance data investigation is carried out once every 10 days. And detecting the field soil water potential every 5d during the drought stress period.
For each F in seedling stage2The individual plants of the group are subjected to tag marking and sampling, DNA is extracted, and chip data is obtained by scanning by adopting a Maize6KSNP chip of China university of agriculture/national corn improvement center. And carrying out QTL initial positioning scanning by combining SNP chip data.
2F population single plant tassel dry-rot grading investigation method
The corn tassel and leaf withering survey standard is divided into 1-5 grades according to the tassel or leaf withering and curling degree:
level 1: the green-keeping property of the whole plant leaves and the tassels is good, the tassels normally bloom and loose powder, and no dry-up symptom exists completely;
and 2, stage: the local position of the tassel has a dry (dry tip) symptom, part of pollen is inactivated, and the top leaves are slightly curled but can still pollinate and fruit;
and 3, level: half branches of the tassel are dry (dry tips), pollen is inactivated, a small amount of tassels can loose powder, and 1-2 leaves begin to dry;
4, level: the male flower is dry and withered in large area (dry tip), the male flower can hardly loose powder, the pollen is inactivated, and 2-3 leaves begin to dry and wither;
and 5, stage: the tassel is completely withered, pollen is dead, pollination and fructification cannot be completed, and 3-4 leaves begin to wither.
The phenotype survey was conducted at Peking time 14:00-16:00, every 7d for 3 surveys, and the detailed survey grade classification criteria are shown in FIG. 4.
Qualitative analysis is carried out on the phenotype data (table 4) and the SNP chip data to obtain a genetic scanning map of the initial positioning of the tassel drought-resistant QTL (figure 5-figure 6) in the flowering period of the corn.
TABLE 4F2Statistical table of phenotype data of two continuous years under drought stress of population progeny
Figure BDA0002415662150000091
The genetic map processing software and the method comprise the following steps:
a genetic linkage map was constructed by the JoinMap4.0 mapping software, and the total length of the linkage map was 1427.82 CM.
QTL initial positioning data analysis
SNP chip for two parents and 187F2The individuals of the population are subjected to genotype analysis. The SNP chip contained 5252 markers in total, and of them, 2187 showing polymorphisms in the parents accounted for 41.64% of the total markers, and the polymorphism was better. These markers were scattered over 10 chromosomes (FIG. 7).
Binding of F for two consecutive years by 2015 and 20162Phenotypic data of the individual plants of the population and genotype data of the SNP chip of 6K are subjected to QTL mapping, and the results are shown in FIGS. 8-9. With LOD2.5 as a threshold value, a major QTL is located on chromosome 3 in two years, and a minor QTL is located on chromosome 9 and is named as QTL-qMd3 and QTL-qMd9 respectively.
The invention combines F through 2015 and 2016 for two consecutive years2Phenotypic data of a single plant of a group and SNP chip genotype data of 6K are used for carrying out initial positioning research on the maize drought-resistant character QTL, effective peak values are repeatedly obtained in the same region of No. 3 and No. 9 chromosomes, QTL scanning positioning regions in two years are basically consistent, and a solid foundation is laid for smooth follow-up fine positioning work.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (3)

1. A construction method of maize flowering period tassel dry-resistant QTL location segregation population is characterized by comprising the following specific steps:
(1) identification and selection of a population parent inbred line: after the drought tolerance identification of the field for many years, the inbred line PHBA6 with strong drought tolerance and dry-fast tassel is taken as P1The inbred line Ji 63 which is highly sensitive to drought and easy to dry tassel is taken as P2；
(2) Preparing a hybridization combination: planting P in Xinjiang1And P2And P is added during the flowering phase1And P2Bagging and pollinating P1×P2Directional hybridization to obtain F1Seeds; f is to be1Planting seeds in Hainan, bagging in flowering period, selfing and pollinating to obtain F2OfA seed;
(3)F2phenotypic characterization of the segregating population: selection of F2Seeds with plump seeds are planted in Xinjiang to obtain F2Group individual plants; for each F in seedling stage2Carrying out plate hanging, marking and sampling on the individual plants of the group offspring, extracting DNA, and carrying out SNP chip scanning to obtain chip data;
(4) survey of tassel dry-rot resistant phenotype: under drought stress conditions on F2Carrying out accurate identification, grading investigation, statistical analysis and data processing on the phenotype of the tassel dry-rot character of a single plant of a group;
(5) according to SNP chip data and F2Constructing a genetic linkage map by using phenotype data of drought resistance of the tassels of the individual plants of the population;
(6) according to F2And carrying out QTL initial positioning on phenotype data and SNP chip data of the individual plants of the group.
2. The method for constructing the maize flowering phase tassel drought-tolerant QTL location segregation population according to claim 1, wherein the soil water potential psi during drought stress in step (2) is-0.5-1.0 MPa.
3. The method for constructing the maize flowering phase tassel drought-resistant QTL location segregation population of claim 1, wherein the QTL of step (6) is initially located on chromosome 3 and chromosome 9.
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