Breeding,of,Super-large-grain,Wheat,Germplasms

Zhanliang YANG Xiaoli BAO Yongshun XU Qiaoxu CHU Fangyun CHU

Abstract ［Objectives］This study was conducted to breed high-yielding wheat varieties.

［Methods］ A new breeding technology of "Artificial mutagenesis to increase genetic differences between parents and to enhance heterosis" was put forward. Longnan 71a and Longnan 71a mutants bred by this technology were crossed with other mutants and normal materials.

［Results］ In the Wudu experimental field of Longnan City, the traits of super-large grain appeared in the obverse and inverse cross with Longnan 71a as parents. The trait of super-large grain was dominantly inherited. The traits of various combinations in the F2 generation were segregated. In the F2 generation, one super-large-grain low-stalk line and other lines with specific traits were selected. In the F3 generation, two super-large-grain low-stalk large-ear lines were selected. These three super-large-grain low-stalk wheat lines had reduced plant height and good lodging resistance, and possessed two high-yielding characters among the three factors of yield, so they were expected to be further bred into high-yielding wheat varieties. One line with the largest grain length of 10.3 mm was selected in the F4 generation. After two years of breeding, in 2022, the largest grain length was measured to be 10.6 mm, and the average 1 000-grain weight and the largest 1 000-grain weight were 75.8 and 100 g, respectively.

［Conclusions］ This study is about the major progress in artificial cultivation of super-large-grain wheat and has considerable practical value.

Key words Artificially induced mutation; Malformed mutation; Genetic difference; Hybridization; Super-large grain

Received: July 16, 2022  Accepted: August 18, 2022

Supported by 2021 Financial Linkage Fund for Promoting Rural Revitalization in Wudu District, Longnan City (WNLF (2021)3).

Zhanliang YANG (1961-), male, P. R. China, middle-school senior teacher, devoted to research about crop breeding.

*Corresponding author.  E-mail: 2643262969@qq.com.

As the worlds second largest crop and a staple food for more than half of the population, wheat is in high demand. In order to meet the food demand of the people, finding new breeding technologies and cultivating high-yield and super-high-yield wheat varieties is an effective means to increase the yield per unit area of wheat, and it is also one of the necessary measures to deal with Chinas food crisis. The components of wheat yield are: grain weight, number of grains per ear, and number of effective ears per unit area. It is estimated that under the condition that the number of grains per ear and the number of effective ears per unit area remain stable, the yield per hectare will increase by 225 kg for every 1 g increase in the thousand-grain weight. Grain weight has the greatest heritability and is also the most stable yield trait that is least affected by the environment［1］. Breeding large-grain and super-large grain wheat varieties is one of the feasible means to achieve high and ultra-high yield. The methods can be as follows (the transgenic technology is beyond the scope of this paper):

First, the aggregation of large-grain genes can be accomplished by crossing two or more parents containing large-grain genes to produce large-grain and super-large-grain wheat varieties. In this conventional method of hybridization, the hybrid progeny should follow Mendelian segregation, and large-grain and super-large-grain varieties with stable inheritance are selected.

Second, radiation mutagenesis［2］, and crossbreeding of mutant deformed plants due to radiation mutagenesis with normal target parents, have also produced germplasm materials for large-grain and super-large-grain wheat［3］.

Third, knocking out the repressor genes of large-grain genes results in the deletion of gene fragments, increase in grain weight and the semi-dwarf phenotype, so that large-grain and dwarf genes can be expressed, and large-grain dwarf wheat varieties can be cultivated［4］.

We used radiation-mutated deformed mutant materials to cross with target parents, and in 2014, we selected the super-large grain wheat "Longnan 71a" and a series of other germplasm materials with specific traits［3］. "Longnan 71a" showed an average thousand-grain weight of 61.5 g, a maximum thousand-grain weight of 84.8 g, a maximum number of grains of 106, and a maximum single-grain weight of 8.3 g in 2015. Its maximum grain length, grain width and grain thickness were 9.6, 4.3 and 4.1 mm, respectively. Because "wheat grain weight is mainly determined by grain length, grain width and grain thickness"［5］, such a grain shape determines a larger thousand-grain weight value. However, although "Longnan 71a" has strong tillering ability and high yield, its plant height is between 80-130 cm and its lodging resistance is poor, so it cannot be used directly as a variety. In order to convert the advantages of super-large grain and strong tillering ability into usable cultivar traits, these materials were crossed as parents with other dwarf materials and deformed dwarf materials, aiming to dwarf super-large-grain germplasm materials to enhance lodging resistance.

Materials and Methods

Materials and combination design

In 2016, in the experimental field in Wudu District, Longnan City, we selected the super-large-grain wheat materials "Longnan 71a" and a dwarf mutant line, extremely dwarf line 1a, short-stalk line No.12 with the flag leaf connected to the ear, No.31, mutant line 10a, and mutant line 64c reappeared in "Longnan 71a", and formed seven combinations: ① 71a dwarf mutant line×extremely dwarf line 1a, numbered 2016-1, ② 71a×extremely dwarf line 1a, numbered 2016-2, ③ short-stalk line No.12 with the flag leaf connected to the ear×71a, numbered 2016-3, ④ No.31×71a, numbered 2016-4, ⑤ mutant line 10a ×71a, numbered 2016-5, ⑥ mutant line 64c×short-stalk line No.12 with the flag leaf connected to the ear, numbered 2016-6, and ⑦ 71a×short-stalk line No.12 with the flag leaf connected to the ear, numbered 2016-7. "Longnan 71a" and Panlin were used as observation control materials.

Experimental methods

Conventional crossbreeding, manual emasculation, and bagging and marking were adopted. Pollination was performed for the first time 2-3 d after emasculation, and the second time was 3-4 d after emasculation.After maturity, seeds of single plants were harvested as F0. In the autumn of 2017, the seeds of the F0 generation were sown. The F0 generation of the three combinations, 2016-1, 2016-2, and 2016-3 with rare seeds, were sown in flowerpots in the campus of Wu Du Ba Yi Middle School, and the F0 generation of the combinations with more seeds was sown in the large experimental field of Chengxian County, which is 100 km away. Among them, 2016-3 obtained many seeds of the F0 generation, which were sown in the flowerpots on the campus of Wu Du Ba Yi Middle School and the large experimental field in Chengxian County. Among them, 2016-3 obtained many seeds of the F0 generation, which were sown in the flowerpots in the campus of Wu Du Ba Yi Middle School and the large experimental field in Chengxian County both. In 2018, the F2 generation was propagated in the large experimental field of Chengxian County. It was also sown in pots in the campus as backups. From the F2 generation onwards, selections were made for individual plants with different performance that appeared in each combination. Individual plants with dwarf stalks, large ears, large grains and disease resistance were mainly selected. The selected individual plants were marked by hanging a board. When marking, plants with a height of less than 65 cm were marked as "dwarf stalk"; plants with a height of 65-85 cm were marked as "medium stalk"; plants with a height of over 85 cm were marked as "tall stalk". The seed size was determined after harvesting. When testing seeds, vernier calipers were used to measure grain data, and an electronic balance was used to measure grain weight. Materials with specific traits and high yield value were selected to continue hybridization, and the selected excellent lines will be optimally propagated from generation to generation.

Results and Analysis

Performance of the hybrid F1 generation

In 2017, Wudu District had less rainfall and sufficient sunshine, and the sown F0 generation hybrid seeds matured well in the F1 generation. Two combinations with 71a as the female parent and one inverse cross combination with 71a as the male parent all showed the super-large grain trait, and the super-large grain trait was dominantly inherited. That year, Chengxian had a lot of rainfall. After entering April, there were four rainy days per week and less than three sunny days on average. Therefore, the wheat seedlings grew wildly, and fell seriously, and the grain filling was insufficient and the maturity was insufficient. Such abnormal weather obscured the true performance of genes of the F1 generation. For example, for 2016-3, the 1 000-grain weight of the F1 generation in Wudu was 68.1 g, while the 1 000-grain weight in Chengxian was 51.3 g, as shown in Table 1［6］. The performance varied greatly in the two different growing environments. It was speculated that the last four combinations would also have advantages in normal growth environment. Comparing the hybrid groups and Longnan 71a with Panlin, significant differences were observed in plant height, grain size and grain weight.

Performance of the hybrid F2 generation

More seeds of the F1 generation were harvested. In 2018, 66.7 m2 was planted for each of the seven combinations in the large experimental field in Chengxian County. The seeds were also sown in pots in the campus of Wu Du Ba Yi Middle School as backups. Due to the large seeding rate for the F2 generation, a large number of various plant shapes appeared in the experimental field in Chengxian County. Among the seven combinations, except 2016-6, the remaining six combinations had the "71a" lineage, and the plant heights were significantly lower than that of "71a" in the control group, exhibiting a significant dwarfing effect. In the F2 generation, there were many "short-stalk" large-ear disease-free single plants with strong tillering ability, as well as "medium-stalk" large-ear single plants. Because the experimental field in Chengxian County encountered heavy rain for three consecutive days during harvest, most of the F2 hybrid materials were soaked and germinated. Fortunately, some important materials were grabbed in the heavy rain. The backups seeded in Wudu guaranteed the integrity of the materials. In the 2016-1 combination, one "medium-stalk large-ear" material was marked, and a medium-stalk large-ear super-large-grain material line was selected from its F3 generation. In the 2016-2 combination, a "medium-stalk large-ear" material was marked, and a super-large-grain medium-stalk large-ear line resistant to powdery mildew was selected from its F3 generation. In the 2016-3 combination, a "dwarf-stalk large-ear" material was marked, with a grain length≤10 mm, and its characters were stably inherited in the F3 generation. In the 2016-5 combination, one "relatively-resistant dwarf-stalk" material was marked.

Performance of the hybrid F3 generation

In 2019, the hybrid F3 generation was sown in Berlin Town, Wudu District and Nanshan Ecological Park, Wudu District. The two experimental fields were both sown with the four lines with specific traits specially noted in the F2 generation. The "super-large-grain large-ear dwarf-stalk (grain length≤10mm)" line selected from the F2 generation of the 2016-3 combination was stably inherited in the F3 generation. From the "medium-stalk large-ear" material marked in the F2 generation of the 2016-1 combination, a large-ear material was selected in the F3 generation. A super-large-grain medium-stalk line resistant to powdery mildew was selected in the F3 generation of the 2016-2 combination. In the F4 generation of the 2016-5 combination, the specific traits of "relatively-high resistance, dwarf stalk" were stably inherited. The inbred lines of the seven combinations exhibited large trait segregation and abundant trait variation.

Agricultural Biotechnology2022

Re-mutation of lines with specific traits

These selected specific large-grain or super-large-grain germplasms had a common feature, their specific traits were stably inherited, and it seemed that Mendelian trait segregation no longer occurs. However, non-Mendelian segregation still occurred, that is, a very small number of re-mutated plants appeared. For example, "71a" appeared in the F1 generation with very few re-mutated individual plants, "71b" and "71c". For the 2016-2 combination (Fig. 1), an individual plant with a grain length of 10.3 mm appeared in the F4 generation (Fig. 2). When propagated to the F6 generation, the largest grain was 10.6 mm long (Fig. 3), and the weight of a single grain was 0.1 g, and the weight of 10 grains was 1.04 g (Fig. 4). Moreover, the largest thousand-grain weight is 100 g, and the average thousand-grain weight was 75.9 g.

Judging from the shape of the grains, they were still in a lean state and not fully matured. The grains would be larger if grain filling was sufficient and the grains were plump and mature under optimal cultivation conditions. The "super-large-grain dwarf-stalk material (grain length ≤10 mm)" selected from the 2016-3 combination had stable inheritance of the "super-large-grain dwarf-stalk" characteristics in the F3 generation in 2019, but two-thirds of the plants were not infected with powdery mildew while one-third of the plants were infected with powdery mildew. Large-grain dwarf disease-resistant individual plants appeared in the 2016-4 combination. It was confirmed by observation and testing that the 2016-4 line had a compact plant type, plump grains, and good resistance, so it had concentrated fine traits and the advantage of high yield. The F4 generation of the 2016-5 combination stably inherited the traits of "relatively high resistance, low stalk and large ear" (Table 2).

Discussion

In this study, according to New Breeding Technology Using Artificial Mutagenesis for Improving Hybrid Vigor by Increasing the Genetic Difference between Parents［6］, the selected new super-large-grain wheat germplasm material "Longnan 71a" was combined with other specific deformed mutant dwarf materials. The hybridization began in the summer of 2016, and the high-stalk super-large-grain wheat germplasm material was successfully dwarfed within 4 years to 2020. The yield traits of the bred wheat germplasm materials with "large grains, large ear and low stalk" achieved two of the three elements of super-high-yielding wheat. As long as the cultivation technology is tackled in increasing effective number of grains per unit area to the best value, it is expected to achieve the goal of super-high-yielding wheat varieties, which also proves the breeding efficiency and practical value of the "new breeding technology".

It is worth noting that when individual plants with very peculiar characters were found in the inbred lines of the test materials or hybrid combinations, they could be inherited stably, and it seemed that the phenomenon of Mendelian trait segregation no longer occurs. Han et al.［8］ believe that "mutagenic breeding has a unique role in improving crop varieties. It can induce gene mutations and produce new types, new traits, and new genes that are not originally available in nature or that are difficult to obtain by conventional methods, and can break gene linkages and improve the recombination rate". It is speculated that artificially induced gene mutations may belong to "point mutations", which occur on a specific chromosome, and its genetic behavior is consistent with the chromosome and chromosome group where it is located. When an artificially induced mutant parent is crossed with a normal plant, or with another type of mutant parent, and individual plants with superparent strange traits appear in the hybrid progeny, and its peculiar superparent traits can be stably inherited in the F2 or F3 generation, which may be due to that bizarre gene recombination events occur in this process, resulting in new genes and new traits. In the 2016-2 combination, a super-large-grain wheat germplasm with a maximum grain length of 10.6 mm, a maximum 1 000-grain weight of 100 g and an average 1 000-grain weight of 75.9 g was selected, which has both theoretical significance and practical value. The wheat germplasm with an average 1 000-grain weight of 65.7 g selected in the 2016-4 combination concentrated excellent traits and showed outstanding high-yielding trait.

We refer to the breeding technology of selecting superior varieties from the offspring by crossing a deformed mutant parent which obtained by artificial mutagenesis with a normal target parent or another deformed mutant parent as a "new technology". Its theoretical

basis is that artificially-induced gene mutation leads to phenotypic deformity mutant plants, which have large genetic differences from the target parent, and are close to extreme types, and strong or super-strong heterosis will appear after hybridization (Table 1)［6］, because "the fundamental reason for the formation of heterosis lies in genetic differences between the parents"［7］. In breeding practice, using deformed mutant plants as a parent for crossing will produce abundant superparent lines in the hybrid progeny. As long as the population of inbred lines is large enough, there are many useful traits to choose from. When an individual plant with very peculiar traits is found, the traits of the selected line will be stably inherited, and a very small number of mutant plants will decrease from generation to generation with propagation, and the line will tend to be stable and consistent relatively quickly. The discovery of this law can shorten the breeding period and speed up the breeding process.

References

［1］ CHEN JH, LAN JH, WANG H, et al. QTL mapping for traits of kernel morphology and grain weight in common wheat［J］. Journal of Triticeae Crops, 2011, 31(6): 1001-1006. (In Chinese).

［2］ WANG CP, ZUI LZ, XU Q, et al. RVA parameter variation among M2 population of radiated wheat variety "Nongda 179"［J］. Journal of Triticeae Crops, 2007, 27(2): 237-240. (In Chinese).

［3］ YANG ZL, ZHU QX, ZHANG YL, et al. The effects of breeding super high yield wheat varieties by mutation and hybridization［J］. Journal of Anhui Agricultural Sciences, 2015, 43(18): 37-40. (In Chinese).

［4］ XU DG, WEN WE, FU LP, et al. Genetic  dissection of a major QTL for kernel weight Spanning the Rht-Bi locus in bread wheat［J］. Theor Appl Genet.2019 sep 12.pii:10.1007/s00122-019-03418-w.doi: PMID:31515582.

［5］ BRESEGHELLOF,SORRELLS ME. Association mapping of kernel  size and milling  quality  in  wheat (Triticum  aestivum L.) cultivars［J］. Genetics, 2006, 172(2): 1173.

［6］ YANG ZL, CHU QX, YOU LJ, et al. A new breeding technique for increasing genetic differences between parents and enhancing heterosis［J］. International Journal of Applied Agricultural Sciences, 2019: 56-61. doi: 10.11648/j.ijaas.20190502.15

［7］ LIU CG. The prospects of studies on cytoplasmic male-sterile line in common wheat［J］. Exploration of Nature, 1994, 13(4): 57-61. (In Chinese).

［8］ HAN WB, LIU LX, GUO HJ, et al. Advance of new techniques in wheat mutation breeding［J］. Journal of Triticeae Crops, 2005, 25(6): 125-129. (In Chinese).